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Sample records for extracellular atp levels

  1. The P2X7 receptor is an important regulator of extracellular ATP levels

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    Andrea eBrandao-Burch

    2012-03-01

    Full Text Available Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signalling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells, possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05-0.5pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are 4-fold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1-100µM AZ10606120, A438079 and KN-62 were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079 and KN-62, at 0.1-10µM, decreased ATP release by mature osteoclasts by up to 70%, 60% and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1-100µM NEM or brefeldin A had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1-10µM also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signalling in bone.

  2. Modulation of Extracellular ATP Content of Mast Cells and DRG Neurons by Irradiation: Studies on Underlying Mechanism of Low-Level-Laser Therapy

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    Lina Wang

    2015-01-01

    Full Text Available Low-level-laser therapy (LLLT is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca2+]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

  3. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

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    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  4. Extracellular ATP induces albuminuria in pregnant rats

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    Faas, M.M.; van der Schaaf, G.; Borghuis, T.; Jongman, R.M.; van Pampus, Maria; de Vos, P.; van Goor, Harry; Bakker, W.W.

    2010-01-01

    BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula.

  5. Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

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    Kowal, Justyna Magdalena

    ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......Y receptors. Previously, our group has shown that cholinergic stimulation of acini caused ATP release into ducts and ATP is an important regulator of ductal functions by being involved in ion and fluid secretion. Pancreatic duct cells are exposed to a number of stimuli, well known to induce ATP...... release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological...

  6. Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena

    ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......Y receptors. Previously, our group has shown that cholinergic stimulation of acini caused ATP release into ducts and ATP is an important regulator of ductal functions by being involved in ion and fluid secretion. Pancreatic duct cells are exposed to a number of stimuli, well known to induce ATP...... release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological...

  7. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU).

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    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.

  8. Extracellular ATP and P2X7 receptors in neurodegeneration.

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    Le Feuvre, Rosalind; Brough, David; Rothwell, Nancy

    2002-07-05

    Neuronal injury and cell death in the central nervous system (CNS) are underlying features of neurodegenerative disorders. However, our understanding of the fundamental mechanisms involved is still limited. Inflammatory processes mediated by cytokines, and interleukin-1 (IL-1) in particular, play a significant role in neuronal death following pathological insults. Despite this growing area of research, very little is known about the factors regulating the expression, cleavage and release of interleukin-1 in the brain. Recent studies on immune cells demonstrate that extracellular ATP can act as a potent stimulus for the maturation and release of interleukin-1beta, via activation of P2X7 receptors. Stimulation of P2X7 receptors with ATP has dramatic cytotoxic properties and a wider role in neurodegenerative processes is possible. This review discusses the potential involvement of extracellular ATP and P2X7 receptors as regulators of interleukin-1-mediated neuropathologies and thus as a mediator of cell death following pathological insults.

  9. Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats

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    Spaans, F.; Melgert, B. N.; Chiang, C.; Borghuis, T.; Klok, P. A.; de Vos, P.; van Goor, H.; Bakker, W.W.; Faas, M. M.

    2014-01-01

    Introduction: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant

  10. Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

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    Wu, Shu-Jing; Wu, Jian-Yong

    2008-01-01

    Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca2+ and H2O2 in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10–500 μM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10–100 μM but decreased from 100–200 μM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPγS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca2+ antagonists, but not affected by a protein kinase inhibitor. ATP also induced H2O2 production, which was dependent on both Ca2+ and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca2+ level, which was dependent on NO but not H2O2. The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca2+ and ROS signalling. PMID:18977749

  11. Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

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    Leigh A. Swayne

    2017-08-01

    Full Text Available Pannexin 1 (Panx1 channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs. Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

  12. Extracellular ATP acts as a damage associated molecular pattern (DAMP signal in plants

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    Kiwamu eTanaka

    2014-09-01

    Full Text Available As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs. ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling role in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor Kinase, which is plant-specific. P2K1 (DORN1 is required for ATP-induced cellular responses (e.g., cytosolic Ca2+ elevation, MAPK phosphorylation, and gene expression. Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of the future research of extracellular ATP as a DAMP signal.

  13. Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

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    Becker Walter

    2011-09-01

    Full Text Available Abstract Background Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells. We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells. Results The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM. UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS. This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin and BDNF (brain derived neurotrophic factor. Conclusion The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein

  14. A receptor that is highly specific for extracellular ATP in developing chick skeletal muscle in vitro.

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    Thomas, S A; Zawisa, M. J.; X. Lin; Hume, R. I.

    1991-01-01

    1. Extracellular adenosine 5'-triphosphate (ATP) activated an early excitatory conductance followed by a late potassium conductance in developing chick skeletal muscle. A series of ATP analogues were tested for their ability to activate these two conductances. All compounds tested were either agonists for both responses or for neither. Furthermore, the potency of agonists was similar for the two responses. 2. The order of potency for agonists was ATP approximately adenosine 5'-O-(3-thiotripho...

  15. Extracellular ATP and adenosine : The Yin and Yang in immune responses?

    NARCIS (Netherlands)

    Faas, M. M.; Saez, T.; de Vos, P.

    Extracellular adenosine 50-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under

  16. Effects of Extracellular ATP and Its Receptors on Peripheral Nerve Regeneration in SD Rats

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    王栓科; 张致英; 洪光祥; 王同光; 王发斌; 康皓

    2003-01-01

    To explore the effect of the extracellular ATP and its receptors on axon regeneration in the sciatic nerve defect in rats, 0. 5 cm defect of the sciatic nerve was made and repaired with long arm silicon tube like a "Y" type. The single arm of the silicon tube was sutured to proximal of the sciatic nerve. 10μl 1 mmol/L ATP in physiological saline was injected into the left chamber of the silicon tube (experimental group) and physiological saline injected into another silicon tube as a control group. In another model 1 mmol/L 10 μl ATP and 1 mmol/L 10μl ATP+0.2 mg/ml suramin were injected respectively into two arms of the silicon tube. After 4 and 8 weeks the specimens were obtained from the silicon tube for examining the axon regeneration histologically and image analysis. All the regeneration axons grew into the silicon tube containing the ATP, but there was no axon regeneration in the silicon tube containing the ATP+Suramin or physiological saline. It was demonstrated that extracellular ATP had a powerful attraction to the regenerated axon of peripheral nerve. The suramin inhibited the axon induction of the extracellular ATP completely via the ATP receptors.

  17. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

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    Shin, Youn Ho; Lee, Seo Jin [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of); Jung, Junyang, E-mail: jjung@khu.ac.kr [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  18. Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

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    Brisevac, Dusica; Adzic, Marija; Laketa, Danijela; Parabucki, Ana; Milosevic, Milena; Lavrnja, Irena; Bjelobaba, Ivana; Sévigny, Jean; Kipp, Markus; Nedeljkovic, Nadezda

    2015-11-01

    Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis.

  19. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

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    Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

    2016-01-01

    Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

  20. Regulation of extracellular ATP in human erythrocytes infected with Plasmodium falciparum.

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    Cora Lilia Alvarez

    Full Text Available In human erythrocytes (h-RBCs various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages. A "3V" mixture containing isoproterenol (β-adrenergic agonist, forskolin (adenylate kinase activator and papaverine (phosphodiesterase inhibitor was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs, t-RBCs (trophozoite-infected RBCs and s-RBCs (schizont-infected RBCs showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe] increased nonlinearly with parasitemia (from 2 to 12.5%. Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an

  1. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

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    M Florencia Leal Denis

    Full Text Available The peptide mastoparan 7 (MST7 triggered in human erythrocytes (rbcs the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe, interacting with P (purinergic receptors, can affect cell volume (Vr, we explored the dynamic regulation between Vr and ATPe.We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors.In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%.Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

  2. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank B; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco(2) showed negligible hemolysis (ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than...... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O(2) saturation did not lead to additional ATP release. An elevation of Pco(2) was also without influence on erythrocyte ATP release. In the saline...

  3. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than...... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco2 was also without influence on erythrocyte ATP release. In the saline...

  4. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than...... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco2 was also without influence on erythrocyte ATP release. In the saline...

  5. ATP, an extracellular signaling molecule in red blood cells: A messenger for malaria?

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    Ghania Ramdani

    2014-10-01

    Full Text Available Adenosine 5′ triphosphate (ATP, discovered in 1929 by Karl Lohmannest, is described as an essential energy source for cells. In the biochemistry of all living organisms, ATP hydrolysis provides the energy required for the chemical reactions of metabolism. It is the precursor of a number of essential enzyme cofactors, such as nicotinamide adenine dinucleotide (NAD + and coenzyme A [NAD + , flavin adenine dinucleotide (FAD, and is ATP coenzyme A are all formed from ATP] and is the source of the phosphoryl group in most kinase-mediated phosphorylation reactions. Another essential, but less known function is that ATP plays a very important role as an extracellular signaling molecule, allowing cells and tissues to communicate. ATP is converted into cAMP, a major second messenger involved in many cellular processes, by adenylyl cyclase, a membrane-associated enzyme. In this review, we describe the role of ATP as a beneficial extracellular molecule released by healthy red blood cells (RBCs in response to hypoxia to mediate a vasodilator signal, by oxidatively stressed RBCs, and by Plasmodium falciparum-infected RBCs (iRBCs, and its similarity with released ATP that by the combined action of the ectonucleotidases CD39 and CD73 is converted to adenosine that mediates sickling in sickle cell disease (SCD.

  6. Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection.

    Science.gov (United States)

    Séror, Claire; Melki, Marie-Thérèse; Subra, Frédéric; Raza, Syed Qasim; Bras, Marlène; Saïdi, Héla; Nardacci, Roberta; Voisin, Laurent; Paoletti, Audrey; Law, Frédéric; Martins, Isabelle; Amendola, Alessandra; Abdul-Sater, Ali A; Ciccosanti, Fabiola; Delelis, Olivier; Niedergang, Florence; Thierry, Sylvain; Said-Sadier, Najwane; Lamaze, Christophe; Métivier, Didier; Estaquier, Jérome; Fimia, Gian Maria; Falasca, Laura; Casetti, Rita; Modjtahedi, Nazanine; Kanellopoulos, Jean; Mouscadet, Jean-François; Ojcius, David M; Piacentini, Mauro; Gougeon, Marie-Lise; Kroemer, Guido; Perfettini, Jean-Luc

    2011-08-29

    Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches. © 2011 Séror et al.

  7. The Danger Signal Extracellular ATP Is an Inducer of Fusobacterium nucleatum Biofilm Dispersal

    Science.gov (United States)

    Ding, Qinfeng; Tan, Kai Soo

    2016-01-01

    Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also showed higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to detach

  8. Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers.

    Science.gov (United States)

    1986-01-01

    ATP may mediate contraction in the urinary bladder of the rat and guinea-pig (53,63,99,238), relaxation in taenia coli (17,63,87,173,380,381) and...receptors. This uncertainty has been generated because of findings in rabbit anococcygeus muscle (405) and guinea-pig taenia coli (457), in which, as...and Holmberg, B. The effects of extracellularly -~ applied ATP and related compounds on electrical and mechanical activity of the smooth muscle taenia

  9. Dynamic increase in extracellular ATP accelerates photoreceptor cell apoptosis via ligation of P2RX7 in subretinal hemorrhage.

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    Shoji Notomi

    Full Text Available Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD. In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV, growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor. Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG, a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.

  10. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    Science.gov (United States)

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  11. Effects of Extracellular ATP on Survival of Sensory Neurons in the Dorsal Root Ganglia of Rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ATP was added to the cultured sensory neurons obtained from the dorsal root ganglia of the neonatal rats and PBS was added to serve as control. MTT assays were conducted to evaluate the survival and activity of the cultured neurons. And the silicone regenerative chamber was used after the sciatic nerve incision of the mature SD rat. 1 mmol/L ATP was injected into the left chamber and 0.09 % natrium chloride was injected into the right chamber as controls. The changes of nitric oxide synthase (NOS) activity in the corresponding dorsal root ganglia were measured histochemically and image analysis was also performed 4 days after the sciatic nerve injury. The results showed that extracellular ATP could enhance the survival of the neurons and the number of NOS positive neurons were significantly different between the ATP and control groups (P<0.05). It was suggested that extracellular ATP had neurotrophic effect on neurons survival and could inhibit the NOS activity of the sensory neurons after the peripheral nerve incision, hence exerting the protective effect on the neurons, which was valuable for nerve regeneration after nerve injury.

  12. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

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    Elizabeth eMedina-Castellanos

    2014-11-01

    Full Text Available The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation. During this response, reactive oxygen species (ROS are produced by the NADPH oxidase (Nox1/NoxR complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK pathways by eATP, Ca2+ and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP. Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

  13. Extracellular ATP Promotes Stomatal Opening of Arabidopsis thaliana through Heterotrimeric G Protein Subunit and Reactive Oxygen Species

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hao; Wei-Xia Wang; Chen Chen; Yu-Fang Wang; Ting Liu; Xia Li; Zhong-Lin Shang

    2012-01-01

    In recent years,adenosine tri-phosphate(ATP)has been reported to exist in apoplasts of plant cells as a signal molecule.Extracellular ATP(eATP)plays important roles in plant growth,development,and stress tolerance.Here,extracellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness.ADP,GTP,and weakly hydrolyzable ATP analogs(ATPγS,Bz-ATP,and 2meATP)showed similar effects,whereas AMP and adenosine did not affect stomatal movement.Apyrase inhibited stomatal opening.ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor(diphenylene iodonium)or deoxidizer(dithiothreitol),and was impaired in null mutant of NADPH oxidase(atrbohD/F).Added ATP triggered ROS generation in guard cells via NADPH oxidase.ATP also induced Ca2+ influx and H+ efflux in guard cells.In atrbohD/F,ATP-induced ion flux was strongly suppressed.In null mutants of the heterotrimeric G protein α subunit,ATP-promoted stomatal opening,cytoplasmic ROS generation,Ca2+ influx,and H+ efflux were all suppressed.These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein,ROS,cytosolic Ca2+,and plasma membrane H+-ATPase.

  14. Extracellular ATP in the lymphohematopoietic system: P2Z purinoceptors and membrane permeabilization

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    Persechini P.M.

    1998-01-01

    Full Text Available The effects of extracellular nucleosides and nucleotides on many organs and systems have been recognized for almost 50 years. The effects of extracellular ATP (ATPo, UTPo, ADPo, and other agonists are mediated by P2 purinoceptors. One of the most dramatic effects of ATPo is the permeabilization of plasma membranes to low molecular mass solutes of up to 900 Da. This effect is evident in several cells of the lymphohematopoietic system and is supposed to be mediated by P2Z, an ATP4--activated purinoceptor. Here, we review some basic information concerning P2 purinoceptors and focus our attention on P2Z-associated phenomena displayed by macrophages. Using fluorescent dye uptake, measurement of free intracellular Ca2+ concentration and electrophysiological recordings, we elucidate some of the events that follow the application of ATP to the extracellular surface of macrophages. We propose a regulatory mechanism for the P2Z-associated permeabilization pore. The presence of P2 purinoceptors in cells of the lymphohematopoietic system makes them potential candidates to mediate immunoregulatory events

  15. Intracellular Assessment of ATP Levels in Caenorhabditis elegans

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    Palikaras, Konstantinos; Tavernarakis, Nektarios

    2017-01-01

    Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans. PMID:28194429

  16. Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

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    Rozan Vroman

    2014-05-01

    Full Text Available Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms, highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form

  17. Extracellular ATP and other nucleotides-ubiquitous triggers of intercellular messenger release.

    Science.gov (United States)

    Zimmermann, Herbert

    2016-03-01

    Extracellular nucleotides, and ATP in particular, are cellular signal substances involved in the control of numerous (patho)physiological mechanisms. They provoke nucleotide receptor-mediated mechanisms in select target cells. But nucleotides can considerably expand their range of action. They function as primary messengers in intercellular communication by stimulating the release of other extracellular messenger substances. These in turn activate additional cellular mechanisms through their own receptors. While this applies also to other extracellular messengers, its omnipresence in the vertebrate organism is an outstanding feature of nucleotide signaling. Intercellular messenger substances released by nucleotides include neurotransmitters, hormones, growth factors, a considerable variety of other proteins including enzymes, numerous cytokines, lipid mediators, nitric oxide, and reactive oxygen species. Moreover, nucleotides activate or co-activate growth factor receptors. In the case of hormone release, the initially paracrine or autocrine nucleotide-mediated signal spreads through to the entire organism. The examples highlighted in this commentary suggest that acting as ubiquitous triggers of intercellular messenger release is one of the major functional roles of extracellular nucleotides. While initiation of messenger release by nucleotides has been unraveled in many contexts, it may have been overlooked in others. It can be anticipated that additional nucleotide-driven messenger functions will be uncovered with relevance for both understanding physiology and development of therapy.

  18. Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells.

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    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Hein, Martina; Petersen, Frank; Thon, Lutz; Adam, Dieter; Bulfone-Paus, Silvia

    2005-04-01

    Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

  19. Trophic actions of extracellular ATP: gene expression profiling by DNA array analysis.

    Science.gov (United States)

    Neary, J T

    2000-07-01

    In addition to Professor Burnstock's work on the short-term signaling actions of extracellular nucleotides and nucleosides, Geoff has had a long-standing interest in trophic actions of purines in development and in pathophysiological conditions which has been instrumental in encouraging my work in this area. The trophic actions of extracellular ATP, alone or in combination with polypeptide growth factors, may play an important role in brain development and may contribute to the reactive gliosis that accompanies brain injury and neurodegeneration. P2Y receptors in astrocytes are coupled to the ERK/MAPK cascade, a signal transduction mechanism crucial for cellular proliferation and differentiation. The mitogenic signaling pathway from P2Y receptors to ERK involves phospholipase D and a calcium-independent PKC isoform, PKCdelta. DNA array analysis reveals a number of changes in gene expression after P2Y receptor occupancy, indicating that this methodology will be a powerful tool in understanding the mechanisms underlying the trophic actions of extracellular nucleotides and nucleosides.

  20. Effect of extracellular adenosine 5'-triphosphate on cryopreserved epididymal cat sperm intracellular ATP concentration, sperm quality, and in vitro fertilizing ability.

    Science.gov (United States)

    Thuwanut, Paweena; Arya, Nlin; Comizzoli, Pierre; Chatdarong, Kaywalee

    2015-09-15

    Intracellular adenosine 5'-triphosphate (ATP) is essential for supporting sperm function in the fertilization process. During cryopreservation, damage of sperm mitochondrial membrane usually leads to compromised production of intracellular ATP. Recently, extracellular ATP (ATPe) was introduced as a potent activator of sperm motility and fertilizing ability. This study aimed to evaluate (1) levels of intracellular ATP in frozen-thawed epididymal cat sperm after incubation with ATPe and (2) effects of ATPe on epididymal cat sperm parameters after freezing and thawing. Eighteen male cats were included. For each replicate, epididymal sperm from two cats were pooled to one sample (N = 9). Each pooled sample was cryopreserved with the Tris-egg yolk extender into three straws. After thawing, the first and second straws were incubated with 0-, 1.0-, or 2.5-mM ATPe for 10 minutes and evaluated for sperm quality at 10 minutes, 1, 3, and 6 hours after thawing and fertilizing ability. The third straw was evaluated for intracellular ATP concentration in control and with 2.5-mM ATPe treatment. Higher concentration of intracellular sperm ATP was observed in the samples treated with 2.5-mM ATPe compared to the controls (0.339 ± 0.06 μg/2 × 10(6) sperm vs. 0.002 ± 0.003 μg/2 × 10(6) sperm, P ≤ 0.05). In addition, incubation with 2.5-mM ATPe for 10 minutes promoted sperm motility (56.7 ± 5.0 vs. 53.3 ± 4.4%, P ≤ 0.05) and progressive motility (3.1 ± 0.2 vs. 2.8 ± 0.4, P ≤ 0.05), mitochondrial membrane potential (36.4 ± 5.5 vs. 28.7 ± 4.8%, P ≤ 0.05), and blastocyst rate (36.1 ± 7.0 and 28.8 ± 7.4%, P ≤ 0.05) compared with the controls. In contrast, ATPe remarkably interfered acrosome integrity after 6 hours of postthawed incubation. In sum, the present finding that optimal incubation time of postthaw epididymal cat sperm under proper ATPe condition might constitute a rationale for the studies on other endangered wild felids regarding sperm quality and embryo

  1. Extracellular ATP mediates necrotic cell swelling in SN4741 dopaminergic neurons through P2X7 receptors.

    Science.gov (United States)

    Jun, Dong-Jae; Kim, Jaeyoon; Jung, Sang-Yong; Song, Ran; Noh, Ji-Hyun; Park, Yong-Soo; Ryu, Sung-Ho; Kim, Joung-Hun; Kong, Young-Yun; Chung, Jun-Mo; Kim, Kyong-Tai

    2007-12-28

    Extracellular ATP has recently been identified as an important regulator of cell death in response to pathological insults. When SN4741 cells, which are dopaminergic neurons derived from the substantia nigra of transgenic mouse embryos, are exposed to ATP, cell death occurs. This cell death is associated with prominent cell swelling, loss of ER integrity, the formation of many large cytoplasmic vacuoles, and subsequent cytolysis and DNA release. In addition, the cleavage of caspase-3, a hallmark of apoptosis, is induced by ATP treatment. However, caspase inhibitors do not overcome ATP-induced cell death, indicating that both necrosis and apoptosis are associated with ATP-induced cell death and suggesting that a necrotic event might override the apoptotic process. In this study we also found that P2X(7) receptors (P2X(7)Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X(7)Rs antagonist, KN62, or by knock-down of P2X(7)Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X(7)Rs.

  2. Inflammasome activation in bovine monocytes by extracellular ATP does not require the purinergic receptor P2X7.

    Science.gov (United States)

    Hussen, Jamal; Düvel, Anna; Koy, Mirja; Schuberth, Hans-Joachim

    2012-10-01

    Extracellular adenosine triphosphate (ATP) is a second signal for the assembly of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, which form a framework to activate caspase 1, leading to the processing and secretion of the pro-inflammatory cytokine interleukin-1β (IL-1β). The aim of the present study was to investigate the role of the ATP-gated ion channel subtype P2X7 receptor in the inflammasome activation of bovine monocytes. ATP-induced inflammasome assembly in bovine monocytes was shown by caspase-1 activation and the release of IL-1β by LPS/ATP-stimulated bovine cells. The IL-1β release depended on potassium efflux but was independent of reactive oxygen generation of bovine monocytes. Unlike in the human system, a P2X7 receptor antagonist did not block the ATP-induced release of IL-1β of LPS-primed bovine cells. P2X7 mediated pore formation was observed in subsets of bovine T lymphocytes (CD4+>CD8+) but not in monocytes. In addition, ATP and 2-MeSATP but not the high affinity P2X7 agonist BzATP induced calcium influx in bovine monocytes. The data indicate that ROS generation plays no role in the ATP-induced activation of inflammasome in bovine monocytes and that P2X7-mediated pore formation is not necessary for the release of Interleukin-1β.

  3. Direct excitation of inhibitory interneurons by extracellular ATP mediated by P2Y1 receptors in the hippocampal slice.

    Science.gov (United States)

    Kawamura, Masahito; Gachet, Christian; Inoue, Kazuhide; Kato, Fusao

    2004-12-01

    ATP is an important cell-to-cell signaling molecule mediating the interactions between astrocytes and neurons in the CNS. In the hippocampal slices, ATP suppresses excitatory transmission mostly through activation of adenosine A1 receptors, because the ectoenzyme activity for the extracellular breakdown of ATP to adenosine is high in slice preparations in contrast to culture environments. Because the hippocampus is also rich in the expression of P2 receptors activated specifically by ATP, we examined whether ATP modulates neuronal excitability in the acute slice preparations independently of adenosine receptors. Although ATP decreased the frequency of spontaneously occurring EPSCs in the CA3 pyramidal neurons through activation of adenosine A1 receptors, ATP concurrently increased the frequency of IPSCs in a manner dependent on action potential generation. This effect was mediated by P2Y1 receptors because (1) 2-methylthio-ATP (2meSATP) was the most potent agonist, (2) 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate diammonium (MRS2179) abolished this effect, and (3) this increase in IPSC frequency was not observed in the transgenic mice lacking P2Y1 receptor proteins. Application of 2meSATP elicited MRS2179-sensitive time- and voltage-dependent inward currents in the interneurons, which depolarized the cell to firing threshold. Also, it increased [Ca2+]i in both astrocytes and interneurons, but, unlike the former effect, the latter was entirely dependent on Ca2+ entry. Thus, in hippocampal slices, in addition to activating A1 receptors of the excitatory terminals after being converted to adenosine, ATP activates P2Y1 receptors in the interneurons, which is linked to activation of unidentified excitatory conductance, through mechanisms distinct from those in the astrocytes.

  4. [Identification of a new pro-invasion factor in tumor microenvironment: progress in function and mechanism of extracellular ATP].

    Science.gov (United States)

    Fang, W G; Tian, X X

    2017-04-18

    Up to 90% of all cancer related morbidity and mortality can be attributed to metastasis. In recent years the study of tumor microenvironment, its cellular and molecular components, and how they can affect neoplastic progression toward metastasis, has become a hot focus in cancer research. Accumulated evidence shows that the formation of metastasis is a multi-step sequential process, in which, the tumor cells continuously interact with the host microenvironment. Host derived factors, i.e. growth factors/inhibitors, angiogenic factors, chemokines, etc. together with different types of host cells, play important roles in the tumor progression towards metastasis. The interaction between the tumor cells and host microenvironment determines the fate of metastasis. The reveal of this interaction mechanism provides us an opportunity to find effective mode of interference and develop novel anti-metastasis drugs. In this review, we have summarized our work on a new pro-invasion factor identified in tumor microenvironment and how it affects tumor invasion and metastass. Adenosine triphosphate (ATP), the key intracellular energy currency, accumulates within the tumor microenvironment and is closely involved in cancer cell metabolism and in antitumor immunity. The established role of ATP as a growth modulator and a proinflammatory mediator endues ATP and other purines with potential players in host-tumor interaction. Our study demonstrated that extracellular ATP stimulated human cancer invasion in in vitro tests. Increased migration and invasive ability across Matrigel was observed in some human carcinoma cell lines, including the prostate, breast, colon, melanoma and lung, when stimulated with ATP or its analogues. ATP enhanced the motility of cancer cells via increasing the amount and length of lamellipodia and filopodia, which were necessary for the cell motility. Significant increase in Rac1 and Cdc42 activities was observed. Using cDNA microarray we found that the

  5. Release of ATP from marginal cells in the cochlea of neonatal rats can be induced by changes in extracellular and intracellular ion concentrations.

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    Yating Peng

    Full Text Available BACKGROUND: Adenosine triphosphate (ATP plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. METHODS: Sprague-Dawley rats aged 1-3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. RESULTS: Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K(+ and intra- and extracellular Ca(2+. Furthermore, changes in the concentration of intracellular Ca(2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. CONCLUSION: We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K(+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A(2.

  6. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L; Kirk, Kevin L

    2016-03-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.

  7. T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP

    Directory of Open Access Journals (Sweden)

    Lisa Perruzza

    2017-03-01

    Full Text Available The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh cell abundance in the Peyer’s patches (PPs of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help.

  8. A possible role for extra-cellular ATP in plant responses to high frequency, low amplitude electromagnetic field.

    Science.gov (United States)

    Roux, David; Faure, Catherine; Bonnet, Pierre; Girard, Sébastien; Ledoigt, Gérard; Davies, Eric; Gendraud, Michel; Paladian, Françoise; Vian, Alain

    2008-06-01

    In parallel to evoking the accumulation of stress-related transcripts, exposure to low level 900 MHz EMF affected the levels of ATP, the main energy molecule of the cell. Its concentration dropped rapidly (27% after 30 min) in response to EMF exposure, along with a 18% decrease in the adenylate energy charge (AEC), a good marker of cell energy status. One could interpret this decrease in ATP and AEC in a classical way, i.e., as the result of an increase in cellular energy usage, but recent work brings exciting new insights in pointing out a signalling function for ATP, especially in the stress physiology context where it could trigger both reactive oxygen species and calcium movement (this latter being involved in plant responses to EMF exposure). In this addendum, we discuss our results within this new perspective for ATP function.

  9. Production of adenosine from extracellular ATP at the striatal cholinergic synapse.

    Science.gov (United States)

    James, S; Richardson, P J

    1993-01-01

    The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC 3.6.1.15) had a Km of 131 microM, whereas the ecto-ADPase (EC 3.6.1.6) had a Km of 58 microM, was Ca(2+)-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC 3.1.3.5) had a Km of 21 microM, was inhibited by AMPPNP and alpha,beta-methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, alpha,beta-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.

  10. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction.

    Science.gov (United States)

    Giniatullin, A; Petrov, A; Giniatullin, R

    2015-01-29

    Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular

  11. Altered extracellular ATP, ADP, and AMP hydrolysis in blood serum of sedentary individuals after an acute, aerobic, moderate exercise session.

    Science.gov (United States)

    Moritz, Cesar Eduardo Jacintho; Teixeira, Bruno Costa; Rockenbach, Liliana; Reischak-Oliveira, Alvaro; Casali, Emerson André; Battastini, Ana Maria Oliveira

    2017-02-01

    Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Exercise promotes distinct adaptations, and can influence purinergic signaling. In the present study, we investigated soluble nucleotidase activities in the blood serum of sedentary young male adults at pre- and post-acute moderate aerobic exercise. In addition, we evaluated how this kind of exercise could influence adenine nucleotide concentrations in the blood serum. Sedentary individuals were submitted to moderate aerobic exercise on a treadmill; blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. The ecto-nucleotide pyrophosphatase/phosphodiesterase was also evaluated, showing an increased activity post-exercise, compared to pre-exercise. Purine levels were analyzed by HPLC in the blood serum, pre- and post-exercise. Decreased levels of ATP and ADP were found post-exercise, in contrast with pre-exercise values. Conversely, post-exercise levels of adenosine and inosine increased compared to pre-exercise levels. Our results indicate an influence of acute exercise on ATP metabolism, modifying enzymatic behavior to promote a protective biological environment.

  12. Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez, F.A.; Rozengurt, E.; Heppel, L.A. (Cornell Univ., Ithaca, NY (USA))

    1989-06-01

    Exposure of Swiss 3T6 mouse fibroblasts to extracellular ATP stimulated the formation of inositol phosphates and mobilized intracellular calcium. The mobilization of intracellular calcium was verified by imaging of fura-2 fluorescence in individual cells and by monitoring the efflux of {sup 45}Ca{sup 2+} from preloaded cells. However, the authors found no activation of protein kinase C as measured by phosphorylation of an 80-kDa acidic protein and by transmodulation of the receptor for epidermal growth factor. A careful examination of the kinetics of the phosphorylation reaction (from 30 sec to 10 min) revealed no activation of protein kinase C by extracellular ATP at any time. The lack of activation of protein kinase C was demonstrated even when a concentration of ATP 10-fold higher than that required to give a strong Ca{sup 2+} signal was used. Extracellular ATP did not inhibit protein kinase C activation by fetal bovine serum, platelet-derived growth factor, or phorbol esters. The effects of ATP were also produced by UTP but not by ADP, AMP, or adenosine. These findings demonstrate that it is possible to induce the mobilization of intracellular calcium by an inositol phosphate-mediated pathway without the activation of protein kinase C.

  13. Effect of visible laser light on ATP level of anaemic red blood cell.

    Science.gov (United States)

    Suardi, Nursakinah; Sodipo, Bashiru Kayode; Mustafa, Mohd Zulkifli; Ali, Zalila

    2016-09-01

    In this work we present influence of visible laser light on ATP level and viability of anaemic red blood cell (RBC). The visible laser lights used in this work are 460nm and 532nm. The responses of ATP level in anaemic and normal RBC before and after laser irradiation at different exposure time (30, 40, 50 and 60s) were observed. Three aliquots were prepared from the ethylenediaminetetraacetic acid (EDTA) blood sample. One served as a control (untreated) and another two were irradiated with 460nm and 560nm lasers. Packed RBC was prepared to study ATP level in the RBC using CellTiter-GloLuminescent cell Viability Assay kit. The assay generates a glow type signal produced by luciferase reaction, which is proportional to the amount of ATP present in RBCs. Paired t-test were done to analyse ATP level before and after laser irradiation. The results revealed laser irradiation improve level of ATP in anaemic RBC. Effect of laser light on anaemic RBCs were significant over different exposure time for both 460nm (p=0.000) and 532nm (p=0.003). The result of ATP level is further used as marker for RBC viability. The influence of ATP level and viability were studied. Optical densities obtained from the data were used to determine cell viability of the samples. Results showed that laser irradiation increased viability of anaemic RBC compared to normal RBC.

  14. ATP consumption of eukaryotic flagella measured at a single-cell level

    CERN Document Server

    Chen, Daniel T N; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-01-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating wavef...

  15. Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine

    Institute of Scientific and Technical Information of China (English)

    Ming-xia WANG; Lei-ming REN

    2006-01-01

    receptors. Conclusion: Extracellular ATP and ADO reduced the cell viability, arrested cell cycle and induced apoptosis in HGC-27 cell line by intracellular uptake of ADO. One of the main routes of ATP-induced apoptosis in HGC-27 cells is through the breakdown to adenosine.

  16. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  17. Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells

    Science.gov (United States)

    Salvestrini, Valentina; Orecchioni, Stefania; Talarico, Giovanna; Reggiani, Francesca; Mazzetti, Cristina; Bertolini, Francesco; Orioli, Elisa; Adinolfi, Elena; Virgilio, Francesco Di; Pezzi, Annalisa; Cavo, Michele

    2017-01-01

    Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2×7R is the most consistently expressed by tumors. P2×7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2×7R. Here, we show that P2×7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2×7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo. Overall, our results demonstrate that P2×7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML. PMID:27980223

  18. Changes in Acetylcholine Extracellular Levels during Cognitive Processes

    Science.gov (United States)

    Pepeu, Giancarlo; Giovannini, Maria Grazia

    2004-01-01

    Measuring the changes in neurotransmitter extracellular levels in discrete brain areas is considered a tool for identifying the neuronal systems involved in specific behavioral responses or cognitive processes. Acetylcholine (ACh) is the first neurotransmitter whose diffusion from the central nervous system was investigated and whose extracellular…

  19. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou (NCSU)

    2016-10-26

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  20. Pravastatin-induced improvement in coronary reactivity and circulating ATP and ADP levels in young adults with type 1 diabetes

    Directory of Open Access Journals (Sweden)

    Tuomas Oskari Kiviniemi

    2012-08-01

    Full Text Available Aims: Extracellular ATP and ADP regulate diverse inflammatory, prothrombotic and vasoactive responses in the vasculature. Statins have been shown to modulate their signaling pathways in vitro. We hypothesized that altered intravascular nucleotide turnover modulates vasodilation in patients with type 1 diabetes (T1DM, and this can be partly restored with pravastatin therapy. Methods: In this randomized double blind study, plasma ATP and ADP levels and echocardiography-derived coronary flow velocity response to cold pressor test (CPT were concurrently assessed in 42 normocholesterolemic patients with T1DM (age 30±6 years, LDL cholesterol 2.5±0.6 mmol/L before and after four-month treatment with pravastatin 40 mg/day or placebo (n=22 and n=20, respectively, and in 41 healthy control subjects. Results: Compared to controls, T1DM patients had significantly higher concentrations of ATP (p<0.01 and ADP (p<0.01 and these levels were partly restored after treatment with pravastatin (p=0.002 and p=0.007, respectively, but not after placebo (p=0.06 and p=0.14, respectively. Coronary flow velocity acceleration was significantly lower in T1DM patients compared to control subjects, and it increased from pre- to post-intervention in the pravastatin (p=0.02, but not in placebo group (p=0.15. Conclusions: Pravastatin treatment significantly reduces circulating ATP and ADP levels of T1DM patients, and concurrently improves coronary flow response to CPT. This study provides a novel insight in purinergic mechanisms involved in pleiotropic effects of pravastatin.

  1. Extracellular vesicles released from mesenchymal stromal cells modulate miRNA in renal tubular cells and inhibit ATP depletion injury.

    Science.gov (United States)

    Lindoso, Rafael S; Collino, Federica; Bruno, Stefania; Araujo, Dayana S; Sant'Anna, Julliana F; Tetta, Ciro; Provero, Paolo; Quesenberry, Peter J; Vieyra, Adalberto; Einicker-Lamas, Marcelo; Camussi, Giovanni

    2014-08-01

    The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.

  2. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...... that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca2+ but, despite subsequent spikes in Ca2+ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes...

  3. Extracellular ATP does not induce P2X7 receptor-dependent responses in cultured renal- and liver-derived swine macrophages

    Directory of Open Access Journals (Sweden)

    Takato Takenouchi

    2014-01-01

    Full Text Available The P2X7 receptor (P2X7R is an ATP-gated cation channel that is abundantly expressed in monocytes/macrophages. P2X7R activation by ATP results in various cellular responses including Ca2+ influx, membrane pore formation, and cytokine secretion. Since P2X7R has low affinity for ATP, high concentrations of ATP (in the mM range are generally required to activate this receptor in vitro. Functional expression of P2X7R has been detected in monocytes/macrophages obtained from different animal species including humans, rodents, dogs, and bovines, but so far it has not been detected in swine (Sus scrofa. In this study, we investigated the expression and functions of P2X7R in swine macrophages, which were isolated from mixed primary cultures of swine kidney or liver tissue. The P2X7R mRNA and protein expression observed in the swine macrophages was comparable to that seen in a c-myc-immortalized mouse kidney-derived clonal macrophage cell line (KM-1. However, extracellular ATP did not induce P2X7R-dependent sustained Ca2+ influx, membrane pore formation, or the secretion of the bioactive cytokine interleukin-1β in the swine macrophages, whereas these responses were clearly observed in the mouse KM-1 cells after stimulation with millimolar concentrations of ATP as a positive control. These findings suggest that the ATP/P2X7R pathway is impaired in swine macrophages at least in the culture conditions used in the present study.

  4. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  5. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  6. Aerobic plate counts and ATP levels correlate with Listeria monocytogenes detection in retail delis.

    Science.gov (United States)

    Hammons, Susan R; Stasiewicz, Matthew J; Roof, Sherry; Oliver, Haley F

    2015-04-01

    Listeria monocytogenes is a foodborne pathogen that causes an estimated 1,591 cases of illness and 255 deaths annually in the United States, the majority of which are attributed to ready-to-eat deli meats processed in retail delis. Because retail delis distribute product directly to consumers, rapid methods to validate cleaning and sanitation are needed to improve retail food safety. This study investigated the relationships among ATP levels, standard aerobic plate count (APC), and L. monocytogenes presence in fully operational delis. Fifteen full-service delis were concurrently sampled for ATP, APC, and L. monocytogenes during preoperational hours once monthly for 3 months. Fifteen additional delis were recruited for 6 months of operational sampling (n = 30). A 1-log increase in APC was equivalent to a 3.3-fold increase in the odds of detecting L. monocytogenes (P < 0.001) and a 1.9-log increase in L monocytogenes population (P = 0.03). An ATP level increase of 1 log relative light unit correlated to a 0.22-log increase in APC (P < 0.001). A preoperational ATP level mean increase by 1 log relative light unit increased the odds of detecting L. monocytogenes concurrently fourfold. A 0.5-log increase in mean ATP level during preoperational sampling corresponded to a 2% increase in the predicted L. monocytogenes prevalence during operation (P < 0.01). Additionally, 10 statistically representative sites were identified and recommended for use in sanitation monitoring programs. Our data support the use of ATP as a rapid method to validate effective cleaning and sanitation to reduce L. monocytogenes in retail delis.

  7. Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels, an increase in glutamate uptake, and a reduction in cytosolic calcium.

    Science.gov (United States)

    Morland, Cecilie; Pettersen, Mi Nguyen; Hassel, Bjørnar

    2016-05-01

    Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10-100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na(+), since substituting excess Na(+) with choline reduced cell death to control levels, whereas gluconate instead of excess Cl(-) did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na(+)] caused reduction in intracellular free [Ca(2+)], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca(2+)] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia.

  8. Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium

    Directory of Open Access Journals (Sweden)

    Yang Yuan

    2012-06-01

    Full Text Available This study explored the reduction of adenosine triphosphate (ATP levels in L-02 hepatocytes by hexavalent chromium (Cr(VI using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%. The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI group (P < 0.05. The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.

  9. Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium.

    Science.gov (United States)

    Yuan, Yang; Peng, Li; Gong-Hua, Hu; Lu, Dai; Xia-Li, Zhong; Yu, Zhou; Cai-Gao, Zhong

    2012-06-01

    This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI) group (P ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.

  10. Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual-color bioluminescence reporter.

    Science.gov (United States)

    Kwon, Hyuck Joon; Ohmiya, Yoshihiro; Yasuda, Kazunori

    2014-12-01

    A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red-emitting Phrixothrix hirtus luciferase (PxRe) and a blue-emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non-mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual-color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes.

  11. Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

    2013-09-13

    Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.

  12. Correlative study of peripheral ATP1A1 gene expression level to anxiety severity score on major depressive disorder patients.

    Science.gov (United States)

    Zhao, Jingjie; Guo, Xu; Du, Yi; Han, Yu; Wang, Yongzhi; Li, Li; Qian, Jialin; Li, Mingzhen; Wu, Huijuan; Golden, Teresa; Wu, Ning

    2016-11-01

    Major depressive disorder (MDD) frequently co-occurs with other psychiatric problems. Our previous study showed that ATP1A1 gene expression level was significantly decreased in MDD patients. This research explores the potential correlations between the ATP1A1 expression level reduction and MDD patients' clinical manifestation. All participant patients were diagnosed by Diagnostic and Statistical Manual of Mental Disorders - 4th edition (DSM-IV). Hamilton rating scale for depression (HAM-D) and anxiety (HAM-A) were applied to group patients into different categories. ATP1A1 expression level was measured by reverse transcript real-time polymerase chain reaction. ATP1A1 expression levels of all MDD subgroups showed significant reduction compared to the control group (p0.05). ATP1A1 expression level reduction is related to MDD anxiety score, which may be an explanation for the clinical manifestations and the underlining physiological mechanisms.

  13. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    Science.gov (United States)

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  14. DYNAMIC ANALYSIS OF CYTOSOLIC GLUCOSE AND ATP LEVELS IN YEAST WITH OPTICAL SENSORS

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Takanaga, Hitomi; Chermak, Diane; Frommer, Wolf B.

    2010-01-01

    Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. Here, genetically encoded Förster Resonance Energy Transfer (FRET)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP at varying external supply and in glucose transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes in intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in an hxt5Δ hexose transporter mutant compared to wild type and to other hxtΔ strains. PMID:20854260

  15. Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Takanaga, Hitomi; Chermak, Diane; Frommer, Wolf B

    2010-12-01

    Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. In the present study, genetically encoded FRET (Förster resonance energy transfer)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP with varying external supply and in glucose-transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes of intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in a hxt5Δ hexose-transporter mutant compared with wild-type and other hxtΔ strains.

  16. Real-time luminescence imaging of cellular ATP release.

    Science.gov (United States)

    Furuya, Kishio; Sokabe, Masahiro; Grygorczyk, Ryszard

    2014-03-15

    Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin-luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin-luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100 μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility.

  17. Effect of LLLT on the level of ATP and ROS from organ of corti cells

    Science.gov (United States)

    Rhee, ChungKu; Chang, So-Young; Ahn, Jin-Chul; Suh, Myung-Whan; Jung, Jae Yun

    2014-03-01

    It is well established that ototoxic antibiotics and acoustic trauma can damage cochlear hair cells and cause hearing loss. Previous studies using transcanal LLLT (Low level laser therapy) showed that LLLT can promote recovery of hearing thresholds and cochlear hair cells. However, its mechanism has not been studied. Aim: The aim of this study is to investigate the mechanism of hearing recovery from gentamicin induced ototoxic hearing loss by LLLT. Methods: HEI- OC1 (House ear institute organ of Corti) cells were cultured for 18 hours and ototoxicity was induced by gentamicin (GM) treatment to the cells. Cultured cells were divided into 6 groups, No treatment control, LLLT only, GM 6.6 mM and GM 13.1 mM, GM 6.6 mM+LLLT and GM 13.1 mM+LLLT cells. LD laser 808 nm, 15 mW, was irradiated to the cultured cells for 15 min, at 4 hours after GM treatment to the cells. ATP was assayed using the ATP assay Kit. ROS was measured using confocal microscope after application of H2DCFDA dye. Results: ATP was decreased in GM 13.1 mM cells and increased in LLLT only cells and GM 13.1 mM+LLLT cells compared to control and 13.1 mM cells. ROS was increased in GM 6.6 mM and GM 13.1 mM cells, and decreased in GM 6.6 mM+LLLT and GM 13.1 mM+LLLT cells compared to GM 6.6 and 13.1 mM cells immediately after laser irradiation. Conclusion: This study demonstrated that LLLT on GM treated HEI-OC1 cells increased ATP and decreased ROS that may contribute to the recovery of hearing.

  18. Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels.

    Science.gov (United States)

    Chinopoulos, Christos; Konràd, Csaba; Kiss, Gergely; Metelkin, Eugeniy; Töröcsik, Beata; Zhang, Steven F; Starkov, Anatoly A

    2011-04-01

    Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ∼ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ∼ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels. © 2011 The Authors Journal compilation © 2011 FEBS.

  19. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

    Directory of Open Access Journals (Sweden)

    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  20. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System

    Directory of Open Access Journals (Sweden)

    Hyuck Joon Kwon

    2015-01-01

    Full Text Available Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  1. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System.

    Science.gov (United States)

    Kwon, Hyuck Joon; Han, Youngbae

    2015-01-01

    Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  2. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    Science.gov (United States)

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

  3. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels.

    Science.gov (United States)

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-10-15

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K(+) (K(ATP)) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP](c)) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of K(ATP) channels in GR neurons, glucose (3 or 15 mM) caused no detectable increase in [ATP](c), monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25-luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mM) with a significant increase in [ATP](c). Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mM glucose with increases in [ATP](c). Further implicating an ATP-independent mechanism of K(ATP) channel closure in hypothalamic neurons, removal of extracellular glucose (10 mM) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mM) of intracellular ATP. Neurons from both brain regions responded to 5 mM lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP](c). High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed K(ATP) channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells.

  4. Extracellular Adenosine Triphosphate Affects Systemic and Kidney Immune Cell Populations in Pregnant Rats

    NARCIS (Netherlands)

    Spaans, Floor; Melgert, Barbro N.; Borghuis, Theo; Klok, Pieter A.; de Vos, Paul; Bakker, Winston W.; van Goor, Harry; Faas, Marijke

    PROBLEM: Changes in the systemic immune response are found in preeclampsia. This may be related to high extracellular adenosine triphosphate (ATP) levels. The question arose whether ATP could affect immune responses in pregnancy. Previously, we investigated whether ATP affected monocyte activation

  5. Transgenic Arabidopsis thaliana containing increased levels of ATP and sucrose is more susceptible to Pseudomonas syringae

    Science.gov (United States)

    Zhang, Renshan; Qi, Hua; Sun, Yuzhe; Xiao, Shi

    2017-01-01

    Disease resistance exerts a fitness cost on plants, presumably due to the extra consumption of energy and carbon. In this study, we examined whether transgenic Arabidopsis thaliana with increased levels of ATP and sucrose is more resistant or susceptible to pathogen infection. Lines of A. thaliana over-expressing purple acid phosphatase 2 (AtPAP2) (OE lines) contain increased levels of ATP and sucrose, with improved growth rate and seed production. Compared to wild type (WT) and pap2 lines, the OE lines were more susceptible to several Pseudomonas syringae pv. tomato (Pst) strains carrying AvrRpm1, AvrRpt2 AvrRps4, AvrPtoB, HrcC and WT strain DC3000. The increased susceptibility of the OE lines to Pst strains cannot solely be attributed to the suppressed expression of R-genes but must also be attributed to the suppression of downstream signaling components, such as MOS2, EDS1 and EDS5. Before infection, the levels of salicylic acid (SA) and jasmonic acid (JA) precursor OPDA were similar in the leaves of OE, pap2 and WT plants, whereas the levels of JA and its derivative JA-Ile were significantly lower in the leaves of OE lines and higher in the pap2 line. The expression of JA marker defense gene PDF1.2 was up-regulated in the OE lines compared to the WT prior to Pst DC3000 infection, but its expression was lower in the OE lines after infection. In summary, high fitness Arabidopsis thaliana exhibited altered JA metabolism and broad suppression of R-genes and downstream genes as well as a higher susceptibility to Pst infections. PMID:28152090

  6. AMPD3-deficient mice exhibit increased erythrocyte ATP levels but anemia not improved due to PK deficiency.

    Science.gov (United States)

    Cheng, Jidong; Morisaki, Hiroko; Toyama, Keiko; Ikawa, Masahito; Okabe, Masaru; Morisaki, Takayuki

    2012-11-01

    AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [A3(-/-)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC-AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double-deficient [PKA(-/-,-/-)] mice by mating A3(-/-) mice with CBA-Pk-1slc/Pk-1slc mice [PK(-/-)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(-/-,-/-) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(-/-) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(-/-) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(-/-) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction.

  7. Extracellular-purine metabolism in blood vessels (part I). Extracellular-purine level in blood of patients with abdominal aortic aneurysm.

    Science.gov (United States)

    Lecka, Joanna; Molski, Stanislaw; Komoszynski, Michal

    2010-09-01

    Adenosine and adenosine derivatives are the main regulators of purinoceptors (P1 and P2) mediated hemostasis and blood pressure. Since impaired hemostasis and high blood pressure lead to atherosclerosis and to the development of aneurysm, in this study we tested and compared the concentration of extracellular purines (e-purines) in the blood in of patients having abdominal aortic aneurysm with that from healthy volunteers. Whereas adenine nucleosides and nucleotides level in human blood plasma was analysed using reverse phase high performance liquid chromatography (HPLC), cholesterol concentration was estimated by an enzymatic assay. We did not find any correlation between e-purines concentration and the age of healthy volunteers. Furthermore, the sum level of e-purines (ATP, ADP, AMP, adenosine, and inosine) in the control group did not exceed 70 microM, while it was nearly two-fold higher in the blood of patients having abdominal aortic aneurysm, (123 microM). In a special case of people with Leriche Syndrome, a disease characterized by deep atherosclerotic changes, the e-purines level had further increased. Additionally, we also report typical atherosclerotic changes in the aorta using histological assays as well as total cholesterol rise. The significant rise in cholesterol concentration in the blood of the patients with abdominal aortas aneurysm, compared with the control groups, was not unique since 23% of the healthy people also exceeded the normal level of cholesterol. Therefore, our results strongly indicate that the estimation of e-purines concentration in the blood may serve as another indicator of atherosclerosis and warrant further consideration as a futuristic diagnostic tool.

  8. Smoking is associated with increased levels of extracellular peptidylarginine deiminase 2 (PAD2) in the lungs

    DEFF Research Database (Denmark)

    Damgaard, Dres; Friberg Bruun Nielsen, Michael; Quisgaard Gaunsbaek, Maria

    2015-01-01

    lavage (BAL) fluid from smokers, but intracellularly located PAD cannot be responsible for citrullination of extracellular self-antigens. We aimed to establish a link between smoking and extracellular PAD2 in the lungs. METHODS: BAL fluid samples were obtained from 13 smokers and 11 nonsmoking controls...... fluids from smokers as compared to non-smokers (p=0.018). The PAD2 content correlated with the overall CRP levels (p=0.009) and cell count (p=0.016). CONCLUSIONS: This first demonstration of increased levels of extracellular PAD2 in the lungs of smokers supports the hypothesis that smoking promotes...

  9. Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette transporters gene enrichment in typhoid fever-infected Nigerian children

    Directory of Open Access Journals (Sweden)

    Resau James H

    2011-09-01

    bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. Conclusions We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

  10. ATP: A Coherent View for School Advanced Level Studies in Biology.

    Science.gov (United States)

    Gayford, Chris

    1986-01-01

    Discusses how instruction of biological concepts as ATP cellular energetics is related to fundamental physical science understandings. Reviews areas of common misconceptions and confusions. Summarizes results of a study which investigated students' knowledge and perception of difficulty associated with the topic of energy and ATP. (ML)

  11. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    Science.gov (United States)

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as (13C)GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as (13C)ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction.

  12. Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular ATP levels and thiol groups

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    The experiments concerned the effect of ethacrynic acid (0.5 mM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 microM). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose...... as well as in presence of 2-deoxyglucose. A23187-induced histamine release was inhibited by ethacrynic acid, and this inhibition was completely reversed by dithiothreitol. These observations may indicate that ethacrynic acid inhibits glycolytic and respiratory energy production in rat mast cells...

  13. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

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    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  14. CalPFl4030 negatively modulates intracellular ATP levels during the development of azole resistance in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    Xin-ming JIA; Ying WANG; Jun-dong ZHANG; Hong-yue TAN; Yuan-yingJIANG; Jun GU

    2011-01-01

    Aim:Widespread and repeated use of azoles, particularly fiuconazole, has led to the rapid development of azole resistance in Candida albicans.We investigated the role of CalPF14030 during the development of azole resistance in C albicans.Methods:The expression of CalPF14030 was measured by quantitative RT-PCR, and CalPF14030 was disrupted by the hisG-URA3-hisG(URA-blaster)method.The sensitivity of C albicans to azoles was examined using a spot assay, and the intracellular ATP concentrations were measured by a luminometer.Results:CalPF14030 expression in C albicans was up-regulated by Ca2+ in a calcineurin-dependent manner, and the protein was overexpressed during the stepwise acquisition of azole resistance.However,disruption or ectopic overexpression of CalPFl4030 did not affect the sensitivity of C albicans to azoles.Finally,we demonstrated that disruption of CalPFll4030 significantly increased intracellular ATP levels.and overexpression significantly decreased intracellular ATP levels jn C albicans.Conclusion:CalPF14030 may negatively modulate intracellular ATP levels during the development of azole resistance in C albicans.

  15. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K+ channels

    Science.gov (United States)

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-01-01

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K+ (KATP) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP]c) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of KATP channels in GR neurons, glucose (3 or 15 mm) caused no detectable increase in [ATP]c, monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25–luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mm) with a significant increase in [ATP]c. Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mm glucose with increases in [ATP]c. Further implicating an ATP-independent mechanism of KATP channel closure in hypothalamic neurons, removal of extracellular glucose (10 mm) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mm) of intracellular ATP. Neurons from both brain regions responded to 5 mm lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP]c. High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed KATP channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells. PMID:12381816

  16. Cystine accumulation attenuates insulin release from the pancreatic β-cell due to elevated oxidative stress and decreased ATP levels.

    Science.gov (United States)

    McEvoy, Bernadette; Sumayao, Rodolfo; Slattery, Craig; McMorrow, Tara; Newsholme, Philip

    2015-12-01

    The pancreatic β-cell has reduced antioxidant defences making it more susceptible to oxidative stress. In cystinosis, a lysosomal storage disorder, an altered redox state may contribute to cellular dysfunction. This rare disease is caused by an abnormal lysosomal cystine transporter, cystinosin, which causes excessive accumulation of cystine in the lysosome. Cystinosis associated kidney damage and dysfunction leads to the Fanconi syndrome and ultimately end-stage renal disease. Following kidney transplant, cystine accumulation in other organs including the pancreas leads to multi-organ dysfunction. In this study, a Ctns gene knockdown model of cystinosis was developed in the BRIN-BD11 rat clonal pancreatic β-cell line using Ctns-targeting siRNA. Additionally there was reduced cystinosin expression, while cell cystine levels were similarly elevated to the cystinotic state. Decreased levels of chronic (24 h) and acute (20 min) nutrient-stimulated insulin secretion were observed. This decrease may be due to depressed ATP generation particularly from glycolysis. Increased ATP production and the ATP/ADP ratio are essential for insulin secretion. Oxidised glutathione levels were augmented, resulting in a lower [glutathione/oxidised glutathione] redox potential. Additionally, the mitochondrial membrane potential was reduced, apoptosis levels were elevated, as were markers of oxidative stress, including reactive oxygen species, superoxide and hydrogen peroxide. Furthermore, the basal and activated phosphorylated forms of the redox-sensitive transcription factor NF-κB were increased in cells with silenced Ctns. From this study, the cystinotic-like pancreatic β-cell model demonstrated that the altered oxidative status of the cell, resulted in depressed mitochondrial function and pathways of ATP production, causing reduced nutrient-stimulated insulin secretion.

  17. Effect of novel atypical antipsychotic, blonanserin, on extracellular neurotransmitter level in rat prefrontal cortex.

    Science.gov (United States)

    Ohoyama, Keiko; Yamamura, Satoshi; Hamaguchi, Tatsuya; Nakagawa, Masanori; Motomura, Eishi; Shiroyama, Takashi; Tanii, Hisashi; Okada, Motohiro

    2011-02-25

    To clarify the mechanisms of action of blonanserin, an atypical antipsychotic drug, we studied the effects of systemic administration of blonanserin and risperidone on extracellular levels of norepinephrine, dopamine, serotonin, GABA and glutamate in the medial prefrontal cortex using microdialysis, and neuronal firing in the ventral tegmental area, locus coeruleus, dorsal raphe nucleus and mediodorsal thalamic nucleus using radiotelemetry. The binding affinities of blonanserin to D(2) and 5-HT(2A) receptors in the rat brain were confirmed and found to be similar. Blonanserin transiently increased neuronal firing in locus coeruleus and ventral tegmental area but not in dorsal raphe nucleus or mediodorsal thalamic nucleus, whereas risperidone increased the firing in locus coeruleus, ventral tegmental area and dorsal raphe nucleus but not in mediodorsal thalamic nucleus. Blonanserin persistently increased frontal extracellular levels of norepinephrine and dopamine but not serotonin, GABA or glutamate, whereas risperidone persistently increased those of norepinephrine, dopamine and serotonin but not GABA or glutamate. These results suggest a pharmacological correlation between the stimulatory effects of these antipsychotics on frontal monoamine release and neuronal activity in monoaminergic nuclei. Inhibition of the α(2) adrenoceptor increased extracellular monoamine levels and enhanced blonanserin-induced increase in extracellular serotonin level. These results indicated that the combination of antagonism of D(2) and 5-HT(2A) receptors contribute to the rise in extracellular levels of norepinephrine and dopamine, and that α(2) adrenoceptors play important roles in frontal serotonin release. They also suggest that blonanserin-induced activation of monoaminergic transmission could be, at least partially, involved in atypical antipsychotic properties of blonanserin. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Evidences of Hfq associates with tryptophanase and affects extracellular indole levels

    Institute of Scientific and Technical Information of China (English)

    Yinghua Zhang; Guofan Hong

    2009-01-01

    In this study, we observed a novel property of Escherichia coil Hfq protein: it possibly influenced extracellular indole levels. The extracellular indole concentrations were increased in Hfq mutant cells and decreased in Hfq overexpression cells in a cell density-dependent manner. The decreased extracellular indole levels in Hfq overexpression cells caused the postpone-ment of entering into stationary phase, lndole was pro-duced by tryptophanase, the gene product of tnaA, which catalyzed tryptophan into indole, ammonia and pyruvate. Further studies showed that at cell density of 0.8 but not at 0.4, tryptophanase activities of total cell extracts were affected by Hfq mutation or overexpression. Protein pull-down assay and co-immunoprecipitation experiments revealed that Hfq associated with trypto-phanase under relatively higher extracellular indole levels, suggesting this was a feedback control of indole production. The association of Hfq and tryptophanase might be indirect because purified Hfq could not affect the values of Km and Vmax of purified tryptophanase.

  19. Evidences of Hfq associates with tryptophanase and affects extracellular indole levels.

    Science.gov (United States)

    Zhang, Yinghua; Hong, Guofan

    2009-08-01

    In this study, we observed a novel property of Escherichia coli Hfq protein: it possibly influenced extracellular indole levels. The extracellular indole concentrations were increased in Hfq mutant cells and decreased in Hfq overexpression cells in a cell density-dependent manner. The decreased extracellular indole levels in Hfq overexpression cells caused the postponement of entering into stationary phase. Indole was produced by tryptophanase, the gene product of tnaA, which catalyzed tryptophan into indole, ammonia and pyruvate. Further studies showed that at cell density of 0.8 but not at 0.4, tryptophanase activities of total cell extracts were affected by Hfq mutation or overexpression. Protein pull-down assay and co-immunoprecipitation experiments revealed that Hfq associated with tryptophanase under relatively higher extracellular indole levels, suggesting this was a feedback control of indole production. The association of Hfq and tryptophanase might be indirect because purified Hfq could not affect the values of Km and Vmax of purified tryptophanase.

  20. Dynamic changes in cytosolic ATP levels in cultured glutamatergic neurons during NMDA-induced synaptic activity supported by glucose or lactate

    DEFF Research Database (Denmark)

    Lange, Sofie Cecilie; Winkler, Ulrike; Andresen, Lars;

    2015-01-01

    We have previously shown that synaptic transmission fails in cultured neurons in the presence of lactate as the sole substrate. Thus, to test the hypothesis that the failure of synaptic transmission is a consequence of insufficient energy supply, ATP levels were monitored employing the ATP biosen...

  1. Cr(VI) induces the decrease of ATP level and the increase of apoptosis rate mediated by ROS or VDAC1 in L-02 hepatocytes.

    Science.gov (United States)

    Yuan, Yang; Ming, Zen; Gong-Hua, Hu; Lan, Guan; Lu, Dai; Peng, Li; Feng, Jing; Cai-Gao, Zhong

    2012-09-01

    The present study explored the ability of the voltage-dependent anion channel 1 (VDAC1) mRNA and ROS levels to modulate the effects of hexavalent chromium Cr(VI) on the adenosine triphosphate (ATP) level and the rate of apoptotic cell death. Cultured L-02 cells were pretreated with 20mM N-acetyl-cysteine (NAC) for 24h or transiently transfected with small interfering RNAs targeting VDAC1 (siVDAC1) for 48h; cells that were not pretreated were used as the control. The cells were subsequently treated with 0, 2, 8, or 32μM Cr(VI) for 24h. Then, levels of VDAC1 mRNA, ROS, and ATP and the apoptosis rate were measured by reverse-transcription quantitative PCR, fluorometry, a bioluminescence assay, and flow cytometry, respectively. The results showed that Cr(VI) at 32μM led to increase in the ROS level, VDAC1 mRNA expression, and the apoptosis rate and a decrease in the ATP level; pretreatment with NAC led to the down-regulation in the levels of ROS, VDAC1 mRNA and apoptosis and the significant up-regulation in the ATP levels. Interestingly, after the pretreatment with siVDAC1 to inhibit VDAC1 mRNA expression, the increased apoptosis rates and decreased ATP levels were reversed as well. These results suggested that changes in the ROS or VDAC1 mRNA levels were associated with changes in the ATP level and apoptosis rate. Furthermore, correlation analysis confirmed the association between both the ROS and VDAC1 levels and both the ATP level and the apoptosis rate. In conclusion, Cr(VI) induced ROS- and VDAC1-mediated decreases in ATP levels and increases in the apoptosis rate.

  2. ATP signals

    DEFF Research Database (Denmark)

    Novak, Ivana

    2016-01-01

    The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas......The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas...

  3. Is the extracellular ATP a key in X-linked Charcot-Marie-Tooth disease and in inherited non-syndromic deafness?

    OpenAIRE

    Mas del Molino, Ezequiel

    2011-01-01

    [spa] El ATP es una molécula ampliamente conocida por su papel en muchas funciones como la homeostasis celular, el mantenimiento de gradientes iónicos, el mantenimiento del pH en gránulos secretores, el almacenamiento energético, regulador de la interacción actina-miosina, etc. Además, el ATP puede actuar como molécula señalizadora a través de los receptores purinérgicos P2. De receptores P2 hay de dos tipos, los P2X, que son ionotrópicos, y los P2Y que son metabotrópicos. Los primeros son un...

  4. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  5. Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

    Science.gov (United States)

    Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

    2007-01-01

    The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures.

  6. Transcranial Direct-Current Stimulation Increases Extracellular Dopamine Levels in the Rat Striatum

    Directory of Open Access Journals (Sweden)

    Tomoko eTanaka

    2013-04-01

    Full Text Available Background: Transcranial direct-current stimulation is a non-invasive procedure that achieves polarity-dependent modulation of neuronal membrane potentials. It has recently been used as a functional intervention technique for the treatment of psychiatric and neurological diseases; however, its neuronal mechanisms have not been fully investigated in vivo. Objective/Hypothesis: To investigate whether the application of cathodal or anodal transcranial direct-current stimulation affects extracellular dopamine and serotonin levels in the rat striatum. Methods: Stimulation and in vivo microdialysis were carried out under urethane anesthesia, and microdialysis probes were slowly inserted into the striatum. After the collection of baseline fractions in the rat striatum, cathodal or anodal transcranial direct-current stimulation was applied continuously for 10 min with a current intensity of 800 µA from an electrode placed on the skin of the scalp. Dialysis samples were collected every 10 min until at least 400 min after the onset of stimulation.Results: Following the application of cathodal, but not anodal, transcranial direct-current stimulation for 10 min, extracellular dopamine levels increased for more than 400 min in the striatum. There were no significant changes in extracellular serotonin levels. Conclusion: These findings suggest that transcranial direct-current stimulation has a direct and/or indirect effect on the dopaminergic system in the rat basal ganglia.

  7. The expression and significance of extracellular ATP in murine acute liver injury model%胞外ATP在小鼠急性肝损伤中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    胡梅琮; 邹玲莉; 黄保军; 王磊

    2014-01-01

    Objective To observe the expression and significance of extracellular adenosine triphosphate (eATP) in concanavalin A (ConA)-induced murine acute liver injury model.Methods A total of 72 mice were randomly classified into control group (saline,36 cases)and ConA group (20 mg/kg ConA,36 cases).The blood specimens and liver tissues were collected at 2,6,12,18,24 and 48 h after injection.The activities of serum alanine aminotransferase (ALT)were measured by Reitman Frankel assay.Hematoxylin-eosin (HE)dyeing was carried out to assess the pathological change of liver tissue.The levels of eATP in serum were detected by chemiluminescence. Western-blot was employed to detect the expression of purinoceptor P2(P2X7).The contents of serum interleukin 1 beta (IL-1β)were assayed by enzyme-linked immunosorbent assay (ELISA).Results The ConA-induced murine acute liver injury model was constructed successfully.The level of eATP increased at 2 h after ConA injection,and reached peak at 18 h (700 nmol /L).Meanwhile,there expressed P2X7 in liver tissues.Compared with control group,the IL-1βlevels in serum of ConA group increased significantly (P<0.01).Conclusions In ConA-induced murine acute liver injury model,eATP releases from the injury liver tissues,and might influence the synthesis and secretion of inflammatory cytokine IL-1βthrough the P2X7 pathway,eventually aggravating the process of acute liver injury.%目的:探讨胞外三磷酸腺苷(eATP)在刀豆蛋白A(ConA)诱导小鼠急性肝损伤中的表达及意义。方法将72只昆明种小鼠随机分为对照组(36只)、ConA组(36只)。ConA组由尾静脉处注射20 mg/kg ConA,对照组注射同体积的无致热原生理盐水。2组分别于注射后2、6、12、18、24、48 h留取血液标本和肝脏标本。采用赖氏法检测血清丙氨酸氨基转移酶(ALT)活性,苏木素-伊红(HE )染色法检查肝组织病理学改变,化学发光技术检测血清eATP水平,免疫印

  8. Specific inhibition of kynurenate synthesis enhances extracellular dopamine levels in the rodent striatum

    Science.gov (United States)

    Amori, L; Wu, H.-Q.; Marinozzi, M; Pellicciari, R; Guidetti, P; Schwarcz, R

    2011-01-01

    Fluctuations in the endogenous levels of kynurenic acid (KYNA), a potent α7 nicotinic and NMDA receptor antagonist, affect extracellular dopamine (DA) concentrations in the rat brain. Moreover, reductions in KYNA levels increase the vulnerability of striatal neurons to NMDA receptor-mediated excitotoxic insults. We now assessed the role of a key KYNA-synthesizing enzyme, kynurenine aminotransferase II (KAT II), in these processes in the rodent striatum, using KAT II KO mice—which have reduced KYNA levels—and the selective KAT II inhibitor (S)-4-(ethylsulfonyl)benzoylalanine (S-ESBA) as tools. S-ESBA (applied by reverse dialysis) raised extracellular DA levels in the striatum of KYNA-deficient mice threefold and caused a much larger, 15-fold increase in wild-type mice. In the rat striatum, S-ESBA produced a 35% reduction in extracellular KYNA, which was accompanied by a 270% increase in extracellular DA. The latter effect was abolished by co-infusion of 100 nM KYNA. Intrastriatal S-ESBA pre-treatment augmented the size of a striatal quinolinate lesion by 370%, and this potentiation was prevented by co-infusion of KYNA. In separate animals, acute inhibition of KAT II reduced the de novo synthesis of KYNA during an early excitotoxic insult without enhancing the formation of the related neurotoxic metabolites 3-hydroxykynurenine and quinolinate. Taken together, these results provide further support for the concept that KAT II is a critical determinant of functionally relevant KYNA fluctuations in the rodent striatum. PMID:19138730

  9. Effect of physical exercise on changes in activities of creatine kinase, cytochrome c oxidase and ATP levels caused by ovariectomy.

    Science.gov (United States)

    Siebert, Cassiana; Kolling, Janaína; Scherer, Emilene B S; Schmitz, Felipe; da Cunha, Maira Jaqueline; Mackedanz, Vanize; de Andrade, Rodrigo B; Wannmacher, Clovis M D; Wyse, Angela T S

    2014-09-01

    The reduction in the secretion of ovarian hormones, principally estrogen, is a consequence of menopause. Estrogens act primarily as female sex hormones, but also exert effects on different physiological systems including the central nervous system. The treatment normally used to reduce the symptoms of menopause is the hormone therapy, which seems to be effective in treating symptoms, but it may be responsible for adverse effects. Based on this, there is an increasing demand for alternative therapies that minimize signs and symptoms of menopause. In the present study we investigated the effect of ovariectomy and/or physical exercise on the activities of energy metabolism enzymes, such as creatine kinase (cytosolic and mitochondrial fractions), pyruvate kinase, succinate dehydrogenase, complex II, cytochrome c oxidase, as well as on ATP levels in the hippocampus of adult rats. Adult female Wistar rats with 90 days of age were subjected to ovariectomy (an animal model widely used to mimic the postmenopausal changes). Thirty days after the procedure, the rats were submitted to the exercise protocol, which was performed three times a week for 30 days. Twelve hours after the last training session, the rats were decapitated for subsequent biochemical analyzes. Results showed that ovariectomy did not affect the activities of pyruvate kinase, succinate dehydrogenase and complex II, but decreased the activities of creatine kinase (cytosolic and mitochondrial fractions) and cytochrome c oxidase. ATP levels were also reduced. Exercise did not produce the expected results since it was only able to partially reverse the activity of creatine kinase cytosolic fraction. The results of this study suggest that estrogen deficiency, which occurs as a result of ovariectomy, affects generation systems and energy homeostasis, reducing ATP levels in hippocampus of adult female rats.

  10. Imaging Adenosine Triphosphate (ATP).

    Science.gov (United States)

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

  11. Optogenetic control of ATP release

    Science.gov (United States)

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

    2013-03-01

    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  12. Effects of a supplement designed to increase ATP levels on muscle strength, power output, and endurance

    Directory of Open Access Journals (Sweden)

    Stout Jeffrey R

    2008-01-01

    Full Text Available Abstract Background The present study examined the acute effects of a nutritional supplement intended to improve adenosine triphosphate (ATP concentrations on vertical jump height, isometric strength of the leg extensors, leg extension endurance, and forearm flexion endurance. Methods Twenty-four healthy men (mean age ± SD = 23 ± 4 yrs, stature = 181 ± 7 cm, and body mass = 82 ± 12 kg volunteered to complete a familiarization trial plus 2 randomly-ordered experimental trials separated by a 7-day washout period. Participants received either 6 (body mass Results There were no differences between the TR and PL trials for CVJ height (P > 0.05, isometric MVC peak torque (P > 0.05, maximal concentric isokinetic peak torque (P > 0.05, percent decline during the leg extension endurance tests (P > 0.05, or repetitions to exhaustion during the forearm flexion endurance tests (P > 0.05. Conclusion These findings indicated no improvements in the measured variables as a result of ingesting this nutritional supplement. Future studies should examine whether chronic supplementation or a loading period is necessary to observe any ergogenic effects of this supplement.

  13. 细胞外ATP和水杨酸对烟草光合指标的影响%Effects of extracellular ATP and salicylic acid on photosynthetic index of tobacco

    Institute of Scientific and Technical Information of China (English)

    冯汉青; 焦青松

    2014-01-01

    研究了细胞外ATP(eATP)和水杨酸(SA)对烟草(Nicotiana tabacum)叶片的气孔导度(GH2 O)、蒸腾速率(E)、光合作用速率(A)与叶绿素荧光参数[包括PSⅡ潜在最大光化学量子效率(Fv/Fm)、PSⅡ光适应下实际光化学效率Y(Ⅱ)、电子传递速率(ETR)、非光化学荧光淬灭(NPQ)和光化学荧光淬灭(qP)]的影响。结果表明:SA能导致A、GH2 O和E的下降,而eATP的处理能缓解SA造成的A、GH2 O和E的下降;但SA未对叶绿素荧光参数Fv/Fm、Y(Ⅱ)、NPQ、qP和ETR造成显著影响,eATP的加入也未改变SA处理下叶片叶绿素荧光参数的水平。这说明SA能导致光合作用的抑制,而eATP能明显缓解SA对光合作用的抑制,但以上作用可能均和光反应阶段无关。并对其内在机理进行了探讨。%The present work studied the effects of extracellular ATP (eATP )and salicylic acid (SA)on transpiration rate (E),stomatal conductance (GH2 O),photosynthesis rate (A)and chlorophyll fluorescence parameters,including maximal photochemical efficiency of PSⅡ(Fv/Fm),effective photochemical quantum yield of PSⅡ(Y(Ⅱ)),PSⅡelec-tron transport rate (ETR),non-photochemical quenching coefficient (NPQ)and the coefficient of photochemical fluo-rescence (qP))of tobacco (Nicotiana tabacum).The results showed that the transpiration rate (E),stomatal con-ductance (GH2 O)and photosynthesis rate (A)decreased under SA stress,while these decreases were anesised by the addition of eATP .However,SA had no significant effects on the chlorophyll fluorescence parameters,and the addition of eATP did not significantly changed the values of the chlorophyll fluorescence parameters under SA stress.These results indicated that SA could inhibit photosynthesis of tobacco leaves and the addition of eATP could alleviate the inhibition of photosynthesis by SA stress,while these effects were not related to the light reaction.We also discussed the possible mechanism based on these observations.

  14. PCR-cloning of tilapia ATP7A cDNA and its mRNA levels in tissues of tilapia following copper administrations.

    Science.gov (United States)

    Chen, Dong Shi; Chan, King Ming

    2011-10-01

    We are studying the toxicity of copper to tilapia and zebrafish and have found that the copper tolerance of tilapia and the sensitivity of zebrafish were due to several proteins' regulation mechanisms that were related to the effects of reactive oxygen species, mitochondrion copper transport, and stress response. To further reveal the mechanism of copper tolerance and sensitivity in tilapia and zebrafish, a full length cDNA of ATP7A was obtained in tilapia. Using real time quantitative PCR, the differential regulations of ATP7A in tilapia and zebrafish were studied. It was found that Cu(2+) gave a higher induction of ATP7A in tilapia than zebrafish, both in vivo and in vitro. These results suggest that the copper tolerance of tilapia may be due to higher expression level of ATP7A.

  15. Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels.

    Science.gov (United States)

    Ramió-Lluch, Laura; Yeste, Marc; Fernández-Novell, Josep M; Estrada, Efrén; Rocha, Luiz; Cebrián-Pérez, José A; Muiño-Blanco, Teresa; Concha, Ilona I; Ramírez, Alfredo; Rodríguez-Gil, Joan E

    2014-01-01

    Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca(2+) levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

  16. ATP as a signaling molecule: the exocrine focus

    DEFF Research Database (Denmark)

    Novak, Ivana

    2003-01-01

    Why and how do cells release ATP? It is not spilled energy. ATP becomes an extracellular regulator. Various cellular responses are initiated by purinergic receptors and signaling processes and are terminated by breakdown of ATP by ectonucleotidases. In epithelia, ATP regulates salt and water...

  17. ATP as a signaling molecule: the exocrine focus

    DEFF Research Database (Denmark)

    Novak, Ivana

    2003-01-01

    Why and how do cells release ATP? It is not spilled energy. ATP becomes an extracellular regulator. Various cellular responses are initiated by purinergic receptors and signaling processes and are terminated by breakdown of ATP by ectonucleotidases. In epithelia, ATP regulates salt and water tran...

  18. Correlation of ATP Citrate Lyase and Acetyl CoA Levels with Trichothecene Production in Fusarium graminearum

    Directory of Open Access Journals (Sweden)

    Naoko Sakamoto

    2013-11-01

    Full Text Available The correlation of ATP citrate lyase (ACL and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II and an enhancer (cobalt chloride of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride.

  19. Correlation of ATP citrate lyase and acetyl CoA levels with trichothecene production in Fusarium graminearum.

    Science.gov (United States)

    Sakamoto, Naoko; Tsuyuki, Rie; Yoshinari, Tomoya; Usuma, Jermnak; Furukawa, Tomohiro; Nagasawa, Hiromichi; Sakuda, Shohei

    2013-11-21

    The correlation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride.

  20. Changes in extracellular levels of amygdala amino acids in genetically fast and slow kindling rat strains.

    Science.gov (United States)

    Shin, Rick S; Anisman, Hymie; Merali, Zul; McIntyre, Dan C

    2002-08-01

    A neurochemical basis for many of the epilepsies has long been suspected to result from an imbalance between excitatory and inhibitory neurotransmitter mechanisms. Data supporting changes in extrasynaptic amino acid levels during epileptogenesis, however, remain controversial. In the present study, we used in vivo microdialysis to measure the levels of extracellular GABA (gamma-aminobutyric acid) and glutamate during seizure development in rats with a genetic predisposition for (Fast), or against (Slow), amygdala kindling. Dialysates were collected from both amygdalae before, during, and up to 12 min after a threshold-triggered amygdala afterdischarge (AD). One hour later, samples were again collected from both amygdalae in response to a hippocampal threshold AD. Daily amygdala kindling commenced the next day but without dialysis. After the rats were fully kindled, the same protocol was again employed. Amino acid levels were not consistently increased above baseline with triggered seizures in either strain. Instead, before kindling, a focal seizure in the Slow rats was associated with a large decrease in GABA in the non-stimulated amygdala, while amino acid levels in the Fast rats remained near baseline in both amygdalae. Similar results were seen after kindling. By contrast, before and after kindling, hippocampal stimulation caused large decreases in all amino acid levels in both amygdalae in both strains. These data suggest that, in response to direct stimulation, extracellular amino acid concentrations remain stable in tissues associated with either greater natural (Fast) or induced (kindled Fast/Slow) excitability, but are lowered with indirect stimulation (hippocampus) and/or low excitability.

  1. Neuropeptide Y infusion into the shell region of the rat nucleus accumbens increases extracellular levels of dopamine

    DEFF Research Database (Denmark)

    Sørensen, Gunnar; Wegener, Gregers; Hasselstrøm, Jørgen;

    2009-01-01

    Increases in extracellular dopamine in the shell region of the nucleus accumbens are centrally involved in mediating reinforcement of addictive drugs. Neuropeptide Y (NPY) and its receptors are present in the nucleus accumbens and have been implicated in addiction mechanisms. This study further...... explored the potential role of NPY in addiction mechanisms using microdialysis to measure extracellular dopamine in vivo after infusion of NPY directly into the accumbal shell region of adult rats. NPY was found to dose-dependently increase extracellular dopamine levels, indicating that NPY could play...

  2. Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

    Science.gov (United States)

    Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

    2015-01-20

    Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration.

  3. Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration.

    Science.gov (United States)

    Taurino, Federica; Giannoccaro, Caterina; Sardanelli, Anna Maria; Cavallo, Alessandro; De Luca, Elisa; Santacroce, Salvatore; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2016-08-15

    ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming phase of liver regeneration, 3 h after partial hepatectomy, liver mitochondria showed a marked lowering of the ATP synthase protein level that was reflected in the impairment of both ATP synthesis and hydrolysis. The ecto-ATP synthase level, in 3 h partial hepatectomized hepatocytes, was decreased similarly to the level of the mitochondrial ATP synthase, associated with a lowering of the ecto-ATP hydrolase activity coupled to proton influx. Noteworthily, the ecto-ATP synthase activity coupled to proton efflux was completely inhibited in 3 h partial hepatectomized hepatocytes, even in the presence of a marked intracellular acidification that would sustain it as in control and sham-operated hepatocytes. At the end of the liver regeneration, 7 days after partial hepatectomy, the level and the catalytic activities of the ecto- and mitochondrial ATP synthase reached the control and sham-operated values. The specific modulation of hepatocyte ecto-ATP synthase catalytic activities during liver regeneration priming phase may modulate the extracellular ADP/ATP levels and/or proton influx/efflux trafficking, making hepatocyte ecto-ATP synthase a candidate for a novel player in the liver regeneration process. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  4. Adenosine triphosphate (ATP) reduces amyloid-β protein misfolding in vitro.

    Science.gov (United States)

    Coskuner, Orkid; Murray, Ian V J

    2014-01-01

    Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aβ and reduction of Aβ misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

  5. High Prolidase Levels may be a Marker of Irreversible Extracellular Matrix Changes in Controlled Acromegaly Patients?

    Science.gov (United States)

    Tabur, S; Sezen, H; Korkmaz, H; Ozkaya, M; Akarsu, E

    2016-02-01

    The present study aimed to evaluate the activity of prolidase in controlled acromegaly patients and its association with oxidative stress. 25 acromegalic patients in remission who were followed in our outpatient clinic and 31 healthy controls were enrolled in the study. Serum growth hormone (GH), insulin-like growth factor 1 (IGF-1), total antioxidative status (TAS), total oxidative stress (TOS), total free sulfhydryl (-SH), paraoxonase (PON), arylesterase (ARE), lipid hydroperoxide (LOOH) and prolidase activity levels were measured. Percent ratio of TOS to TAS level was accepted as oxidative stress index (OSI). Serum prolidase activity, TOS, OSI, and LOOH levels were significantly higher in acromegaly patients compared to the healthy control group (pacromegaly patients compared to the healthy control group (p=0.002). Prolidase activity were positively correlated with TOS, OSI, LOOH and negatively correlated with -SH in patients with acromegaly (r=0.471, pacromegaly patients. These results suggest that extracellular matrix changes continue eventhough the disease is controlled, and elevated oxidative stress is involved in the increased prolidase activity in acromegaly patients.

  6. Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area.

    Science.gov (United States)

    Sotomayor-Zárate, Ramón; Abarca, Jorge; Araya, Katherine A; Renard, Georgina M; Andrés, María E; Gysling, Katia

    2015-11-01

    A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20μM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10μM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100μM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Effect of nitrogen narcosis on extracellular levels of dopamine and its metabolites in the rat striatum, using intracerebral microdialysis.

    Science.gov (United States)

    Barthelemy-Requin, M; Semelin, P; Risso, J J

    1994-12-19

    In man, nitrogen narcosis is characterized by euphoria, impaired cognitive function, neuromuscular incoordination and, ultimately, loss of consciousness. Because of the motor movement disorders, we chose to study the nigrostriatal system, whose major function is to regulate the extrapyramidal nervous system. The purpose of this investigation was to monitor changes in extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum of conscious rats, using intracerebral microdialysis. Results show a 40% decrease in extracellular DA concentration, a 59% increase in extracellular DOPAC and an increase in HVA starting with exposure to the nitrogen mixture. Thirty minutes after the beginning of the exposure, a compensation phase took place. HVA returns to its initial basal value, and levels of DOPAC and DA returned towards normal but never reached their initial values. These results contrast with those observed during the High Pressure Neurological Syndrome (HPNS, 5.1 MPa of helium pressure) in which there is a significant increase in extracellular DA. Therefore, some of the symptoms of nitrogen narcosis may be linked with the decrease in the extracellular DA levels.

  8. The effects of thioperamide on extracellular levels of glutamate and GABA in the rat prefrontal cortex.

    Science.gov (United States)

    Welty, Natalie; Shoblock, James R

    2009-12-01

    Histamine H3 receptors (H3R) are presynaptic heteroreceptors that negatively modulate the release of histamine and other neurotransmitters such as acetylcholine. Blocking H3 receptors with antagonists/inverse agonists has been shown to be procognitive and this effect has often been associated with increases in acetylcholine transmission. H3 receptors are abundantly expressed in the prefrontal cortex, an area associated with cognitive performance. While the procognitive effects of H3 receptor antagonists/inverse agonists may depend on alterations to acetylcholine or histamine release, other transmitters involved in cognitive processing such as glutamate and gamma-aminobutyric acid (GABA) may also be involved. The purpose of the present study was to examine the effects of thioperamide, an H3 receptor antagonist, on extracellular levels of glutamate and GABA in the prefrontal cortex. By means of in vivo microdialysis on freely moving Sprague Dawley rats, samples were collected and assayed via high-performance liquid chromatography coupled to electrochemical detection. Replacement of calcium with magnesium revealed that the release of GABA, but not glutamate, was calcium-dependent. Thioperamide (10-20 mg/kg) did not affect basal glutamate or GABA release. Perfusion with a high concentration of potassium (100 mM) increased GABA, but not glutamate, release and thioperamide (20 mg/kg) attenuated the effects of high potassium on GABA release. These data indicate that H3 receptors in the prefrontal cortex can enhance stimulated GABA release, but do not regulate basal levels of glutamate or GABA.

  9. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea.

    Science.gov (United States)

    Pearman, John K; Irigoien, Xabier; Carvalho, Susana

    2016-04-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore-offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales.

  10. LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

    Directory of Open Access Journals (Sweden)

    Rossana Migheli

    Full Text Available The leucine-rich repeat kinase 2 (LRRK2 gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD. LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

  11. LRRK2 Affects Vesicle Trafficking, Neurotransmitter Extracellular Level and Membrane Receptor Localization

    Science.gov (United States)

    Spissu, Ylenia; Sanna, Giovanna; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.; Galioto, Manuela; Rocchitta, Gaia; Biosa, Alice; Serra, Pier Andrea; Carri, Maria Teresa; Crosio, Claudia; Iaccarino, Ciro

    2013-01-01

    The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells. PMID:24167564

  12. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea

    KAUST Repository

    Pearman, John K.

    2015-11-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore–offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales.

  13. High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

    Science.gov (United States)

    Adam, Marion; Urbanski, Henryk F.; Garyfallou, Vasilios T.; Welsch, Ulrich; Köhn, Frank M.; Schwarzer, J. Ullrich; Strauss, Leena; Poutanen, Matti; Mayerhofer, Artur

    2011-01-01

    Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signaling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild-type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through prepubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of Tumor necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signaling of the testis in health and disease. PMID:22413766

  14. Acute dosing of latrepirdine (Dimebon™, a possible Alzheimer therapeutic, elevates extracellular amyloid-β levels in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Sano Mary

    2009-12-01

    Full Text Available Abstract Background Recent reports suggest that latrepirdine (Dimebon™, dimebolin, a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD. However, the mechanism(s underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-β (Aβ peptide in the brain, and Aβ-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Aβ using in vitro and in vivo experimental systems. Results We evaluated extracellular levels of Aβ in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 μM. Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 μM or 10 μM. Drug-naïve Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg. Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Aβ in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01, while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01. Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 μM and led to more modest

  15. Characterization of Intracellular and Extracellular Saxitoxin Levels in Both Field and Cultured Alexandrium spp. Samples from Sequim Bay, Washington

    Directory of Open Access Journals (Sweden)

    Vera L. Trainer

    2008-05-01

    Full Text Available Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs, intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004- 2007 and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA and an enzyme-linked immunosorbent assay (ELISA. Characterization of the PST toxin profile in the Sequim Bay isolates by preMar. column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

  16. Effects of Altered Levels of Extracellular Superoxide Dismutase and Irradiation on Hippocampal Neurogenesis in Female Mice

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Yani [Department of Neurology and Neurological Sciences, Stanford University, Stanford, California (United States); Leu, David [Department of Neurology and Neurological Sciences, Stanford University, Stanford, California (United States); Palo Alto Institute of Research and Education, Palo Alto, California (United States); Chui, Jennifer [Department of Neurology and Neurological Sciences, Stanford University, Stanford, California (United States); Fike, John R. [Departments of Neurosurgery and Radiation Oncology, University of California, San Francisco, California (United States); Huang, Ting-Ting, E-mail: tthuang@stanford.edu [Department of Neurology and Neurological Sciences, Stanford University, Stanford, California (United States); VA Palo Alto Health Care System, Palo Alto, California (United States)

    2013-11-15

    Purpose: Altered levels of extracellular superoxide dismutase (EC-SOD) and cranial irradiation have been shown to affect hippocampal neurogenesis. However, previous studies were only conducted in male mice, and it was not clear if there was a difference between males and females. Therefore, female mice were studied and the results compared with those generated in male mice from an earlier study. Methods and Materials: Female wild-type, EC-SOD-null (KO), and EC-SOD bigenic mice with neuronal-specific expression of EC-SOD (OE) were subjected to a single dose of 5-Gy gamma rays to the head at 8 weeks of age. Progenitor cell proliferation, differentiation, and long-term survival of newborn neurons were determined. Results: Similar to results from male mice, EC-SOD deficiency and irradiation both resulted in significant reductions in mature newborn neurons in female mice. EC-SOD deficiency reduced long-term survival of newborn neurons whereas irradiation reduced progenitor cell proliferation. Overexpression of EC-SOD corrected the negative impacts from EC-SOD deficiency and irradiation and normalized the production of newborn neurons in OE mice. Expression of neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 were significantly reduced by irradiation in wild-type mice, but the levels were not changed in KO and OE mice even though both cohorts started out with a lower baseline level. Conclusion: In terms of hippocampal neurogenesis, EC-SOD deficiency and irradiation have the same overall effects in males and females at the age the studies were conducted.

  17. Deltorphin II enhances extracellular levels of dopamine in the nucleus accumbens via opioid receptor-independent mechanisms.

    NARCIS (Netherlands)

    Murakawa, K.; Hirose, N.; Takada, K.; Suzuki, T.; Nagase, H.; Cools, A.R.; Koshikawa, N.

    2004-01-01

    The effects of the delta2-opioid receptor agonist, deltorphin II, on extracellular levels of dopamine in the rat nucleus accumbens were investigated in awake animals by in vivo brain microdialysis. In agreement with previous studies, perfusion of deltorphin II (50.0 nmol) into the nucleus accumbens

  18. Effects of the 5-HT(4) receptor agonist RS67333 and paroxetine on hippocampal extracellular 5-HT levels

    DEFF Research Database (Denmark)

    Licht, Cecilie Löe; Knudsen, Gitte Moos; Sharp, Trevor

    2010-01-01

    The 5-HT(4) receptor modulates activity of serotonergic neurons and is a new potential target for antidepressant treatment. This microdialysis study evaluated the effect of the 5-HT(4) receptor agonist, RS67333, on extracellular serotonin (5-hydroxytryptamine, 5-HT) and 5-HIAA levels in rat ventral...... on extracellular 5-HT or 5-HIAA levels, while acute paroxetine (0.5mg/kg i.v.) increased 5-HT levels by 299+/-16% and decreased 5-HIAA levels by 25+/-4%. Administration of RS67333 80 min after paroxetine caused an additional transient increase in 5-HT levels (to 398+/-52% of baseline). Subchronic RS67333...... administration (1.5mg/kg i.p.) increased basal 5-HT levels by 73+/-15% and decreased 5-HIAA levels by 27+/-13%. In conclusion, the 5-HT(4) receptor agonist RS67333 augmented the acute effect of paroxetine on extracellular 5-HT levels in the ventral hippocampus, and after 3 days increased basal hippocampal 5-HT...

  19. New insights on transcriptional responses of genes involved in carbon central metabolism, respiration and fermentation to low ATP levels in Escherichia coli.

    Science.gov (United States)

    Soria, Sandra; de Anda, Ramón; Flores, Noemí; Romero-Garcia, Susana; Gosset, Guillermo; Bolívar, Francisco; Báez-Viveros, José Luis

    2013-04-01

    Adenosine-5-triphosphate (ATP) plays a fundamental role in many cellular processes such as transport, central carbon metabolism, biosynthetic reactions, macromolecular synthesis, signal transduction and cellular division. In addition, the intracellular [ATP]/[ADP] ratio in Escherichia coli plays an important role in controlling the specific rates of growth (µ), glucose consumption (qGlc ) and oxygen uptake (qO2), as well as the transcriptome pattern in the cell, as was recently reported. In the current study, the energetic level (expressed as [ATP]/[ADP] ratio) was substantially reduced in E. coli strains by either over-expressing the F1 -ATPase activity (JMAGD(+)) or inactivating ATP synthase (JMat(-)). The physiological characterization of the wild-type JM101 strain and its derivative JMAGD(+) and JMatp(-) strains was conducted in bioreactors containing minimal medium with glucose. The inactivation of the atp operon and F1 -ATPase overexpression significantly diminished the energetic level and cAMP concentration in derivative strains. Relative transcription levels of 105 genes involved in glucose transport, glycolysis, tricarboxylic acid (TCA) cycle, fermentation, respiration, transcriptional regulators, transcription and genes involved in stress were determined by using qPCR. Interestingly, in the JMAGD(+) and JMatp(-) strains, having a reduced energetic level, many transcripts of glycolysis, TCA cycle and respiratory genes were down-regulated when compared to wild type JM101. The transcriptional responses, detected in the strains with reduced energetic level show down-regulation of genes involved in central carbon metabolism and respiration, these results are apposite to the observed trends of increased metabolic fluxes in glucose consumption, glycolysis, acetate synthesis, TCA cycle and respiration. Regulation mediated by CRP-cAMP complex may explain some observed transcriptional responses of TCA cycle genes, since cAMP concentration and crp transcript level

  20. Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death.

    Science.gov (United States)

    Fennessey, Christine M; Ivie, Susan E; McClain, Mark S

    2012-08-01

    Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.

  1. Low-level laser (light) therapy increases mitochondrial membrane potential and ATP synthesis in C2C12 myotubes with a peak response at 3-6 hours

    Science.gov (United States)

    Ferraresi, Cleber; Kaippert, Beatriz; Avci, Pinar; Huang, Ying-Ying; de Sousa, Marcelo Victor Pires; Bagnato, Vanderlei Salvador; Parizotto, Nivaldo Antonio; Hamblin, Michael R

    2014-01-01

    Low level laser (light) therapy has been used before exercise to increase muscle performance in both experimental animals and in humans. However uncertainty exists concerning the optimum time to apply the light before exercise. The mechanism of action is thought to be stimulation of mitochondrial respiration in muscles, and to increase adenosine triphosphate (ATP) needed to perform exercise. The goal of this study was to investigate the time course of the increases in mitochondrial membrane potential (MMP) and ATP in myotubes formed from C2C12 mouse muscle cells and exposed to light-emitting diode therapy (LEDT). LEDT employed a cluster of LEDs with 20 red (630 ± 10 nm, 25 mW) and 20 near-infrared (850 ± 10 nm, 50 mW) delivering 28 mW/cm2 for 90 sec (2.5 J/cm2) with analysis at 5 min, 3 h, 6 h and 24 h post-LEDT. LEDT-6h had the highest MMP, followed by LEDT-3h, LEDT-24h, LEDT-5min and Control with significant differences. The same order (6h>3h>24h>5min>Control) was found for ATP with significant differences. A good correlation was found (r=0.89) between MMP and ATP. These data suggest an optimum time window of 3-6 h for LEDT stimulate muscle cells. PMID:25443662

  2. Effect of copper on extracellular levels of key pro-inflammatory molecules in hypothalamic GN11 and primary neurons.

    Science.gov (United States)

    Spisni, Enzo; Valerii, Maria Chiara; Manerba, Marcella; Strillacci, Antonio; Polazzi, Elisabetta; Mattia, Toni; Griffoni, Cristiana; Tomasi, Vittorio

    2009-07-01

    Copper dyshomeostasis is responsible for the neurological symptoms observed in the genetically inherited copper-dependent disorders (e.g., Menkes' and Wilson's diseases), but it has been also shown to have an important role in neurodegenerative diseases such as Alzheimer disease, prion diseases, Parkinson's disease and amyotrophic lateral sclerosis. It is widely accepted that increased extracellular copper levels contribute to neuronal pathogenic process by increasing the production of dangerous radical oxygen species, but the existence of other molecular mechanisms explaining copper neurotoxicity has not been investigated yet. By using a cellular model based on hypothalamic GN11 cultured neurons exposed to copper supplementation and by analysing the cell conditioned media, we try here to identify new molecular events explaining the association between extracellular copper accumulation and neuronal damages. We show here that increased extracellular copper levels produce a wide complex of alterations in the neuronal extracellular environment. In particular, copper affects the secretion of molecules involved in the protection of neurons against oxidative stress, such as cyclophilin A (CypA), or of molecules capable of shifting neuronal cells towards a pro-inflammatory state, such as IL-1alpha, IL-12, Rantes, neutrophil gelatinase-associated lipocalin (NGAL) and secreted protein acidic and rich in cysteine (SPARC). Copper pro-inflammatory properties have been confirmed by using primary neurons.

  3. Low intracellular ATP levels exacerbate carcinogen-induced inflammatory stress response and inhibit in vitro tubulogenesis in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth Tahanian

    2011-01-01

    Full Text Available Elizabeth Tahanian, Sabrina Peiro, Borhane AnnabiLaboratoire d'Oncologie Moléculaire, Centre de Recherche BioMED, Département de Chimie, Université du Québec à Montréal, Montréal, Québec, CanadaAbstract: Solid tumor development requires angiogenesis and is correlated to the expression of inflammatory markers through cellular metabolic and energetic adaptation. While high glycolysis rates enable the cancer cell compartment to generate adenosine triphosphate (ATP, very little is known about the impact of low intracellular ATP concentrations within the vascular endothelial cell compartment, which is responsible for tumor angiogenesis. Here, we investigated the effect of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis through intracellular ATP depletion, on human brain microvascular endothelial cell (HBMEC angiogenic properties. While preformed capillaries remained unaffected, we found that in vitro tubulogenesis was dose-dependently decreased by 2-DG and that this correlated with reduced intracellular ATP levels. Procarcinogenic signaling was induced with phorbol 12-myristate 13-acetate (PMA and found to trigger the proinflammatory marker cyclooxygenase-2 (COX-2 and endoplasmic reticulum (ER stress marker GRP78 expression, whose inductions were potentiated when PMA was combined with 2-DG treatment. Inversely, PMA-induced matrix-metalloproteinase-9 (MMP-9 gene expression and protein secretion were abrogated in the presence of 2-DG, and this can be partially explained by reduced nuclear factor-κB signaling. Collectively, we provide evidence for an intracellular ATP requirement in order for tubulogenesis to occur, and we link increases in ER stress to inflammation. A better understanding of the metabolic adaptations of the vascular endothelial cells that mediate tumor vascularization will help the development of new drugs and therapies.Keywords: endoplasmic reticulum stress, MMP-9, COX-2, 2-deoxy-D-glucose, endothelial

  4. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  5. NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV Nonstructural Protein 5A (NS5A via MEK/Extracellular Signal Regulated Kinase (ERK Pathway

    Directory of Open Access Journals (Sweden)

    Xuesong Gao

    2013-05-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein 5A (NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

  6. Potassium Aspartate Attenuates Brain Injury Induced by Controlled Cortical Impact in Rats Through Increasing Adenosine Triphosphate (ATP) Levels, Na+/K+-ATPase Activity and Reducing Brain Edema.

    Science.gov (United States)

    Gu, Yi; Zhang, Jie; Zhao, Yumei; Su, Yujin; Zhang, Yazhuo

    2016-12-13

    BACKGROUND Potassium aspartate (PA), as an electrolyte supplement, is widely used in clinical practice. In our previous study, we found PA had neuroprotective effects against apoptosis after cerebral ischemia/reperfusion in rats. In this study, we examine whether PA has protective effects on traumatic brain injury (TBI). MATERIAL AND METHODS TBI was induced by controlled cortical impact (CCI) in rats. Vehicle treatment (control) or PA treatment was administered intraperitoneally at 30 minutes after CCI. The modified neurological severity score (mNSS) and cortical lesion volume were examined. Brain edema and blood-brain barrier (BBB) integrity were measured, as well as brain ATP contents, lactic acid levels, and Na+/K+-ATPase activities. RESULTS We found that CCI induced cortical injury in rats. Acute PA treatment at the dose of 62.5 mg/kg and 125 mg/kg significantly improved neurological deficits (pATP (pATP levels, Na+/K+-ATPase activity, and reducing brain edema. It provides experimental evidence for the clinical application of PA.

  7. Morphine decreases extracellular levels of glutamate in the anterior cingulate cortex: an in vivo microdialysis study in freely moving rats

    Institute of Scientific and Technical Information of China (English)

    YueHAO; Jing-yuYANG; MingGUO; Chun-fuWU; Ming-fanWU

    2004-01-01

    AIM: The anterior cingulate cortex (ACC), an important region of prefrontal cortex for cognitive functions, has been implicated in drug abuse and addiction. In the present study, we intended to investigate the effect of morphine on the extracellular levels of glutamate in the ACC in freely moving rats. METHODS: In vivo microdialysis coupled to high performance liquid chromatography and electrochemical detection had been used for the

  8. Association of Levels of Antibodies from Patients with Inflammatory Bowel Disease with Extracellular Proteins of Food and Probiotic Bacteria

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    Arancha Hevia

    2014-01-01

    Full Text Available Inflammatory bowel disease (IBD is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n=50, Crohn's disease patients (CD, n=37, and ulcerative colitis patients (UC, n=15. Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH were significantly higher in the IBD group (P<0.002; n=52. The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria.

  9. Extracellular matrix protein fibulin-1 plasma levels are associated with increased cardiovascular risk in chronic kidney disease

    DEFF Research Database (Denmark)

    Scholze, Alexandra

    INTRODUCTION AND AIMS: Fibulin-1 is one of the few extracellular matrix proteins present in blood in high concentrations. We aimed to define the relationship between plasma fibulin-1 levels and risk markers of cardiovascular disease in patients with chronic kidney disease. METHODS: Plasma fibulin-1...... of plasma fibulin-1. CONCLUSIONS: Increased plasma fibulin-1 levels were associated with impaired kidney function and diabetes. Fibulin-1 levels were also associated with hemodynamic cardiovascular risk markers. We conclude, that fibulin-1 is involved in the pathogenesis of cardiovascular disease observed...

  10. Comparative effects of parathion and chlorpyrifos on extracellular endocannabinoid levels in rat hippocampus: Influence on cholinergic toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK (United States); Parsons, Loren [Committee on Neurobiology of Affective Disorders, The Scripps Research Institute, La Jolla, CA (United States); Pope, Carey, E-mail: carey.pope@okstate.edu [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK (United States)

    2013-11-01

    Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides (OPs) that elicit acute toxicity by inhibiting acetylcholinesterase (AChE). Endocannabinoids (eCBs, N-arachidonoylethanolamine, AEA; 2-arachidonoylglycerol, 2AG) can modulate neurotransmission by inhibiting neurotransmitter release. We proposed that differential inhibition of eCB-degrading enzymes (fatty acid amide hydrolase, FAAH, and monoacylglycerol lipase, MAGL) by PS and CPF leads to differences in extracellular eCB levels and toxicity. Microdialysis cannulae were implanted into hippocampus of adult male rats followed by treatment with vehicle (peanut oil, 2 ml/kg, sc), PS (27 mg/kg) or CPF (280 mg/kg) 6–7 days later. Signs of toxicity, AChE, FAAH and MAGL inhibition, and extracellular levels of AEA and 2AG were measured 2 and 4 days later. Signs were noted in PS-treated rats but not in controls or CPF-treated rats. Cholinesterase inhibition was extensive in hippocampus with PS (89–90%) and CPF (78–83%) exposure. FAAH activity was also markedly reduced (88–91%) by both OPs at both time-points. MAGL was inhibited by both OPs but to a lesser degree (35–50%). Increases in extracellular AEA levels were noted after either PS (about 2-fold) or CPF (about 3-fold) while lesser treatment-related 2-AG changes were noted. The cannabinoid CB1 receptor antagonist/inverse agonist AM251 (3 mg/kg, ip) had no influence on functional signs after CPF but markedly decreased toxicity in PS-treated rats. The results suggest that extracellular eCBs levels can be markedly elevated by both PS and CPF. CB1-mediated signaling appears to play a role in the acute toxicity of PS but the role of eCBs in CPF toxicity remains unclear. - Highlights: • Chlorpyrifos and parathion both extensively inhibited hippocampal cholinesterase. • Functional signs were only noted with parathion. • Chlorpyrifos and parathion increased hippocampal extracellular anandamide levels. • 2-Arachidonoylglycerol levels were

  11. Sleep, brain energy levels, and food intake: Relationship between hypothalamic ATP concentrations, food intake, and body weight during sleep-wake and sleep deprivation in rats.

    Science.gov (United States)

    Dworak, M; Kim, T; McCarley, R W; Basheer, R

    2011-06-01

    The feeling of hunger and feeding, a wake-state-dependent behavior, is regulated by specific centers within the hypothalamus. While paraventricular nucleus (PVN), arcuate nucleus (ARC), and dorso- and ventromedial hypothalamus (DMH/VMH) regulate feeding, the lateral hypothalamus (LH) is associated both with feeding and wake/REM sleep regulation. In order to examine the effects of sleep and wakefulness on food intake and body weight, we also measured hypothalamic ATP concentrations, which are known to be involved in feeding behavior and sleep-wake regulation. In rats, food intake and body weight was measured during a 24-h light-dark cycle and during 6 h of sleep deprivation (SD) performed by gentle handling. Tissue samples from the PVN, ARC/DMH/VMH, and LH were collected after 6 h of SD and from time-matched diurnal controls. ATP was measured by luciferin-luciferase bioluminescence assay. Across the 24-h light-dark period, rats consumed approximately 28.13±4.48 g of food and gained 5.22±1.65 g with a positive correlation between food intake and body weight. During SD, while food intake increased significantly +147.31±6.13%, they lost weight significantly (-93.29±13.64%) when compared to undisturbed controls. SD resulted in a significant decrease in ATP levels only in LH (-44.60±21.13%) with no change in PVN, ARC/DMH/VMH region when compared with undisturbed controls. The results indicate a strong overall correlation between ATP concentrations in the LH and individual food intake and suggest a sleep-wake dependent neuronal control of food intake and body weight.

  12. Increased extracellular dopamine and 5-hydroxytryptamine levels contribute to enhanced subthalamic nucleus neural activity during exhausting exercise

    Directory of Open Access Journals (Sweden)

    Y Hu

    2015-09-01

    Full Text Available The purpose of the study was to explore the mechanism underlying the enhanced subthalamic nucleus (STN neural activity during exhausting exercise from the perspective of monoamine neurotransmitters and changes of their corresponding receptors. Rats were randomly divided into microdialysis and immunohistochemistry study groups. For microdialysis study, extracellular fluid of the STN was continuously collected with a microdialysis probe before, during and 90 min after one bout of exhausting exercise. Dopamine (DA and 5-hydroxytryptamine (5-HT levels were subsequently detected with high-performance liquid chromatography (HPLC. For immunohistochemistry study, the expression of DRD 2 and HT 2C receptors in the STN, before, immediately after and 90 min after exhaustion was detected through immunohistochemistry technique. Microdialysis study results showed that the extracellular DA and 5-HT neurotransmitters increased significantly throughout the procedure of exhausting exercise and the recovery period (P0.05. Our results suggest that the increased extracellular DA and 5-HT in the STN might be one important factor leading to the enhanced STN neural activity and the development of fatigue during exhausting exercise. This study may essentially offer useful evidence for better understanding of the mechanism of the central type of exercise-induced fatigue.

  13. Ascorbate reduces morphine-induced extracellular DOPAC level in the nucleus accumbens: A microdialysis study in rats.

    Science.gov (United States)

    Rajaei, Z; Alaei, H; Nasimi, A; Amini, H; Ahmadiani, A

    2005-08-16

    Most drugs of abuse increase dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the shell of the nucleus accumbens. The effects of ascorbate, which is known to modulate dopamine neurotransmission, on the extracellular level of DOPAC in the nucleus accumbens of naive rats and of rats treated acutely with morphine were studied by using in vivo microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-ECD). Acute morphine (20 mg/kg ip) treatment increased the level of DOPAC in the nucleus accumbens to approximately 170% of basal level. Acute treatment with ascorbate (500 mg/kg ip) alone did not alter nucleus accumbens' DOPAC level, but pretreatment with ascorbate (500 mg/kg ip) 30 min before morphine administration attenuated the effects of acute morphine on the level of DOPAC. These results suggest that ascorbate modulates the mesolimbic dopaminergic pathway.

  14. Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality.

    Science.gov (United States)

    Iaffaldano, N; Paventi, G; Pizzuto, R; Di Iorio, M; Bailey, J L; Manchisi, A; Passarella, S

    2016-08-01

    This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions.

  15. Modulation of memory with septal injections of morphine and glucose: effects on extracellular glucose levels in the hippocampus.

    Science.gov (United States)

    McNay, Ewan C; Canal, Clinton E; Sherwin, Robert S; Gold, Paul E

    2006-02-28

    The concentration of glucose in the extracellular fluid (ECF) of the hippocampus decreases substantially during memory testing on a hippocampus-dependent memory task. Administration of exogenous glucose, which enhances task performance, prevents this decrease, suggesting a relationship between hippocampal glucose availability and memory performance. In the present experiment, spontaneous alternation performance and task-related changes in hippocampal ECF glucose were assessed in rats after intraseptal administration of morphine, which impairs memory on a spontaneous alternation task, and after co-administration of intraseptal glucose, which attenuates that impairment. Consistent with previous findings, spontaneous alternation testing resulted in a decrease in hippocampal ECF glucose levels in control rats. However, rats that received intraseptal morphine prior to testing showed memory impairments and an absence of the task-related decrease in hippocampal ECF glucose levels. Intraseptal co-administration of glucose with morphine attenuated the memory impairment, and ECF glucose levels in the hippocampus decreased in a manner comparable to that seen in control rats. These data suggest that fluctuations in hippocampal ECF glucose levels may be a marker of mnemonic processing and support the view that decreases in extracellular glucose during memory testing reflect increased glucose demand during memory processing.

  16. High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris.

    Science.gov (United States)

    Eom, Gyeong Tae; Lee, Seung Hwan; Song, Bong Keun; Chung, Keun-Wo; Kim, Young-Wun; Song, Jae Kwang

    2013-08-01

    The gene encoding lipase B from Candida antarctica (CalB) was expressed in Pichia pastoris after it was synthesized by the recursive PCR and cloned into the Pichia expression plasmid, pPICZαA. The CalB was successfully secreted in the recombinant P. pastoris strain X-33 with an apparent molecular weight of 34 kDa. For 140 h flask culture, the dry cell weight and the extracellular lipase activity reached at 5.4 g/l and 57.9 U/l toward p-nitrophenyl palmitate, respectively. When we performed the fed-batch fermentation using a methanol feeding strategy for 110 h, the dry cell weight and the extracellular lipase activity were increased to 135.7 g/l and 11,900 U/l; the CalB protein concentration was 1.18 g/l of culture supernatant. The characteristics of CalB recovered from the P. pastoris culture were compared with the commercial form of CalB produced in Aspergillus oryzae. The kinetic constants and specific activity, the effects of activity and stability on temperature and pH, the glycosylation extent, the degree of immobilization on macroporous resin and the yield of esterification reaction between oleic acid and n-butanol were almost identical to each other. Therefore, we successfully proved that the Pichia-based expression system for CalB in this study was industrially promising compared with one of the most efficient production systems.

  17. Mitochondrial diaphorases as NAD+ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition

    Science.gov (United States)

    Kiss, Gergely; Konrad, Csaba; Pour-Ghaz, Issa; Mansour, Josef J.; Németh, Beáta; Starkov, Anatoly A.; Adam-Vizi, Vera; Chinopoulos, Christos

    2014-01-01

    Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD+ supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD+ pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD+ derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.—Kiss, G., Konrad, C., Pour-Ghaz, I., Mansour, J. J., Németh, B., Starkov, A. A., Adam-Vizi, V., Chinopoulos, C. Mitochondrial diaphorases as NAD+ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition. PMID:24391134

  18. Detecting ATP release by a biosensor method.

    Science.gov (United States)

    Hayashi, Seiji; Hazama, Akihiro; Dutta, Amal K; Sabirov, Ravshan Z; Okada, Yasunobu

    2004-11-09

    Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

  19. The effects of LPM570065, a novel triple reuptake inhibitor, on extracellular serotonin, dopamine and norepinephrine levels in rats.

    Directory of Open Access Journals (Sweden)

    Renyu Zhang

    Full Text Available Triple reuptake inhibitors (TRIs are currently being developed as a new class of promising antidepressants that block serotonin (5-HT, dopamine (DA and norepinephrine (NE transporters, thereby increasing extracellular monoamine concentrations. The purpose of this study was to investigate the effects of LPM570065, a novel TRI and a desvenlafaxine prodrug, on extracellular 5-HT, DA and NE levels in the rat striatum after acute and chronic administration relative to desvenlafaxine, using High Performance Liquid Chromatography (HPLC and microdialysis. Acute administration was performed by providing rodents with oral solutions (0.06 mmol·kg(-1 p.o., oral suspensions (0.06 mmol·kg(-1 p.o. and intravenous solutions (0.04 mmol·kg(-1 i.v. of LPM570065 and desvenlafaxine. Oral suspensions (0.06 mmol·kg(-1·day(-1 of the two drugs were also administered for a 14-day chronic period. HPLC analysis revealed that LPM570065 rapidly penetrated the rat striatum, converted into desvenlafaxine and exhibited larger total exposure compared with the administration of desvenlafaxine. Microdialysis revealed that acute and chronic administration of oral suspension of LPM570065 increased the 5-HT, DA and NE levels more than the relative administration of desvenlafaxine. Unlike desvenlafaxine, acute administration of an intravenous LPM570065 solution did not induce the undesirable 90% decrease in extracellular 5-HT levels. In contrast to the fully dose-dependent elevation of 5-HT induced by desvenlafaxine, the acute administration of LPM570065 showed a capped increase in extracellular 5-HT levels when combined with WAY-100635. Additionally, forced swim test demonstrated that acute and chronic administration of LPM570065 reduced the immobility time more than the relative administration of desvenlafaxine. These data suggest that LPM570065 may have greater efficacy and/or a more rapid onset of antidepressant action than desvenlafaxine and also counterbalance the harmful

  20. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Amstrup, Jan; Henriksen, Katrine Lütken

    2003-01-01

    ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock...... and, most importantly, cholinergic stimulation released 5-20nM ATP into the medium, as monitored by luminescence of the luciferin/luciferase reaction. ATP release was visualized at the single acinus level as luciferin consumption detected by confocal laser scanning microscopy (CLSM). The estimated ATP...... concentrations were higher, about 10µM, around acinar cells after cholinergic stimulation. Fluorescence of quinacrine and MANT-ATP indicated that some ATP is stored in secretory granules. Pancreatic acini have transcripts for P2X1, P2X4, P2Y2, and P2Y4 receptors, but measurements of Ca2+ signals in isolated...

  1. Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

    Science.gov (United States)

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems.

  2. Local Control of Extracellular Dopamine Levels in the Medial Nucleus Accumbens by a Glutamatergic Projection from the Infralimbic Cortex.

    Science.gov (United States)

    Quiroz, César; Orrú, Marco; Rea, William; Ciudad-Roberts, Andrés; Yepes, Gabriel; Britt, Jonathan P; Ferré, Sergi

    2016-01-20

    It is generally assumed that infralimbic cortex (ILC) and prelimbic cortex, two adjacent areas of the medial prefrontal cortex (mPFC) in rodents, provide selective excitatory glutamatergic inputs to the nucleus accumbens (NAc) shell and core, respectively. It is also generally believed that mPFC influences the extracellular levels of dopamine in the NAc primarily by an excitatory collateral to the ventral tegmental area (VTA). In the present study, we first established the existence of a selective functional connection between ILC and the posteromedial portions of the VTA (pmVTA) and the mNAc shell (pmNAc shell), by measuring striatal neuronal activation (immunohistochemical analysis of ERK1/2 phosphorylation) and glutamate release (in vivo microdialysis) upon ILC electrical stimulation. A novel optogenetic-microdialysis approach allowed the measurement of extracellular concentrations of glutamate and dopamine in the pmNAc shell upon local light-induced stimulation of glutamatergic terminals from ILC. Cortical electrical and local optogenetic stimulation produced significant increases in the extracellular concentrations of glutamate and dopamine in the pmNAc shell. Local blockade of glutamate release by perfusion of an adenosine A2A receptor antagonist in the pmNAc shell blocked the dopamine release induced by local optogenetic stimulation but only partially antagonized dopamine release induced by cortical electrical stimulation. The results demonstrate that ILC excitatory afferents directly modulate the extracellular concentration of dopamine in the pmNAc shell, but also support the involvement of an indirect mechanism of dopamine control, through a concomitant ILC-mediated activation of the pmVTA. Significance statement: We established the existence of a functional connection between the infralimbic cortex (ILC) and the posteromedial portions of the ventral tegmental area (pmVTA) and the medial nucleus acumbens shell (pmNAc shell). A novel optogenetic

  3. The astrocyte-derived alpha7 nicotinic receptor antagonist kynurenic acid controls extracellular glutamate levels in the prefrontal cortex.

    Science.gov (United States)

    Wu, Hui-Qiu; Pereira, Edna F R; Bruno, John P; Pellicciari, Roberto; Albuquerque, Edson X; Schwarcz, Robert

    2010-01-01

    The cognitive deficits seen in schizophrenia patients are likely related to abnormal glutamatergic and cholinergic neurotransmission in the prefrontal cortex. We hypothesized that these impairments may be secondary to increased levels of the astrocyte-derived metabolite kynurenic acid (KYNA), which inhibits alpha7 nicotinic acetylcholine receptors (alpha7AChR) and may thereby reduce glutamate release. Using in vivo microdialysis in unanesthetized rats, we show here that nanomolar concentrations of KYNA, infused directly or produced in situ from its bioprecursor kynurenine, significantly decrease extracellular glutamate levels in the prefrontal cortex. This effect was prevented by the systemic administration of galantamine (3 mg/kg) but not by donepezil (2 mg/kg), indicating that KYNA blocks the allosteric potentiating site of the alpha7AChR, which recognizes galantamine but not donepezil as an agonist. In separate rats, reduction of prefrontal KYNA formation by (S)-4-ethylsulfonyl benzoylalanine, a specific inhibitor of KYNA synthesis, caused a significant elevation in extracellular glutamate levels. Jointly, our results demonstrate that fluctuations in endogenous KYNA formation bidirectionally influence cortical glutamate concentrations. These findings suggest that selective attenuation of cerebral KYNA production, by increasing glutamatergic tone, might improve cognitive function in individuals with schizophrenia.

  4. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer

    National Research Council Canada - National Science Library

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-01-01

    ...) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC...

  5. The Role of ATP in the Regulation of NCAM Function

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a direct...... ATP-NCAM interaction and discuss its functional implications. The ectodomain of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP...... overlaps with the site of NCAM-FGFR interaction, and ATP is capable of disrupting NCAM-FGFR binding. This implies that NCAM signaling through FGFR can be regulated by ATP, which is supported by the observation that ATP can abrogate NCAM-induced neurite outgrowth. Finally, ATP can induce NCAM ectodomain...

  6. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Liu Bin

    2012-09-01

    Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  7. ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

    Science.gov (United States)

    Song, Shanshan; Jacobson, Krista N; McDermott, Kimberly M; Reddy, Sekhar P; Cress, Anne E; Tang, Haiyang; Dudek, Steven M; Black, Stephen M; Garcia, Joe G N; Makino, Ayako; Yuan, Jason X-J

    2016-01-15

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. Copyright

  8. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our...... aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan......-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient...

  9. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    2008-12-18

    mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca2+ signaling through gap junctions seems more effective.

  10. A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.

    Science.gov (United States)

    Vanegas, Diana C; Clark, Greg; Cannon, Ashley E; Roux, Stanley; Chaturvedi, Prachee; McLamore, Eric S

    2015-12-15

    The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment.

  11. Antiphospholipid Antibodies Bind ATP: A putative Mechanism for the Pathogenesis of Neuronal Dysfunction

    Directory of Open Access Journals (Sweden)

    J. Chapman

    2005-01-01

    Full Text Available Antiphospholipid antibodies (aPL generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA. Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS. IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (>50% inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound β2-glycoprotein-I (β2-GPI, also known as apolipoprotein H suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of β2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of β2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through β2-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters.

  12. Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28 regulator during early antiviral response mediated by IFNβ in mouse pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    Wieke Freudenburg

    Full Text Available Autoimmune destruction of insulin producing pancreatic β-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28 activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic β-cells. Of special interest to immunoproteasome activation in β-cells are the effects of IFNβ, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic β1(i, β2(i and β5(i, and 11S mRNAs upon exposure to IFNβ or IFNγ. Higher concentrations of IFNβ than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNβ-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic β-cells exposed to IFNβ, but primarily in cells with reduced ATP levels that stimulate the

  13. Extracellular citrulline levels in the nucleus accumbens during the acquisition and extinction of a classical conditioned reflex with pain reinforcement.

    Science.gov (United States)

    Savel'ev, S A; Saul'skaya, N B

    2007-03-01

    Studies on Sprague-Dawley rats using in vivo microdialysis and HPLC showed that the acquisition and performance of a classical conditioned reflex with pain reinforcement was accompanied by increases in the concentrations of citrulline (a side product of nitric oxide formation) and arginine (the substrate of NO synthase) in the intercellular space of the nucleus accumbens. During extinction of the reflex, there was a decrease in the elevation of extracellular citrulline in this brain structure, which correlated with the extent of extinction of the reflex. Recovery of the reflex led to increases in arginine and citrulline levels in the nucleus accumbens. These data suggest that there is an increase in nitric oxide production in the nucleus accumbens during the acquisition and performance of a classical conditioned reflex with pain reinforcement, which decreases as the reflex is extinguished and recovers with recovery of the reflex.

  14. Acute iboga alkaloid effects on extracellular serotonin (5-HT) levels in nucleus accumbens and striatum in rats.

    Science.gov (United States)

    Wei, D; Maisonneuve, I M; Kuehne, M E; Glick, S D

    1998-08-03

    The iboga alkaloid, ibogaine, its metabolite, noribogaine, and the congener, 18-methoxycoronaridine (18-MC) have all been claimed to have anti-addictive properties in animal models, but the mechanisms underlying these effects are unclear. Ibogaine and noribogaine were shown to have affinity for the serotonin transporter, and inhibition of serotonin reuptake has been proposed to be involved in their anti-addictive actions. It is not known yet if 18-MC also has this property. In vivo microdialysis and HPLC (microbore) were used to determine acute changes in extracellular serotonin levels in nucleus accumbens (NAC) and striatum (STR) after both i.p. (40 mg/kg for all drugs) and i.v. (1-10 mg/kg for ibogaine and noribogaine) drug administration in awake freely moving female Sprague-Dawley rats (250-275 g). After i.p. administration, ibogaine, noribogaine and 18-MC had very different effects on extracellular serotonin levels in both NAC and STR: ibogaine elicited large increases (up to 25-fold in NAC and 10- fold in STR), noribogaine produced moderate increases (up to 8-fold in NAC and 5-fold in STR), and 18-MC had no effect in either brain region. These and other data suggest that (1) the serotonergic system may not be an essential factor in the anti-addictive actions of these drugs; (2) ibogaine (or an unidentified metabolite) may release serotonin as well as inhibit its reuptake; (3) stimulation of the ascending serotonergic system may mediate ibogaine's hallucinogenic effect; and (4) 18-MC probably has no affinity for the serotonin transporter, and is unlikely to be a hallucinogen.

  15. A new classification paradigm of extracellular polymeric substances (EPS) in activated sludge: separation and characterization of exopolymers between floc level and microcolony level.

    Science.gov (United States)

    Wang, Bin-Bin; Chang, Qing; Peng, Dang-Cong; Hou, Yin-Ping; Li, Hui-Juan; Pei, Li-Ying

    2014-11-01

    Extracellular polymeric substances (EPS) play a crucial role in the formation of activated sludge flocs. However, until now, the EPS are rather classified by the method used for extraction than by a theoretical consideration of their function and composition. In this paper, a new classification paradigm of EPS was proposed, which offered a novel approach to identify the role of EPS in the formation of activated sludge flocs. The current study gave an exploration to distinguish the EPS in the floc level (extra-microcolony polymers, EMPS) and in the microcolony level (extra-cellular polymers, ECPS). It was found that cation exchange resin treatment is efficient to disintegrate the flocs for EMPS extraction, however, inefficient to disaggregate the microcolonies for ECPS harvesting. A two-steps extraction strategy (cation exchange resin treatment followed by ultrasonication-high speed centrifugation treatment) was suggested to separate these two types of EPS in activated sludge flocs and the physicochemical characteristics of EMPS and ECPS were compared. The protein/polysaccharide ratio of ECPS was higher than that of EMPS and the molecular weight of proteins in EMPS and ECPS were found to be different. The ECPS contained higher molecular weight proteins and more hydrophobic substances than the EMPS contained. The result of excitation-emission matrix fluorescence spectroscopy analysis also showed that the EMPS and the ECPS have different fluorescent expressions and the components of EMPS were more diverse than that of ECPS. All results reported herein demonstrated that two different types of exopolymers exist in the activated sludge flocs and the inter-particle forces for aggregation of activated sludge flocs are not identical between the floc level and the microcolony level. It suggested that cation bridging interactions are more crucial in floc level flocculation, while the entanglement and hydrophobic interactions are more important in microcolony level cohesion.

  16. Action of Al-ATP on the isolated working rat heart.

    Science.gov (United States)

    Korchazhkina, O; Wright, G; Exley, C

    1998-02-15

    ATP is an important extracellular messenger in the coronary vasculature of the heart. To be effective its extracellular concentration must be tightly controlled and this is achieved via ectonucleotidases located in the luminal surface of the coronary endothelium. Al-ATP is a potent inhibitor of the hydrolysis of ATP and we speculated that Al-ATP released by cells into the blood would disrupt the signalling function of extracellular ATP. We tested this hypothesis by perfusing isolated working Wistar rat hearts with buffers containing either ATP or Al-ATP. The functional parameters measured were, coronary flow, heart rate and pulsatile power. A number of control perfusions including adenosine, ATP-gamma-S and Al were used to identify those effects which might be specific to ATP and Al-ATP. Al-ATP did not appear to inhibit the function of the endothelial ectonucleotidases. Both ATP and Al-ATP produced a significant increase in coronary flow and this could be attributed to a coronary vasodilation. Interestingly, whilst the effect of ATP was reversible that of Al-ATP was not. ATP caused a reduction in heart rate which was potentiated by aluminium. The negatively chronotropic effect of Al-ATP was mediated via a mechanism which was either distinct from or in addition to the similar response known to be caused by adenosine. We have demonstrated for the first time an influence of Al-ATP on heart function. Perhaps more pertinently we present the first evidence that Al-ATP may influence the function of ATP-specific receptors.

  17. External Dentin Stimulation Induces ATP Release in Human Teeth.

    Science.gov (United States)

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

  18. Elevated levels of mitochonrial respiratory complexes activities and ATP production in 17-β-estradiol-induced prolactin-secretory tumor cells in male rats are inhibited by melatonin in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    WANG Bao-qiang; YANG Quan-hui; XU Rong-kun; XU Jian-ning

    2013-01-01

    Background Our earlier studies indicate that melatonin inhibits the proliferation of prolactinoma and induces apoptosis of pituitary prolactin-secreting tumor in rats.Melatonin has also been shown to induce apoptosis and to reduce the production of ATP in breast tumor cells.This study analyzed the levels of the four mitochondrial respiratory complexes and the production of ATP and also the effects of melatonin treatment of prolactinoma.Methods In the in vivo study,mitochondria were harvested from control pituitaries or prolactinoma collected from the pituitaries of melatonin-and 17-β-estradiol (E2)-treated male rats.In the in vitro study,prolactinoma cells mitochondria were harvested.Activities of the four mitochondrial respiratory complexes were assayed using fluorometer.ATP production of prolactinoma cells was estimated using bioluminescent methods.Results Elevated levels of four mitochondrial respiratory complexes activities and ATP production were recorded in prolactinoma cells.Moreover,in both in vivo and in vitro studies,melatonin inhibited the activities of mitochondrial respiratory complexes and the production of ATP in prolactinoma cells.Conclusions There is a link between mitochondrial function increase and tumorigenesis.Melatonin induces apoptosis of pituitary prolactin-secreting tumor of rats via the induction of mitochondrial dysfunction and inhibition of energy metabolism.

  19. Cellular ATP release in the lung and airway

    Directory of Open Access Journals (Sweden)

    Satoru Ito

    2016-11-01

    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  20. Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

    Science.gov (United States)

    Xia, Jingsheng; Lim, Jason C; Lu, Wennan; Beckel, Jonathan M; Macarak, Edward J; Laties, Alan M; Mitchell, Claire H

    2012-05-15

    Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.

  1. Relationship between serum HER2 extracellular domain levels,tissue HER2 expression,and clinico-pathological parameters in early stage breast cancer

    Institute of Scientific and Technical Information of China (English)

    MA Li; YANG Hong-ying; HAN Xiao-hong; LI Jia; WANG Fang; ZHANG Chun-ling; YAO Jia-rui; SHI Yuan-kai

    2012-01-01

    Background Measurement of human epidermal growth factor receptor 2(HER2)protein in the serum of metastatic breast cancer patients has previously been reported,but there are no consistent data to support the clinical utility of serum HER2 extracellufar domain for patients with early stage breast cancer.We aimed to evaluate the correlation between serum extracellular domain levels and tissue HER2 expression,and analyzed their relationship with clinico-pathological parameters in patients with early stage disease.Methods A prospective study was conducted on 232 breast cancer patients with stage Ⅰ-Ⅲ?prior to treatment.Preoperative serum samples were measured by enzyme-linked immunosorbent assay.Tissue HER2 status was analyzed by immunohistochemistry and fluorescence in situ hybridization assays.Results The median serum extracellular domain concentration was 6.8 ng/ml.The best diagnostic cut-off value was 7.4 ng/ml,with 62.9% sensitivity and 85.3% specificity.High serum extracellular domain levels were reported in 89 patients(38.3%),and HER2-positive expression was observed in 77 patients(33.2%).Multivariate analysis showed that elevated serum extracellular domain correlated with postmenopausal status(P<0.001),high histological grade(P<0.001),negativity of both estrogen(P=0.012)and progesterone receptors(P<0.001),and high levels of carcinoembryonic antigen 153(P=0.048).Conclusions We recommend that 7.4 ng/ml should be used as the cut-off value when evaluating serum extracellular domain levels in early stage of breast cancer.Patients with high serum extracellular domain levels have a certain clinicopathological characteristics,may provide a basis for clinical practice.

  2. Alterations in brain extracellular dopamine and glycine levels following combined administration of the glycine transporter type-1 inhibitor Org-24461 and risperidone.

    Science.gov (United States)

    Nagy, Katalin; Marko, Bernadett; Zsilla, Gabriella; Matyus, Peter; Pallagi, Katalin; Szabo, Geza; Juranyi, Zsolt; Barkoczy, Jozsef; Levay, Gyorgy; Harsing, Laszlo G

    2010-12-01

    The most dominant hypotheses for the pathogenesis of schizophrenia have focused primarily upon hyperfunctional dopaminergic and hypofunctional glutamatergic neurotransmission in the central nervous system. The therapeutic efficacy of all atypical antipsychotics is explained in part by antagonism of the dopaminergic neurotransmission, mainly by blockade of D(2) dopamine receptors. N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia can be reversed by glycine transporter type-1 (GlyT-1) inhibitors, which regulate glycine concentrations at the vicinity of NMDA receptors. Combined drug administration with D(2) dopamine receptor blockade and activation of hypofunctional NMDA receptors may be needed for a more effective treatment of positive and negative symptoms and the accompanied cognitive deficit in schizophrenia. To investigate this type of combined drug administration, rats were treated with the atypical antipsychotic risperidone together with the GlyT-1 inhibitor Org-24461. Brain microdialysis was applied in the striatum of conscious rats and determinations of extracellular dopamine, DOPAC, HVA, glycine, glutamate, and serine concentrations were carried out using HPLC/electrochemistry. Risperidone increased extracellular concentrations of dopamine but failed to influence those of glycine or glutamate measured in microdialysis samples. Org-24461 injection reduced extracellular dopamine concentrations and elevated extracellular glycine levels but the concentrations of serine and glutamate were not changed. When risperidone and Org-24461 were added in combination, a decrease in extracellular dopamine concentrations was accompanied with sustained elevation of extracellular glycine levels. Interestingly, the extracellular concentrations of glutamate were also enhanced. Our data indicate that coadministration of an antipsychotic with a GlyT-1 inhibitor may normalize hypofunctional NMDA receptor-mediated glutamatergic neurotransmission with reduced

  3. Mitochondrial diaphorases as NAD⁺ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition.

    Science.gov (United States)

    Kiss, Gergely; Konrad, Csaba; Pour-Ghaz, Issa; Mansour, Josef J; Németh, Beáta; Starkov, Anatoly A; Adam-Vizi, Vera; Chinopoulos, Christos

    2014-04-01

    Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD(+) supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD(+) pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD(+) derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.

  4. ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Koch-Nolte, Friedrich; Haag, Friedrich; Bulfone-Paus, Silvia

    2009-04-01

    Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

  5. Extraction and Analysis of Extracellular Polymeric Substances (EPS): Comparison of Methods and EPS Levels in Salmonella pullorum SA 1685

    Science.gov (United States)

    The extracellular polymeric substances (EPS) production and composition for Salmonella pullorum SA 1685 exposed to artificial groundwater (AGW) has been examined utilizing three EPS extraction methods: lyophilization, ethanol, and sonication. Experiments were carried out to evaluate the robustness...

  6. Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

    Directory of Open Access Journals (Sweden)

    Junnan Xu

    2014-01-01

    Full Text Available This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+ after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

  7. Role of Glycogenolysis in Stimulation of ATP Release from Cultured Mouse Astrocytes by Transmitters and High K+ Concentrations

    Directory of Open Access Journals (Sweden)

    Junnan Xu

    2013-12-01

    Full Text Available This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+ after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+ -dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

  8. Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations.

    Science.gov (United States)

    Xu, Junnan; Song, Dan; Bai, Qiufang; Zhou, Lijun; Cai, Liping; Hertz, Leif; Peng, Liang

    2014-01-13

    This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

  9. Convulsant and Subconvulsant Doses of Norfloxacin in the Presence and Absence of Biphenylacetic Acid Alter Extracellular Hippocampal Glutamate but Not Gamma-Aminobutyric Acid Levels in Conscious Rats

    Science.gov (United States)

    Smolders, I.; Gousseau, C.; Marchand, S.; Couet, W.; Ebinger, G.; Michotte, Y.

    2002-01-01

    Fluoroquinolones are antibiotics with central excitatory side effects. These adverse effects presumably result from inhibition of γ-aminobutyric acid (GABA) binding to GABAA receptors. This GABA antagonistic effect is greatly potentiated by the active metabolite of fenbufen, biphenylacetic acid (BPAA). Nevertheless, it remains questionable whether GABA receptor antagonism alone can explain the convulsant activity potentials of these antimicrobial agents. The present study was undertaken to investigate the possible effects of norfloxacin, both in the absence and in the presence of BPAA, on the extracellular hippocampal levels of GABA and glutamate, the main central inhibitory and excitatory amino acid neurotransmitters, respectively. This in vivo microdialysis approach with conscious rats allows monitoring of behavioral alterations and concomitant transmitter modulation in the hippocampus. Peroral administration of 100 mg of BPAA per kg of body weight had no effect on behavior and did not significantly alter extracellular GABA or glutamate concentrations. Intravenous perfusion of 300 mg of norfloxacin per kg did not change the rat's behavior or the concomitant neurotransmitter levels in about half of the experiments, while the remaining animals exhibited severe seizures. These norfloxacin-induced convulsions did not affect extracellular hippocampal GABA levels but were accompanied by enhanced glutamate concentrations. Half of the rats receiving both 100 mg of BPAA per kg and 50 mg of norfloxacin per kg displayed lethal seizures, while the remaining animals showed no seizure-related behavior. In the latter subgroup, again no significant alterations in extracellular GABA levels were observed, but glutamate overflow remained significantly elevated for at least 3 h. In conclusion, norfloxacin exerts convulsant activity in rats, accompanied by elevations of extracellular hippocampal glutamate levels but not GABA levels, even in the presence of BPAA. PMID:11796360

  10. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Masaki Nakano

    2017-08-01

    Full Text Available Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances, which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

  11. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O

    2000-01-01

    to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure...... of ATP A2 was solved to 1.45 A resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p...

  12. The P2X7 loss-of-function Glu496Ala polymorphism affects ex vivo cytokine release and protects against the cytotoxic effects of high ATP-levels

    Directory of Open Access Journals (Sweden)

    Wesselius Anke

    2012-12-01

    Full Text Available Abstract Background The P2X7 receptor plays an important role in cytokine release during the inflammatory response in vivo. Polymorphisms within the P2X7 receptor gene that lead to loss of receptor function may contribute to impaired cytokine release by immune cells. Therefore, we investigated whether a known loss-of-function polymorphism (Glu496Ala in the P2X7 receptor gene leads to alterations in cytokine release in response to ATP. Results An ex vivo whole blood model was used to induce an inflammatory reaction with the pro-inflammatory stimuli LPS and PHA (phytohemagglutinin. Blood from n=9 subjects with the Glu496Ala P2X7 SNP (P2X7MUT and n=7 ‘wild-type’ subjects (no P2X7 SNP; P2X7WT was used. Addition of ATP (0.9-3 mM to LPS/PHA-stimulated whole blood induced an increase in IL-1β release in P2X7MUT subjects, whereas decreased release was observed in P2X7WT subjects. Decreased levels of IL-6 and TNF-α in response to ATP were shown in both P2X7MUT and P2X7WT subjects, which was less pronounced in P2X7MUT subjects. ATP at 3 mM also significantly decreased levels of lactate dehydrogenase (LDH in P2X7MUT subjects compared to P2X7WT subjects. Conclusions The presence of the non-synonymous Glu496Ala loss-of-function polymorphism within the P2X7 receptor gene is likely to be of importance in the release of cytokines during inflammation. Furthermore, this study suggests that carriers of the Glu496Ala loss-of-function polymorphism are protected against the cytotoxic effects of high ATP-levels.

  13. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key proinflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

  14. Microglial migration mediated by ATP-induced ATP release from lysosomes

    Institute of Scientific and Technical Information of China (English)

    Ying Dou; Qing-ming Luo; Shumin Duan; Hang-jun Wu; Hui-quan Li; Song Qin; Yin-er Wang; Jing Li; Hui-fang Lou; Zhong Chen; Xiao-ming Li

    2012-01-01

    Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system.Attracted by factors released from damaged cells,microglia are recruited towards the damaged or infected site,where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris.ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury.However,the mechanisms of the long-range migration of microglia remain to be clarified.Here,we found that lysosomes in microglia contain abundant ATP and exhibit Ca2+-dependent exocytosis in response to various stimuli.By establishing an efficient in vitro chemotaxis assay,we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia,a response that was significantly inhibited in microglia treated with an agent inducing iysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice),a small GTPase required for the trafficking and exocytosis of secretory iysosomes.These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis,thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

  15. Neuropeptide Y infusion into the shell region of the rat nucleus accumbens increases extracellular levels of dopamine

    DEFF Research Database (Denmark)

    Sørensen, Gunnar; Wegener, Gregers; Hasselstrøm, Jørgen;

    2009-01-01

    Increases in extracellular dopamine in the shell region of the nucleus accumbens are centrally involved in mediating reinforcement of addictive drugs. Neuropeptide Y (NPY) and its receptors are present in the nucleus accumbens and have been implicated in addiction mechanisms. This study further...

  16. The second extracellular loop of pore-forming subunits of ATP-binding cassette transporters for basic amino acids plays a crucial role in interaction with the cognate solute binding protein(s).

    Science.gov (United States)

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-04-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)(2) complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP(2) of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P(2) variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM.

  17. The Second Extracellular Loop of Pore-Forming Subunits of ATP-Binding Cassette Transporters for Basic Amino Acids Plays a Crucial Role in Interaction with the Cognate Solute Binding Protein(s)▿

    Science.gov (United States)

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-01-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)2 complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP2 of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P2 variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM. PMID:20154136

  18. Levels of Circulating MMCN-151, a Degradation Product of Mimecan, Reflect Pathological Extracellular Matrix Remodeling in Apolipoprotein E Knockout Mice

    DEFF Research Database (Denmark)

    Barascuk, N; Vassiliadis, E; Zheng, Qiuju;

    2011-01-01

    Arterial extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) is one of the major hallmarks of atherosclerosis. Mimecan, also known as osteoglycin has been implicated in the integrity of the ECM. This study assessed the validity of an enzyme-linked immunosorbent assay (ELISA)......) developed to measure a specific MMP12-derived fragment of mimecan, MMCN-151, in apolipoprotein-E knockout (ApoE-KO) mice....

  19. Morphine sensitization increases the extracellular level of glutamate in CA1 of rat hippocampus via μ-opioid receptor.

    Science.gov (United States)

    Farahmandfar, Maryam; Karimian, Seyed Morteza; Zarrindast, Mohammad-Reza; Kadivar, Mehdi; Afrouzi, Hossein; Naghdi, Nasser

    2011-04-25

    Repeated administration of abuse drugs such as morphine elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect plastic changes requiring regulation of glutamatergic system in the brain. In this study, we investigated the effect of morphine sensitization on extracellular glutamate concentration in the hippocampus, a brain region rich in glutamatergic neurons. Sensitization was induced by subcutaneous (s.c.) injection of morphine, once daily for 3 days followed by 5 days free of the opioid treatment. The results showed that extracellular glutamate concentration in the CA1 was decreased following administration of morphine in non-sensitized rats. However, morphine-induced behavioral sensitization significantly increased the extracellular glutamate concentration in this area. The enhancement of glutamate in morphine sensitized rats was prevented by administration of naloxone 30 min before each of three daily doses of morphine. These results suggest an adaptation of the glutamatergic neuronal transmission in the hippocampus after morphine sensitization and it is postulated that opioid receptors may play an important role in this effect.

  20. Hydrogen-rich saline attenuates isoflurane-induced caspase-3 activation and cognitive impairment via inhibition of isoflurane-induced oxidative stress, mitochondrial dysfunction, and reduction in ATP levels

    Science.gov (United States)

    Li, Cheng; Hou, Lengchen; Chen, Dan; Lin, Fuqing; Chang, Tao; Li, Mengzhu; Zhang, Lingling; Niu, Xiaoyin; Wang, Huiying; Fu, Shukun; Zheng, Junhua

    2017-01-01

    Objectives: The inhaled general anesthetic isoflurane has been shown to induce caspase-3 activation in vitro and in vivo. The underlying mechanisms and functional consequences of this activity remain unclear. Isoflurane can induce caspase-3 activation by causing accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, and reduction in adenosine triphosphate (ATP) levels. This study aimed to investigate the protective effect of hydrogen, a novel antioxidant, against isoflurane-induced caspase-3 activation and cognitive impairment. Methods: H4 human neuroglioma cells overexpressing human amyloid precursor protein were treated with saline or hydrogen-rich saline (HS, 300 μM), with or without 2% isoflurane, for 6 h or 3 h. Western blot analysis, fluorescence assays, and a mitochondrial swelling assay were used to evaluate caspase-3 activation, levels of ROS and ATP, and mitochondrial function. The effect of the interaction of isoflurane (1.4% for 2 h) and HS (5 mL/kg) on cognitive function in mice was also evaluated using a fear conditioning test. Results: We found that HS attenuated isoflurane-induced caspase-3 activation. Moreover, HS treatment mitigated isoflurane-induced ROS accumulation, opening of mitochondrial permeability transition pores, reduction in mitochondrial membrane potential, and reduction in cellular ATP levels. Finally, HS significantly alleviated isoflurane-induced cognitive impairment in mice. Conclusions: Our results suggest that HS attenuates isoflurane-induced caspase-3 activation and cognitive impairment via inhibition of isoflurane-induced oxidative stress, mitochondrial dysfunction, and reduction in ATP levels. These findings warrant further research into the underlying mechanisms of this activity, and indicate that HS has the potential to attenuate anesthesia neurotoxicity.

  1. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Jason Alan Gruenhagen

    2003-12-12

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

  2. ATP and potassium ions: a deadly combination for astrocytes

    Science.gov (United States)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

  3. Rapid fluctuations in extracellular brain glucose levels induced by natural arousing stimuli and intravenous cocaine: fueling the brain during neural activation

    Science.gov (United States)

    Lenoir, Magalie

    2012-01-01

    Glucose, a primary energetic substrate for neural activity, is continuously influenced by two opposing forces that tend to either decrease its extracellular levels due to enhanced utilization in neural cells or increase its levels due to entry from peripheral circulation via enhanced cerebral blood flow. How this balance is maintained under physiological conditions and changed during neural activation remains unclear. To clarify this issue, enzyme-based glucose sensors coupled with high-speed amperometry were used in freely moving rats to evaluate fluctuations in extracellular glucose levels induced by brief audio stimulus, tail pinch (TP), social interaction with another rat (SI), and intravenous cocaine (1 mg/kg). Measurements were performed in nucleus accumbens (NAcc) and substantia nigra pars reticulata (SNr), which drastically differ in neuronal activity. In NAcc, where most cells are powerfully excited after salient stimulation, glucose levels rapidly (latency 2–6 s) increased (30–70 μM or 6–14% over baseline) by all stimuli; the increase differed in magnitude and duration for each stimulus. In SNr, where most cells are transiently inhibited by salient stimuli, TP, SI, and cocaine induced a biphasic glucose response, with the initial decrease (−20–40 μM or 5–10% below baseline) followed by a reboundlike increase. The critical role of neuronal activity in mediating the initial glucose response was confirmed by monitoring glucose currents after local microinjections of glutamate (GLU) or procaine (PRO). While intra-NAcc injection of GLU transiently increased glucose levels in this structure, intra-SNr PRO injection resulted in rapid, transient decreases in SNr glucose. Therefore, extracellular glucose levels in the brain change very rapidly after physiological and pharmacological stimulation, the response is structure specific, and the pattern of neuronal activity appears to be a critical factor determining direction and magnitude of physiological

  4. Decreased extracellular adenosine levels lead to loss of hypoxia-induced neuroprotection after repeated episodes of exposure to hypoxia.

    Directory of Open Access Journals (Sweden)

    Mei Cui

    Full Text Available Achieving a prolonged neuroprotective state following transient ischemic attacks (TIAs is likely to effectively reduce the brain damage and neurological dysfunction associated with recurrent stroke. HPC is a phenomenon in which advanced exposure to mild hypoxia reduces the stroke volume produced by a subsequent TIA. However, this neuroprotection is not long-lasting, with the effects reaching a peak after 3 days. Therefore, in this study, we investigated the use of multiple episodes of hypoxic exposure at different time intervals to induce longer-term protection in a mouse stroke model. C57BL/6 mice were subjected to different hypoxic preconditioning protocols: a single episode of HPC or five identical episodes at intervals of 3 days (E3d HPC or 6 days (E6d HPC. Three days after the last hypoxic exposure, temporary middle cerebral artery occlusion (MCAO was induced. The effects of these HPC protocols on hypoxia-inducible factor (HIF regulated gene mRNA expression were measured by quantitative PCR. Changes in extracellular adenosine concentrations, known to exert neuroprotective effects, were also measured using in vivo microdialysis and high pressure liquid chromatography (HPLC. Neuroprotection was provided by E6d HPC but not E3d HPC. HIF-regulated target gene expression increased significantly following all HPC protocols. However, E3d HPC significantly decreased extracellular adenosine and reduced cerebral blood flow in the ischemic region with upregulated expression of the adenosine transporter, equilibrative nucleoside transporter 1 (ENT1. An ENT1 inhibitor, propentofylline increased the cerebral blood flow and re-established neuroprotection in E3d HPC. Adenosine receptor specific antagonists showed that adenosine mainly through A1 receptor mediates HPC induced neuroprotection. Our data indicate that cooperation of HIF-regulated genes and extracellular adenosine is necessary for HPC-induced neuroprotection.

  5. Increases in extracellular fluid glucose levels in the rat hippocampus following an anesthetic dose of pentobarbital or ketamine-xylazine: an in vivo microdialysis study.

    Science.gov (United States)

    Canal, Clinton E; McNay, Ewan C; Gold, Paul E

    2005-02-15

    Using in vivo microdialysis, we examined glucose levels in the extracellular fluid (ECF) of the hippocampus and in the blood prior to and during pentobarbital (45 mg/kg) or ketamine-xylazine (66 mg/kg, 7 mg/kg) anesthesia. Anesthesia with either pentobarbital or ketamine-xylazine significantly increased hippocampal ECF glucose levels (mean peak increases of +71% and +85%, respectively). In addition, there were substantial increases in blood glucose levels (mean peak increases of +24% and +30%, respectively). The increased levels of hippocampal ECF glucose during anesthesia complement past evidence for decreases in ECF glucose in the hippocampus observed while rats perform a memory task sensitive to hippocampal damage, providing further support for the view that ECF glucose levels in the hippocampus are dynamically coupled to local neural activity.

  6. Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

    Science.gov (United States)

    Kennedy, H J; Pouli, A E; Ainscow, E K; Jouaville, L S; Rizzuto, R; Rutter, G A

    1999-05-01

    Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.

  7. Local release of ATP into the arterial inflow and venous drainage of human skeletal muscle: insight from ATP determination with the intravascular microdialysis technique

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Thaning, Pia; Nyberg, Michael Permin

    2011-01-01

    Intraluminal ATP could play an important role in the local regulation of skeletal muscle blood flow, but the stimuli that cause ATP release and the levels of plasma ATP in vessels supplying and draining human skeletal muscle remain unclear. To gain insight into the mechanisms by which ATP...... is released into plasma, we measured plasma [ATP] with the intravascular microdialysis technique at rest and during dynamic exercise (normoxia and hypoxia), passive exercise, thigh compressions and arterial ATP, tyramine and ACh infusion in a total of 16 healthy young men. Femoral arterial and venous[ATP...

  8. Monitoring In Vivo Changes in Tonic Extracellular Dopamine Level by Charge-Balancing Multiple Waveform Fast-Scan Cyclic Voltammetry.

    Science.gov (United States)

    Oh, Yoonbae; Park, Cheonho; Kim, Do Hyoung; Shin, Hojin; Kang, Yu Min; DeWaele, Mark; Lee, Jeyeon; Min, Hoon-Ki; Blaha, Charles D; Bennet, Kevin E; Kim, In Young; Lee, Kendall H; Jang, Dong Pyo

    2016-11-15

    Dopamine (DA) modulates central neuronal activity through both phasic (second to second) and tonic (minutes to hours) terminal release. Conventional fast-scan cyclic voltammetry (FSCV), in combination with carbon fiber microelectrodes, has been used to measure phasic DA release in vivo by adopting a background subtraction procedure to remove background capacitive currents. However, measuring tonic changes in DA concentrations using conventional FSCV has been difficult because background capacitive currents are inherently unstable over long recording periods. To measure tonic changes in DA concentrations over several hours, we applied a novel charge-balancing multiple waveform FSCV (CBM-FSCV), combined with a dual background subtraction technique, to minimize temporal variations in background capacitive currents. Using this method, in vitro, charge variations from a reference time point were nearly zero for 48 h, whereas with conventional background subtraction, charge variations progressively increased. CBM-FSCV also demonstrated a high selectivity against 3,4-dihydroxyphenylacetic acid and ascorbic acid, two major chemical interferents in the brain, yielding a sensitivity of 85.40 ± 14.30 nA/μM and limit of detection of 5.8 ± 0.9 nM for DA while maintaining selectivity. Recorded in vivo by CBM-FSCV, pharmacological inhibition of DA reuptake (nomifensine) resulted in a 235 ± 60 nM increase in tonic extracellular DA concentrations, while inhibition of DA synthesis (α-methyl-dl-tyrosine) resulted in a 72.5 ± 4.8 nM decrease in DA concentrations over a 2 h period. This study showed that CBM-FSCV may serve as a unique voltammetric technique to monitor relatively slow changes in tonic extracellular DA concentrations in vivo over a prolonged time period.

  9. Extracellular nucleotide signaling in plants

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  10. Elevated extracellular calcium increases fibroblast growth factor-2 gene and protein expression levels via a cAMP/PKA dependent pathway in cementoblasts.

    Science.gov (United States)

    Kanaya, Sousuke; Nemoto, Eiji; Ebe, Yukari; Somerman, Martha J; Shimauchi, Hidetoshi

    2010-09-01

    Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the

  11. Cr(Ⅵ)干涉VDAC1 mRNA表达与细胞内ATP水平的联系%Interference of hexavalent chromium on VDAC1 mRNA expression or ATP level and their potential association

    Institute of Scientific and Technical Information of China (English)

    杨渊; 邹悦; 李鹏; 罗磊; 戴璐; 钟才高

    2012-01-01

    目的 探讨Cr(Ⅵ)对细胞内电压依赖性离子通道(VDAC1) mRNA表达和三磷酸腺苷(ATP)水平的干涉效应及其之间的联系.方法 实验共分成6个处理组,分别用O、2、4、8、16和32μmol/L Cr(Ⅵ)染毒处理12、24和36 h,然后用逆转录荧光定量聚合酶链反应(RT-qPCR)和荧光素生物发光法分别检测细胞内VDAC1mRNA和能量ATP水平.结果 (1)细胞内VDACl mRNA表达在12h明显低于对照组水平,在24h表达水平有所增加,染毒36h后,各剂量组均明显增加,平均增加至对照组的2.65倍;(2)细胞内ATP含量在12h增高,高剂量组(2μmol/L)尤为明显,在24h后细胞内ATP水平明显下降,染毒36h后,低剂量(2μmol/L和4μmol/L)组ATP含量又回升至对照水平,在高浓度(8、16和32μ mol/L)组,ATP仍处较低水平;(3)相关分析显示,细胞内VDAC1 mRNA表达与ATP水平之间呈中度负相关(r=0.604,P<0.05).结论 Cr(Ⅵ)对细胞内ATP水平的干涉效应与VDAC1 mRNA表达异常有关,VDAC1 mRNA表达增加是Cr(Ⅵ)诱导细胞内能量ATP水平降低的分子机制之一.%Objective To explore the interference of hexavalent chromium-Cr( VI) on voltage-dependent anion channel ( VDAC1) mRNA expression and intracellular adenosine triphosphate( ATP) level in cells and their potential association. Methods Cultured L-02 hepatocytes were treated with 2,4,8,16 and 32n,mol/L of Cr(VI)for 12,24 and 36 hours (h).The expression of VDAC1 mRNA was measured by reverse transcription quantitative polymerase chain reaction ( RT-qPCR ) , whereas the levels of intracellular ATP was determined by an ATP-specific bioluminescence assay. Results ( 1 ) The expression of VDAC1 mRNA in cells of treated groups was less than that of control group at 12h,and then showed slight increase at 24h,and increased significantly after 36 h of treatment. (2) After being treated by Cr(VI)for 12 h,ATP level in cells increased, especially in higher dose groups, and the ATP level then decreased significantly at

  12. Hemopexin activity and extracellular ATP in the pathogenesis of preeclampsia

    NARCIS (Netherlands)

    Spaans, Floor

    2014-01-01

    Pre-eclampsie (zwangerschapsvergiftiging), de meest voorkomende zwangerschapscomplicatie, wordt gekenmerkt door hoge bloeddruk en eiwitten in de urine. We hebben geen behandeling, behalve het inleiden van de bevalling. Na de bevalling verdwijnt de aandoening. De exacte oorzaak van pre-eclampsie is n

  13. Hemopexin activity and extracellular ATP in the pathogenesis of preeclampsia

    NARCIS (Netherlands)

    Spaans, Floor

    2014-01-01

    Pre-eclampsie (zwangerschapsvergiftiging), de meest voorkomende zwangerschapscomplicatie, wordt gekenmerkt door hoge bloeddruk en eiwitten in de urine. We hebben geen behandeling, behalve het inleiden van de bevalling. Na de bevalling verdwijnt de aandoening. De exacte oorzaak van pre-eclampsie is

  14. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer.

    Science.gov (United States)

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-01-01

    Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC), promoting a microenvironment favorable for tumor growth. Serum samples obtained from 79 subjects [26 lean (LN) and 53 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC). Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN), chitinase-3-like protein 1 (YKL-40), tenascin C (TNC) and lipocalin-2 (LCN-2) were determined by ELISA. Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (Pcolon cancer (Pobesity (Pobese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics.

  15. Cloning and high level expression of the biologically active extracellular domain of Macaca mulatta CD40 in Pichia pastoris.

    Science.gov (United States)

    Zhu, Shengyun; Wan, Lin; Yang, Hao; Cheng, Jingqiu; Lu, Xiaofeng

    2016-03-01

    The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model.

  16. Adenovirus-induced extracellular signal-regulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    during the late phase of infection. Pharmacologic inhibition of ERK phosphorylation reduced virus recovery by >100-fold. Blocking MEK/ERK signaling affected virus DNA replication and mRNA levels only weakly but strongly reduced the amount of viral proteins, independently of the kinases MNK1 and PKR....... Hence, adenovirus induces the oncogenic Raf/MEK/ERK signaling pathway to enhance viral progeny by sustaining the levels of viral proteins. Concerning therapy, our results suggest that the use of Raf/MEK/ERK inhibitors will interfere with the propagation of oncolytic adenoviruses.......The Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling cascade enhances tumor cell proliferation in many cases. Here, we show that adenovirus type 5, a small DNA tumor virus used in experimental cancer therapy, strongly induces ERK phosphorylation...

  17. Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival.

    Science.gov (United States)

    Hucthagowder, Vishwanathan; Morava, Eva; Kornak, Uwe; Lefeber, Dirk J; Fischer, Björn; Dimopoulou, Aikaterini; Aldinger, Annika; Choi, Jiwon; Davis, Elaine C; Abuelo, Dianne N; Adamowicz, Maciej; Al-Aama, Jumana; Basel-Vanagaite, Lina; Fernandez, Bridget; Greally, Marie T; Gillessen-Kaesbach, Gabriele; Kayserili, Hulya; Lemyre, Emmanuelle; Tekin, Mustafa; Türkmen, Seval; Tuysuz, Beyhan; Yüksel-Konuk, Berrin; Mundlos, Stefan; Van Maldergem, Lionel; Wevers, Ron A; Urban, Zsolt

    2009-06-15

    Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse-chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.

  18. Serum HER 2 extracellular domain level is correlated with tissue HER 2 status in metastatic gastric or gastro-oesophageal junction adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Shu-Qin Dai

    Full Text Available BACKGROUND: To explore the association between serum human epidermal growth factor receptor 2 (HER 2 extracellular domain (ECD levels and tissue HER 2 status in metastatic gastric cancer. PATIENTS AND METHODS: HER 2 status was retrospectively analyzed in 219 advanced gastric or gastroesophageal junction (GEJ patients. Serum HER 2 ECD was measured by chemiluminescent assay and tissue HER 2 was assessed by fluorescent in situ hybridisation (FISH and immunohistochemistry (IHC assay. RESULTS: Significant associations were found between serum HER 2 ECD levels and tissue HER 2 status. Twenty-four patients had HER 2 ECD levels >16.35 ng/mL, which has a sensitivity of 51.4% and a specificity of 97.3% to predict tissue HER 2 status. When the cut-off value was increased to 22 ng/mL, then all 12 patients with serum HER 2 ECD levels>22 ng/mL were tissue HER 2 positive, corresponding to a specificity of 100% and a sensitivity of 32.4%. High serum HER 2 ECD levels were strongly associated with the intestinal histological type (Lauren's classification, liver metastasis, multiple metastasis (>2 and increased LDH levels, but not with overall survival. CONCLUSIONS: The high specificity of the serum HER 2 ECD assay in predicting tissue HER 2 status suggests its potential as a surrogate marker of the HER 2 status in gastric cancer.

  19. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer

    Science.gov (United States)

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-01-01

    Background Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC), promoting a microenvironment favorable for tumor growth. Methods Serum samples obtained from 79 subjects [26 lean (LN) and 53 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC). Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN), chitinase-3-like protein 1 (YKL-40), tenascin C (TNC) and lipocalin-2 (LCN-2) were determined by ELISA. Results Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01) and colon cancer (P<0.05). LCN-2 levels were affected by obesity (P<0.05), but no differences were detected regarding the presence or not of CC. A positive association (P<0.05) with different inflammatory markers was also detected. Conclusions To our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics. PMID:27612200

  20. Mitochondrial flashes regulate ATP homeostasis in the heart.

    Science.gov (United States)

    Wang, Xianhua; Zhang, Xing; Wu, Di; Huang, Zhanglong; Hou, Tingting; Jian, Chongshu; Yu, Peng; Lu, Fujian; Zhang, Rufeng; Sun, Tao; Li, Jinghang; Qi, Wenfeng; Wang, Yanru; Gao, Feng; Cheng, Heping

    2017-07-10

    The maintenance of a constant ATP level ('set-point') is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart.

  1. Regulation of adenosine levels during cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Stephanie CHU; Wei XIONG; Dali ZHANG; Hanifi SOYLU; Chao SUN; Benedict C ALBENSI; Fiona E PARKINSON

    2013-01-01

    Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events,and attenuates the excitotoxic neuronal injury.Adenosine is produced both intracellularly and extracellularly,and nucleoside transport proteins transfer adenosine across plasma membranes.Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption,cellular release of ATP,metabolism of extracellular ATP (and other adenine nucleotides),adenosine influx,adenosine efflux and adenosine metabolism.Recent studies have used genetically modified mice to investigate the relative contributions of intra-and extracellular pathways for adenosine formation.The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase.From these studies,we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures,but not in hippocampal slices or in vivo mice exposed to ischemic conditions.

  2. ATP Synthesis in the Extremely Halophilic Bacteria

    Science.gov (United States)

    Hochstein, Lawrence I.; Morrison, David (Technical Monitor)

    1994-01-01

    The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other

  3. Respiratory infections cause the release of extracellular vesicles: implications in exacerbation of asthma/COPD.

    Directory of Open Access Journals (Sweden)

    Suffwan Eltom

    Full Text Available Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations.To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis.Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1β/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia.This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

  4. KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

    Directory of Open Access Journals (Sweden)

    Javier Pizarro-Delgado

    Full Text Available Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1. ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering and second (amplifying phases of glucose-induced insulin secretion.

  5. Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts

    Science.gov (United States)

    Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

    2017-01-01

    ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1–P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury. PMID:28163685

  6. Proteome-level display by 2-dimensional chromatography of extracellular matrix-dependent modulation of the phenotype of bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Singh Anil

    2006-06-01

    Full Text Available Abstract Background The extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype. Results Depending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1 – 4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway. Conclusion The system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype

  7. Virus-induced dilated cardiomyopathy is characterized by increased levels of fibrotic extracellular matrix proteins and reduced amounts of energy-producing enzymes.

    Science.gov (United States)

    Nishtala, Krishnatej; Phong, Truong Q; Steil, Leif; Sauter, Martina; Salazar, Manuela G; Kandolf, Reinhard; Kroemer, Heyo K; Felix, Stephan B; Völker, Uwe; Klingel, Karin; Hammer, Elke

    2011-11-01

    The most relevant clinical phenotype resulting from chronic enteroviral myocarditis is dilated cardiomyopathy (DCM). Mice of the susceptible mouse strain A.BY/SnJ mimick well human DCM since they develop as a consequence of persistent infection and chronic inflammation a dilation of the heart ventricle several weeks after coxsackievirus B3 (CVB3) infection. Therefore, this model is well suited for the analysis of changes in the heart proteome associated with DCM. Here, we present a proteomic survey of the dilated hearts based on differential fluorescence gel electrophoresis and liquid chromatography-mass spectrometric centered methods in comparison to age-matched non-infected hearts. In total, 101 distinct proteins, which belong to categories immunity and defense, cell structure and associated proteins, energy metabolism and protein metabolism/modification differed in their levels in both groups. Levels of proteins involved in fatty acid metabolism and electron transport chain were found to be significantly reduced in infected mice suggesting a decrease in energy production in CVB3-induced DCM. Furthermore, proteins associated with muscle contraction (MLRV, MLRc2, MYH6, MyBPC3), were present in significantly altered amounts in infected mice. A significant increase in the level of extracellular matrix proteins in the dilated hearts indicates cardiac remodeling due to fibrosis.

  8. Real-time imaging of inflation-induced ATP release in the ex vivo rat lung.

    Science.gov (United States)

    Furuya, Kishio; Tan, Ju Jing; Boudreault, Francis; Sokabe, Masahiro; Berthiaume, Yves; Grygorczyk, Ryszard

    2016-11-01

    Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function. Copyright © 2016 the American Physiological Society.

  9. Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

    Science.gov (United States)

    Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

  10. 异丙酚在不同pH值环境下对苯甲酰苯甲酸ATP诱发的巨噬细胞嘌呤2X7受体电流的影响%Effect of propofol on BzATP induced P2X7-gated currents in RAW264.7 macrophages under different extracellular pH values

    Institute of Scientific and Technical Information of China (English)

    刘红亮; 戴体俊

    2011-01-01

    To investigate the sensitivity of P2X7 receptor to its specific agonist BZATP and the effect of propofol on P2X7-gated currents in RAW264. 7 macrophages under different extracellular pH values. METHODS-RAW264. 7 cells were cultured, and whole-cell patch clamp technique was used. BZATP (10 - 10000 pimol/L) was applied for 5 seconds, the currents were recorded, and the EC50 value of BZATP was achieved under the extracellular pH value of 7. 4 or 6. 0. Propofol (1 -100 jumol/L) was applied to the cells for 1 min, then BZATP with two times EC50 value was applied, the IC50 or EC50 value of propofol was achieved under different extracellular pH values. To investigate the effect of propofol on the dose-response curve of B2ATP under different extracellular pH values, propofol with IC50 value was applied, and BZATP (10-10000 fimol/L) was applied for 5 seconds, the EC50 value of BZATP was achieved. RESULTS :BZATP could induce the inward cur-rents in a dose-dependent manner, and the EC50 value of BZATP was (112 ±26) μmol/L and (643 ±87) μmol/L at extracellular pH value of 7. 4 or 6. 0. Propofol could inhibit P2X7-gated currents at pH 7. 4, and the IC50 value was (31 ±6) μmol/L; but propofol increased P2X7-ga-ted currents at pH 6. 0, and the EC50 value was (38 ±6) fxmol/L. The IC50 value of propofol made the dose-response curve of BZATP shifted rightward at pH 7.4, or leftward at pH 6.0. CONCLUSION: The sensitivity of P2X7 receptor to BZATP decreases when extracellular pH value changes from 7. 4 to 6. 0. Propofol with clinically related concentrations could inhibit P2X7-ga-ted currents at pH 7. 4, and increase P2X7-gated currents at pH 6. 0 extracellularly.%目的:观察在不同pH值条件下巨噬细胞膜嘌呤2X7(P2X7)受体电流对其特异性激动剂苯甲酰苯甲酸ATP (BZATP)的敏感性及异丙酚对P2X7受体电流的影响.方法:应用培养的RAW264.7巨噬细胞,采用全细胞膜片钳技术,向细胞施加BZ ATP(10~10000 μmol/L)5 s,记录电流,

  11. 水稻叶绿体ATP合成酶基因转录丰度受赤霉素诱导调节%The mRNA Expression Level of Rice Chloroplast ATP Synthase Response to Gibberellin

    Institute of Scientific and Technical Information of China (English)

    娄沂春; 董海涛; 李德葆

    2001-01-01

    采用mRNA差异显示技术分离和鉴定了水稻受赤霉素诱导的差异表达基因。经50个引物组合差异显示,获得21个诱导表达差异的cDNA片段。经反向Northern初步筛选对其中5个阳性片段进行克隆及序列分析。其序列经国际联网BLAST查询表明编号为GA21C的为水稻叶绿体ATP合成酶基因片段。Southern杂交结果证实此基因为单拷贝。Norrhern杂交结果显示确受赤霉素诱导表达且在诱导16 h后达到高峰,表明赤霉素诱导水稻产生生理反应过程涉及叶绿体基因表达。%By using mRNA differential display,gene expression patterns in rice induced by plant hormone-gibberellin were investigated.From 50 combinations of anchor and arbitrary primers,twenty one tagged eDNA fragments were obtained and screened the fragments by reverse-Northern.Five positive eDNA fragments were cloned and sequenced.One of which was shown to encode sequences for rice chloroplast ATP synthase.Northern blot analysis indicated that the upregulation of this gene occurs at the transcriptional level in rice after gibberellin treatment for 16 h,suggesting that chloroplast ATP synthase may play a role in rice response to gibberellin.

  12. Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.

    Science.gov (United States)

    Tu, Q; Yu, L; Zhang, P; Zhang, M; Zhang, H; Jiang, J; Chen, C; Zhao, S

    2000-04-01

    A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome.

  13. Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa.

    Directory of Open Access Journals (Sweden)

    Young-Joo Yi

    Full Text Available Inorganic pyrophosphate (PPi is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1 in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.

  14. High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders.

    Science.gov (United States)

    Caivano, Antonella; Laurenzana, Ilaria; De Luca, Luciana; La Rocca, Francesco; Simeon, Vittorio; Trino, Stefania; D'Auria, Fiorella; Traficante, Antonio; Maietti, Maddalena; Izzo, Tiziana; D'Arena, Giovanni; Mansueto, Giovanna; Pietrantuono, Giuseppe; Laurenti, Luca; Musto, Pellegrino; Del Vecchio, Luigi

    2015-12-01

    Many cell types release extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, which play a role in physiology and diseases. Presence and phenotype of circulating EVs in hematological malignancies (HMs) remain largely unexplored.The aim of this study was to characterize EVs in peripheral blood of HM patients compared to healthy subjects (controls). We isolated serum EVs from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia (WM), Hodgkin's lymphoma (HL), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and controls. EVs were isolated from serum of peripheral blood by ultracentrifuge steps and analyzed by flow cytometry to define count, size, and immunophenotype. MV levels were significantly elevated in WM, HL, MM, AML, and some MPNs and, though at a lesser degree, in CLL and NHL as compared to healthy controls. HL, MM, and MPNs generated a population of MVs characterized by lower size (below 0.3 μm) when compared to controls. MVs from patients specifically expressed tumor-related antigens, such as CD19 in B cell neoplasms, CD38 in MM, CD13 in myeloid tumors, and CD30 in HL. Both total and antigen-specific count of MVs significantly correlated with different HM clinical features such as Rai stage in CLL, International Prognostic Scoring System in WM, International Staging System in MM, and clinical stage in HL. MVs may represent a novel biomarker in HMs.

  15. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  16. A higher response of plasma neuropeptide Y, growth hormone, leptin levels and extracellular glycerol levels in subcutaneous abdominal adipose tissue to Acipimox during exercise in patients with bulimia nervosa: single-blind, randomized, microdialysis study

    Directory of Open Access Journals (Sweden)

    Smitka Kvido

    2011-11-01

    Full Text Available Abstract Background Neuropeptide Y (NPY is an important central orexigenic hormone predominantly produced by the hypothalamus, and recently found to be secreted in adipose tissue (AT. Acipimox (Aci inhibits lipolysis in AT and reduces plasma glycerol and free fatty acid (FFA levels. Exercise and Aci are enhancers of growth hormone (GH and NPY secretion and exercise may alter leptin levels. We expect to find abnormal neuropeptidergic response in plasma and AT in patients with bulimia nervosa (BN. We hypothesize that Aci influences these peptides via a FFA-independent mechanism and that Aci inhibits lipolysis through a cyclic adenosine monophosphate (cAMP-dependent pathway. Dysregulations of the AT-brain axis peptides might be involved in binge eating as is the case in BN. Methods The objective of this study was to determine the responses of plasma NPY, GH, leptin, FFA and glycerol levels to exercise in BN patients and healthy women (C given the anti-lipolytic drug Aci or placebo. The secondary objective of this study was to compare the responses of extracellular glycerol levels and plasma glycerol levels to exercise alone or together with Aci administration in BN patients and C women. Extracellular glycerol was measured in vivo in subcutaneous (sc abdominal AT using microdialysis. Eight BN and eight C women were recruited for this single-blind, randomized study. Aci or placebo was given 1 hour before the exercise (45 min, 2 W/kg of lean body mass [LBM]. NPY, GH, leptin, FFA, glycerol plasma and AT glycerol levels were measured using commercial kits. Results The primary outcome of this study was that the exercise with Aci administration resulted in plasma NPY and GH increase (after a 45-minute exercise and leptin (after a 90-minute post-exercise recovering phase increased more in BN patients. The secondary outcomes of this study were that the exercise with Aci administration induced a higher decrease of extracellular glycerol in BN patients

  17. ATP Is Required and Advances Cytokine-Induced Gap Junction Formation in Microglia In Vitro

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    Pablo J. Sáez

    2013-01-01

    Full Text Available Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs and gap junction channels (GJCs, affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-α/IFN-γ-induced dye coupling, probably through the induction of IL-1β release. Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.

  18. Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

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    Ip Virginia

    2010-09-01

    Full Text Available Abstract Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H. High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non

  19. Interdependence of ATP signalling and pannexin channels; the servant was really the master all along?

    Science.gov (United States)

    Jackson, Michael F

    2015-12-15

    Pannexin channels are recognized as important conduits for the release of ATP, which contributes to purinergic signalling. Pathologically, ATP release via these channels acts as a find-me signal for apoptotic cell clearance. Accordingly, there is considerable and growing interest in understanding the function and regulation of pannexin channels. In a recent issue of the Biochemical Journal, Boyce et al. provide evidence that the surface expression of pannexin channels is regulated by extracellular ATP. They propose a model in which ATP triggers pannexin channel internalization through a pathway involving clathrin- and caveolin-independent entry into early endosomes. Intriguingly, their evidence suggests that internalization is initiated through the association of ATP with pannexin channels themselves as well as ionotropic purinergic receptor 7 (P2X7) receptors.

  20. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Novak, Ivana

    2001-01-01

    The energy providing substrate ATP can be released from various cells and act extracellularly to regulate the same cells or neighboring cells. However, the pathway for ATP release and the eliciting physiological stimulus are unclear. Recently, we showed that ATP activates P2X and P2Y purinergic...... overlapping with those marked by acridine orange and LysoTracker Red. In functional studies we show that native pancreatic acini release ATP in response to various stimuli but most importantly to cholinergic stimulation, a very likely physiological stimulus in this epithelium. In a close vicinity of acini we...

  1. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  2. Molecular mechanism for H(2)S-induced activation of K(ATP) channels.

    Science.gov (United States)

    Jiang, Bo; Tang, Guanghua; Cao, Kun; Wu, Lingyun; Wang, Rui

    2010-05-15

    Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

  3. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    Science.gov (United States)

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Intrarenal localization of the plasma membrane ATP channel pannexin1.

    Science.gov (United States)

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T X; Yu, Alan; Peti-Peterdi, János

    2012-11-15

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

  5. Phosphocreatine, an Intracellular High-Energy Compound, is Found in the Extracellular Fluid of the Seminal Vesicles in Mice and Rats

    Science.gov (United States)

    Lee, H. J.; Fillers, W. S.; Iyengar, M. R.

    1988-10-01

    High levels of phosphocreatine, a compound known to serve as an intracellular energy reserve, were found in the fluid contained in seminal vesicle glands. The concentrations of phosphocreatine in the extracellular fluid in the mouse and rat were found to be 5.6 ± 1.6 and 2.2 ± 0.8 μ mol/g, respectively, which are higher than the intracellular levels reported for smooth muscles. The creatine concentrations in the seminal vesicular fluid from these two species were 22.8 ± 3.1 and 13.0 ± 5.3 μ mol/g, respectively. These creatine levels are approximately 100 and 65 times higher than the creatine levels in mammalian blood. Smaller amounts of ATP (phosphocreatine/ATP ratio of 20-40) and traces of ADP were also found. Comparison of the pattern of distribution of macromolecules (proteins and DNA) with the distribution of phosphocreatine between the cells and the fluid of the seminal vesicle indicates that cell lysis did not account for the phosphocreatine in the seminal vesicle fluid. Rather, the available evidence strongly suggests that this high-energy compound is actively secreted. We found that in the testes, the sperm are exposed to the highest known creatine concentration in any mammalian tissue studied. Based on these results and other recent reports, we propose that the extracellular phosphocreatine, ATP, and creatine are involved in sperm metabolism.

  6. Follow the ATP: tumor energy production: a perspective.

    Science.gov (United States)

    Oronsky, Bryan T; Oronsky, Neil; Fanger, Gary R; Parker, Christopher W; Caroen, Scott Z; Lybeck, Michelle; Scicinski, Jan J

    2014-01-01

    As early as the 1920s, the eminent physician and chemist, Otto Warburg, nominated for a second Nobel Prize for his work on fermentation, observed that the core metabolic signature of cancer cells is a high glycolytic flux. Warburg averred that the prime mover of cancer is defective mitochondrial respiration, which drives a switch to an alternative energy source, aerobic glycolysis in lieu of Oxidative Phosphorylation (OXPHOS), in an attempt to maintain cellular viability and support critical macromolecular needs. The cell, deprived of mitochondrial ATP production, must reprogram its metabolism as a secondary survival mechanism to maintain sufficient ATP and NADH levels for macromolecule production, membrane integrity and DNA synthesis as well as maintenance of membrane ionic gradients. A time-tested method to identify and disrupt criminal activity is to "follow the money" since the illicit proceeds from crime are required to underwrite it. By analogy, strategies to target cancer involve following and disrupting the flow of ATP and NADH, the energetic and redox "currencies" of the cell, respectively, since the tumor requires high levels of ATP and NADH, not only for metastasis and proliferation, but also, on a more basic level, for survival. Accordingly, four broad ATP reduction strategies to impact and potentially derail cancer energy production are highlighted herein: 1) small molecule energy-restriction mimetic agents (ERMAs) that target various aspects of energy metabolism, 2) reduction of energy 'subsidization' with autophagy inhibitors, 3) acceleration of ATP turnover to increase energy inefficiency, and 4) dietary energy restriction to limit the energy supply.

  7. ATP as a biomarker of viable microorganisms in clean-room facilities

    Science.gov (United States)

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  8. ATP as a biomarker of viable microorganisms in clean-room facilities

    Science.gov (United States)

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  9. Serum, liver, and lung levels of the major extracellular matrix components at the early stage of BCG-induced granulomatosis depending on the infection route.

    Science.gov (United States)

    Kim, L B; Shkurupy, V A; Putyatina, A N

    2015-01-01

    Experiments on the model of mouse BCG-induced granulomatous showed that the content of glycosaminoglycans and proteoglycans in the extracellular matrix of the liver and lungs are changed at the early stages of inflammation (days 3 and 30 postinfection) before cell destruction in the organs begins. This is related to degradation of extracellular matrix structures. Their high content in the blood and interstitium probably contributes to the formation of granulomas, fibroblast proliferation and organ fibrosis. These processes depend on the infection route that determines different conditions for generalization of the inflammation process. Intravenous method of vaccine injection is preferable to use when designing the experiments simulating tuberculosis granulomatosis, especially for the analysis of its early stages.

  10. Substantia nigra osmoregulation: taurine and ATP involvement.

    Science.gov (United States)

    Morales, Ingrid; Dopico, Jose G; Sabate, Magdalena; Gonzalez-Hernandez, Tomas; Rodriguez, Manuel

    2007-05-01

    An extracellular nonsynaptic taurine pool of glial origin was recently reported in the substantia nigra (SN). There is previous evidence showing taurine as an inhibitory neurotransmitter in the SN, but the physiological role of this nonsynaptic pool of taurine has not been explored. By using microdialysis methods, we studied the action of local osmolarity on the nonsynaptic taurine pool in the SN of the rat. Hypoosmolar pulses (285-80 mosM) administered in the SN by the microdialysis probe increased extrasynaptic taurine in a dose-dependent way, a response that was counteracted by compensating osmolarity with choline. The opposite effect (taurine decrease) was observed when osmolarity was increased. Under basal conditions, the blockade of either the AMPA-kainate glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dionine disodium or the purinergic receptors with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid modified the taurine concentration, suggesting that both receptors modulate the extrasynaptic pool of taurine. In addition, these drugs decreased the taurine response to hypoosmolar pulses, suggesting roles for glutamatergic and purinergic receptors in the taurine response to osmolarity. The participation of purinergic receptors was also supported by the fact that ATP (which, under basal conditions, increased the extrasynaptic taurine in a dose-dependent way) administered in doses saturating purinergic receptors also decreased the taurine response to hypoosmolarity. Taken together, present data suggest osmoregulation as a role of the nonsynaptic taurine pool of the SN, a function that also involves glutamate and ATP and that could influence the nigral cell vulnerability in Parkinson's disease.

  11. Variation in Extracellular Protease Production among Clinical Isolates of Staphylococcus aureus Due to Different Levels of Expression of the Protease Repressor sarA

    OpenAIRE

    Karlsson, Anna; Arvidson, Staffan

    2002-01-01

    Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 s...

  12. Construction of high level prokaryotic expression and purification system of PD-L1 extracellular domain by using Escherichia coli host cell machinery.

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-10-01

    Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1 was cloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion. Sequence analysis of cloned targeted genes showed 100% homology of original sequence. The recombinant protein was expressed using 1mM/mL IPTG and purified by affinity chromatography on a column of Ni-NTA and confirmed by SDS-PAGE and western blot analysis. Results showed that our constructed pET30(+)/PDL1-ECD system efficiently produces desired recombinant protein with molecular weight of 38.1kDa. The prokaryotic expression system provides an easy method to express PD-L1 extracellular domain that further facilitate the role of PD-1/PD-L1 binding inhibition and helps in valuable drug and antibodies production. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  13. BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells

    Science.gov (United States)

    Yoshida, Tomoki; Kakizuka, Akira; Imamura, Hiromi

    2016-01-01

    ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed “BTeam”. BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the ε subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7–4.1 mM) and mitochondrial matrix (2.4–2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations. PMID:28000761

  14. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    Science.gov (United States)

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  15. The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

    OpenAIRE

    Suzuki, Haruka; Kuroda, Hiroshi; Yukawa, Yasushi; Sugiura, Masahiro

    2011-01-01

    The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the ...

  16. Developmental changes in the expression of ATP7A during a critical period in postnatal neurodevelopment.

    Science.gov (United States)

    Niciu, M J; Ma, X-M; El Meskini, R; Ronnett, G V; Mains, R E; Eipper, B A

    2006-01-01

    ATP7A is a P-type ATPase that transports copper from cytosol into the secretory pathway for loading onto cuproproteins or efflux. Mutations in Atp7a cause Menkes disease, a copper-deficiency disorder fatal in the postnatal period due to severe neurodegeneration. Early postnatal copper injections are known to diminish degenerative changes in some human patients and mice bearing mutations in Atp7a. In situ hybridization studies previously demonstrated that ATP7A transcripts are expressed widely in the brain. ATP7A-specific antibody was used to study the neurodevelopmental expression and localization of ATP7A protein in the mouse brain. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in neocortex and cerebellum. In the developing and adult brain, ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. ATP7A expression decreases in most neuronal subpopulations from birth to adulthood. In contrast, ATP7A expression increases in CA2 hippocampal pyramidal and cerebellar Purkinje neurons. ATP7A is expressed in a subset of astrocytes, microglia, oligodendrocytes, tanycytes and endothelial cells. ATP7A is largely localized to the trans-Golgi network, adopting the cell-specific and developmentally-regulated morphology of this organelle. The presence of ATP7A in the axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this enzyme. In sum, the precisely-regulated neurodevelopmental expression of ATP7A correlates well with the limited therapeutic window for effective treatment of Menkes disease.

  17. How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings?

    Directory of Open Access Journals (Sweden)

    Navid Omidbakhsh

    Full Text Available BACKGROUND: Meters based on adenosine triphosphate (ATP bioluminescence measurements in relative light units (RLU are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? OBJECTIVE: This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals' interference in their readings. METHODS: First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter's ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings. RESULTS: All four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated. CONCLUSIONS: Apart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries.

  18. Stimulation-dependent release, breakdown, and action of endogenous ATP in mouse hemidiaphragm preparation: the possible role of ATP in neuromuscular transmission.

    Science.gov (United States)

    Vizi, E S; Nitahara, K; Sato, K; Sperlágh, B

    2000-07-01

    In this study the in vitro mouse phrenic nerve- hemidiaphragm preparation was utilized to study the release and extracellular catabolism of endogenous ATP and its action on the postsynaptic site, i.e. on the contraction force evoked by nerve stimulation. ATP, measured by the luciferin-luciferase assay, was released stimulation-dependently from the mouse hemidiaphragm in response to electrical field stimulation at 10 Hz. Blockade of the Na(+) channel activity by tetrodotoxin inhibited the majority of the release of ATP in response to stimulation, showing that it is related to neuronal activity. The nicotinic receptor antagonists d-tubocurarine, and alpha-bungarotoxin and cooling the bath temperature to 7 degrees C also reduced stimulation-induced ATP outflow, suggesting that nicotinic receptors are responsible for the part of the release of ATP that is released from postsynaptic sites in a carrier-mediated manner. Exogenous ATP (20-500 microM) added to the bath was degraded to ADP and AMP by the action of ectoATPase and ectoATPdiphosphohydrolase; the K(m) and v(max) values of these enzymes were 185.8 microM and 55.16 nmol/min.g respectively. However, the total amount of nucleotides ([ATP+ADP+AMP]) was increased after the addition of ATP, indicating that ATP itself promoted further adenine nucleotide release. Twitch contractions of the rat hemidiaphragm preparation evoked by low frequency electrical stimulation was blocked concentration-dependently by the non-depolarizing muscle relaxants d-tubocurarine and pancuronium. Suramin (100 microM-1 mM) reversed neuromuscular blockade by d-tubocurarine and pancuronium; i.e., it shifted their concentration-response curves to the right Taken together our data, that endogenous ATP is released by stimulation and subsequently catabolized in the hemidiaphragm preparation and that suramin inhibits ecto-ATPase activity could be interpreted as meaning that suramin prolongs the action of endogenous ATP to elicit twitch contraction

  19. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects.

    Science.gov (United States)

    Kojima, Shuji; Ohshima, Yasuhiro; Nakatsukasa, Hiroko; Tsukimoto, Mitsutoshi

    2017-01-01

    Adenosine triphosphate (ATP) serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy) induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  20. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects

    Directory of Open Access Journals (Sweden)

    Shuji Kojima

    2017-02-01

    Full Text Available Adenosine triphosphate (ATP serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  1. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects

    Science.gov (United States)

    Ohshima, Yasuhiro; Nakatsukasa, Hiroko; Tsukimoto, Mitsutoshi

    2017-01-01

    Adenosine triphosphate (ATP) serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy) induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  2. Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks

    Directory of Open Access Journals (Sweden)

    Daniel eGoldman

    2012-07-01

    Full Text Available Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100ms that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2. The model further predicts how insulin, at concentrations found in prediabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

  3. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  4. A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation.

    Science.gov (United States)

    Al Adhami, Hala; Evano, Brendan; Le Digarcher, Anne; Gueydan, Charlotte; Dubois, Emeric; Parrinello, Hugues; Dantec, Christelle; Bouschet, Tristan; Varrault, Annie; Journot, Laurent

    2015-03-01

    Genomic imprinting is an epigenetic mechanism that restrains the expression of ∼ 100 eutherian genes in a parent-of-origin-specific manner. The reason for this selective targeting of genes with seemingly disparate molecular functions is unclear. In the present work, we show that imprinted genes are coexpressed in a network that is regulated at the transition from proliferation to quiescence and differentiation during fibroblast cell cycle withdrawal, adipogenesis in vitro, and muscle regeneration in vivo. Imprinted gene regulation is not linked to alteration of DNA methylation or to perturbation of monoallelic, parent-of-origin-dependent expression. Overexpression and knockdown of imprinted gene expression alters the sensitivity of preadipocytes to contact inhibition and adipogenic differentiation. In silico and in cellulo experiments showed that the imprinted gene network includes biallelically expressed, nonimprinted genes. These control the extracellular matrix composition, cell adhesion, cell junction, and extracellular matrix-activated and growth factor-activated signaling. These observations show that imprinted genes share a common biological process that may account for their seemingly diverse roles in embryonic development, obesity, diabetes, muscle physiology, and neoplasm.

  5. Decellularized matrices as in vitro models of extracellular matrix in tumor tissues at different malignant levels: Mechanism of 5-fluorouracil resistance in colorectal tumor cells.

    Science.gov (United States)

    Hoshiba, Takashi; Tanaka, Masaru

    2016-11-01

    Chemoresistance is a major barrier for tumor chemotherapy. It is well-known that chemoresistance increases with tumor progression. Chemoresistance is altered by both genetic mutations and the alteration of extracellular microenvironment. Particularly, the extracellular matrix (ECM) is remodeled during tumor progression. Therefore, ECM remodeling is expected to cause the acquisition of chemoresistance in highly malignant tumor tissue. Here, we prepared cultured cell-derived decellularized matrices that mimic native ECM in tumor tissues at different stages of malignancy, and 5-fluorouracil (5-FU) resistance was compared among these matrices. 5-FU resistance of colorectal tumor cells increased on the matrices derived from highly malignant tumor HT-29 cells, although the resistance did not increase on the matrices derived from low malignant tumor SW480 cells and normal CCD-841-CoN cells. The resistance on HT-29 cell-derived matrices increased through the activation of Akt and the upregulation of ABCB1 and ABCC1 without cell growth promotion, suggesting that ECM remodeling plays important roles in the acquisition of chemoresistance during tumor progression. It is expected that our decellularized matrices, or "staged tumorigenesis-mimicking matrices", will become preferred cell culture substrates for in vitro analysis of comprehensive ECM roles in chemoresistance and the screening and pharmacokinetic analysis of anti-cancer drugs.

  6. High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

    Science.gov (United States)

    Yu, Zhenxiao; Zheng, Hongchen; Zhao, Xingya; Li, Shufang; Xu, Jianyong; Song, Hui

    2016-08-01

    The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications.

  7. Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells.

    Science.gov (United States)

    Takahara, Norihiro; Ito, Satoru; Furuya, Kishio; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-12-01

    Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca(2+)-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

  8. Involvement of connexin43 hemichannel in ATP release after γ-irradiation

    Science.gov (United States)

    Ohshima, Yasuhiro; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Kojima, Shuji

    2012-01-01

    Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that γ-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X7 receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X7 receptor-dependent ATP release after 0.5 Gy γ-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked γ-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy γ-irradiation tyrosine phosphorylation was significantly blocked by P2X7 receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X7 receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X7 receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X7 receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43. PMID:22843620

  9. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  10. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  11. ATP formation and ATP hydrolysis during fatiguing, intermittent stimulation of different types of single muscle fibres from Xenopus laevis.

    Science.gov (United States)

    Nagesser, A S; Van der Laarse, W J; Elzinga, G

    1993-12-01

    This report describes changes of the rate of ATP hydrolysis in single, intact muscle fibres during the development of fatigue induced by intermittent tetanic stimulation. High (type 3) and low (type 1) oxidative muscle fibres dissected from the iliofibularis muscle of Xenopus laevis were studied at 20 degrees C. The rate of ATP hydrolysis was calculated during different time intervals from changes in the content of nucleotides, creatine compounds and lactate, as well as lactate efflux and oxygen uptake. During the first phase of intermittent stimulation, phosphocreatine is fully reduced while the rate of oxygen consumption increases to its maximum, the lactate content increases to a maximum level, and a small amount of IMP is formed; the rate of ATP hydrolysis in type 3 fibres is constant while force decreases, whereas the rate decreases approximately in proportion to force in type 1 fibres. After the first phase, the rate of ATP hydrolysis in type 3 fibres decreases slightly and the fibres reach a steady metabolic state in which the rates of ATP formation and hydrolysis are equal; in type 1 fibres a drastic change of the rate of ATP hydrolysis occurs and a steady metabolic state is not reached. On the basis of the time courses of the metabolic changes, it is concluded that the rate of ATP hydrolysis in type 3 fibres is reduced by acidification and/or a reduced calcium efflux from the sarcoplasmic reticulum, whereas in type 1 fibres inorganic phosphate and/or acidification inhibit the rate initially and ADP is a likely candidate to explain the drastic fall of the rate of ATP hydrolysis during late phases of fatiguing stimulation.

  12. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space

    NARCIS (Netherlands)

    Stuart, Rosemary A.; Gruhler, Albrecht; Klei, Ida van der; Guiard, Bernard; Koll, Hans; Neupert, Walter

    1994-01-01

    The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted.

  13. Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2011-01-01

    The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mut

  14. A secreted tyrosine kinase acts in the extracellular environment.

    Science.gov (United States)

    Bordoli, Mattia R; Yum, Jina; Breitkopf, Susanne B; Thon, Jonathan N; Italiano, Joseph E; Xiao, Junyu; Worby, Carolyn; Wong, Swee-Kee; Lin, Grace; Edenius, Maja; Keller, Tracy L; Asara, John M; Dixon, Jack E; Yeo, Chang-Yeol; Whitman, Malcolm

    2014-08-28

    Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.

  15. Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells

    DEFF Research Database (Denmark)

    Gromada, J; Dissing, S; Kofod, Hans

    1995-01-01

    The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open...... probability of single ATP-sensitive K(+)-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (cell patch clamp configuration. A half......-maximal steady-state inhibition of the ATP-sensitive K+ currents is observed at 89 pmol/l repaglinide and at 47 pmol/l glibenclamide in whole-cell experiments of longer duration (30 min). Applying digital Ca2+ imaging on single beta TC3 cells we found that repaglinide and glibenclamide induced a concentration...

  16. Nerve growth factor treatment of sensory neuron primary cultures causes elevated levels of the mRNA encoding the ATP synthase beta-subunit as detected by a novel PCR-based differential cloning method.

    Science.gov (United States)

    Kendall, G; Ensor, E; Crankson, H D; Latchman, D S

    1996-03-01

    The mRNA encoding the rat ATP synthase beta-subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase beta-subunit cDNA clones were isolated from a lambda library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA-derived probes from untreated and nerve-growth-factor-treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR-based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed.

  17. ATPase Activity Measurements Using Radiolabeled ATP

    NARCIS (Netherlands)

    Swarts, H.G.; Koenderink, J.B.

    2016-01-01

    ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [gamma-(32)P]ATP, which is hydrolyzed by P-type ATPases

  18. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...

  19. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz;

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...

  20. 2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

    Science.gov (United States)

    Schachter, Julieta; Valadao, Ana Luiza Chaves; Aguiar, Renato Santana; Barreto-de-Souza, Victor; Rossi, Atila Duque; Arantes, Pablo Ricardo; Verli, Hugo; Quintana, Paula Gabriela; Heise, Norton; Tanuri, Amilcar; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis

    2014-01-01

    The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

  1. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O;

    2000-01-01

    to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown......-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant...

  2. Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells.

    Science.gov (United States)

    Szade, Krzysztof; Zukowska, Monika; Jozkowicz, Alicja; Dulak, Jozef

    2016-01-01

    The metabolism of quiescent adult stem cells differs from the metabolism of differentiated cells. The metabolic processes are tightly regulated and their alterations disturb function of stem cells. One of the indicators of metabolic status of cells is the ATP level. While the method of measuring the ATP levels has been known for many years, estimating ATP levels in small population of defined stem cells isolated directly from the tissue has remained challenging. Here, we show our method of measuring the ATP levels in hematopoietic stem cells sorted from murine bone marrow. We used magnetic sorting as well as cell sorter and adopted the commonly used bioluminescence-based detection kits in described protocol. Our strategy allows to measure ATP levels in 1000 highly purified HSC.

  3. Effects of the 5-HT1B receptor antagonist NAS-181 on extracellular levels of acetylcholine, glutamate and GABA in the frontal cortex and ventral hippocampus of awake rats: a microdialysis study.

    Science.gov (United States)

    Hu, Xiao Jing; Wang, Fu-Hua; Stenfors, Carina; Ogren, Sven Ove; Kehr, Jan

    2007-09-01

    The purpose of this study was to investigate the effects of the 5-HT(1B) receptor antagonist NAS-181 ((R)-(+)-2-(3-morpholinomethyl-2H-chromen-8-yl) oxymethyl-morpholine methanesulfonate) on cholinergic, glutamatergic and GABA-ergic neurotransmission in the rat brain in vivo. Extracellular levels of acetylcholine, glutamate and GABA were monitored by microdialysis in the frontal cortex (FC) and ventral hippocampus (VHipp) in separate groups of freely moving rats. NAS-181 (1, 5 or 10 mg/kg, s.c.) caused a dose-dependent increase in ACh levels, reaching the maximal values of 500% (FC) and 230% (VHipp) of controls at 80 min post-injection. On the contrary, NAS-181 injected at doses of 10 or 20 mg/kg s.c. had no effect on basal extracellular levels of Glu and GABA in these areas. The present data suggest that ACh neurotransmission in the FC and VHipp, the brain structures strongly implicated in cognitive function, is under tonic inhibitory control of 5-HT(1B) heteroreceptors localized at the cholinergic terminals in these areas.

  4. Extracellular poly(ADP-ribose) is a neurotrophic signal that upregulates glial cell line-derived neurotrophic factor (GDNF) levels in vitro and in vivo.

    Science.gov (United States)

    Nakajima, Hidemitsu; Itakura, Masanori; Sato, Keishi; Nakamura, Sunao; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2017-03-04

    Synthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease.

  5. Effect of ATP on intracellular pH in pancreatic ducts involves P2X7 receptors

    DEFF Research Database (Denmark)

    Henriksen, Katerine L; Novak, Ivana

    2003-01-01

    Pancreatic acini release ATP, which can stimulate HCO3--secreting ducts that express purinergic receptors from both P2X and P2Y families. The aim of this study was to investigate whether extracellular ATP affects HCO3- or H+ transport across the plasma membrane of intralobular ducts, and determine......, experiments were performed in solutions with or without bicarbonate buffers (+BIC or -BIC), with amiloride derivative EIPA, or with low extracellular Cl- concentrations. Although these transporters contributed to pHi recovery from acidosis, ATP had no effect. Nevertheless, ATP induced a small and reversible...... decrease in pHi by 0.07+/-0.02 pH-units and BzATP decreased pHi by 0.29+/-0.07 pH-units in -BIC (n=10, 11). These effects were abolished by Brilliant Blue and in Ca2+-free solutions. Our study shows that the pHi effect of ATP is mediated by P2X7 receptors. However, ATP does not affect H+/HCO3- transporters...

  6. Subunit movements in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis.

    Science.gov (United States)

    Bienert, Roland; Rombach-Riegraf, Verena; Diez, Manuel; Gräber, Peter

    2009-12-25

    Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

  7. A1Ao-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions.

    Science.gov (United States)

    McMillan, Duncan G G; Ferguson, Scott A; Dey, Debjit; Schröder, Katja; Aung, Htin Lin; Carbone, Vincenzo; Attwood, Graeme T; Ronimus, Ron S; Meier, Thomas; Janssen, Peter H; Cook, Gregory M

    2011-11-18

    An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(o)-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA(1)A(o) was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na(+)-binding signature made up of identical amino acid residues. The purified MbbrA(1)A(o) was stimulated by sodium ions, and Na(+) provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A(1)A(o)-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions.

  8. Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.

    LENUS (Irish Health Repository)

    Pitceathly, Robert D S

    2012-09-11

    Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

  9. De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

    DEFF Research Database (Denmark)

    Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki

    2012-01-01

    in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without...... affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3....

  10. Pharmacological and molecular comparison of K(ATP) channels in rat basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Edvinsson, Lars; Olesen, Jes;

    2006-01-01

    ATP-sensitive potassium (K(ATP)) channels play an important role in the regulation of cerebral vascular tone. In vitro studies using synthetic K(ATP) channel openers suggest that the pharmacological profiles differ between rat basilar arteries and rat middle cerebral arteries. To address this issue....... Densitometric measurements of the Western blot signals further showed higher expression levels of Kir6.1 and SUR2B proteins in rat middle cerebral arteries than was found in rat basilar arteries. In conclusion, our in vitro pharmacological studies showed no evidence for functional endothelial K(ATP) channels...

  11. Microcystin-LR acute exposure does not alter in vitro and in vivo ATP, ADP and AMP hydrolysis in adult zebrafish (Danio rerio brain membranes

    Directory of Open Access Journals (Sweden)

    Luiza Wilges Kist

    2012-10-01

    Full Text Available Microcystins (MCs are toxins produced by cyanobacteria during the blooms that could accumulate in aquatic animals and be relocated to higher trophic levels. Adenosine triphosphate (ATP acts as an excitatory neurotransmitter and/or a neuromodulator in the extracellular space playing important roles in physiological and pathological conditions. The aim of this study was, therefore, to evaluate the acute effects of different concentrations of MC-LR on nucleoside triphosphate diphosphohydrolases and 5’-nucleotidade in adult zebrafish (Danio rerio brain membranes. The results have shown no significant changes in ATP, adenosine diphosphate (ADP and adenosine monophosphate (AMP hydrolysis in zebrafish brain membranes. MC-LR in vitro also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidase activity in the conditions tested. However, additional studies including chronic exposure should be performed in order to achieve a better understanding about MC-LR toxicity mechanisms in the central nervous system.

  12. Hypophosphatemia promotes lower rates of muscle ATP synthesis

    DEFF Research Database (Denmark)

    Pesta, Dominik H; Tsirigotis, Dimitrios N; Befroy, Douglas E

    2016-01-01

    Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [(31)P]-magnetic resonance spectroscopy....... By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in NaPi2a knockout mice (NaPi2a(-/-)) compared with their wild-type littermate controls...... hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum Pi content and muscle VATP After oral Pi repletion and normalization of serum Pi levels, muscle VATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP...

  13. ATP Production in Chlamydomonas reinhardtii Flagella by Glycolytic Enzymes

    DEFF Research Database (Denmark)

    Mitchell, Beth F; Pedersen, Lotte B; Feely, Michael

    2005-01-01

    Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half...... of glycolysis (phosphoglycerate mutase, enolase, and pyruvate kinase). These enzymes can generate one ATP molecule for every substrate molecule consumed. Flagellar fractionation shows that enolase is at least partially associated with the axoneme, whereas phosphoglycerate mutase and pyruvate kinase primarily...... reside in the detergent-soluble (membrane + matrix) compartments. We further show that axonemal enolase is a subunit of the CPC1 central pair complex and that reduced flagellar enolase levels in the cpc1 mutant correlate with the reduced flagellar ATP concentrations and reduced in vivo beat frequencies...

  14. Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

    Science.gov (United States)

    Bertrand, C A; Danahay, H; Poll, C T; Laboisse, C; Hopfer, U; Bridges, R J

    2004-02-01

    ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

  15. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  16. Carbon and energy metabolism of atp mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    . The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production...

  17. Extracellular calcium sensing and extracellular calcium signaling

    Science.gov (United States)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  18. Elucidation of the ATP7B N-domain Mg2+-ATP coordination site and its allosteric regulation.

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    Claude Hercend

    Full Text Available The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain of the Mg(2+-ATP coordination site and answer to the controversial role of the Mg(2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg(2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process.

  19. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution......, since the method is very sensitive (detects 0.5 ng ATP/L) and results are obtained within minutes. When calculating the ATP value a number of parameters need to be considered. These were investigate by use of two different reagent kits (PCP-kit and Lumin(ATE)/Lumin(EX)-kit), internal standard...... and an Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...

  20. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution......, since the method is very sensitive (detects 0.5 ng ATP/L) and results are obtained within minutes. When calculating the ATP value a number of parameters need to be considered. These were investigate by use of two different reagent kits (PCP-kit and Lumin(ATE)/Lumin(EX)-kit), internal standard...... and an Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...

  1. In vivo imaging demonstrates ATP release from murine keratinocytes and its involvement in cutaneous inflammation after tape stripping.

    Science.gov (United States)

    Takahashi, Toshiya; Kimura, Yutaka; Niwa, Kazuki; Ohmiya, Yoshihiro; Fujimura, Taku; Yamasaki, Kenshi; Aiba, Setsuya

    2013-10-01

    Adenosine 5'-triphosphate (ATP) release from keratinocytes has been observed in various stress models in vitro, but studies demonstrating epidermal ATP release in vivo are limited. To visualize extracellular ATP (eATP) in vivo, we developed enhanced green-emitting luciferase immobilized on agarose beads (Eluc-agarose). Subcutaneous injection of Eluc-agarose together with ATP into the dorsal skin of BALB/c mice following intraperitoneal luciferin injection produced detectable and measurable bioluminescence using an in vivo imaging system. Using Eluc-agarose, we demonstrated in vivo that bright bioluminescence was observed from 1 to 20 minutes after repeated tape stripping of murine skin. This bioluminescence was suppressed by the local administration of apyrase. Eluc-agarose bioluminescence was observed only in tape-stripped skin with transepidermal water loss (TEWL) between 100 and 140 g m(2) h(-1), indicating a loss of bioluminescence with excessive tape stripping (TEWL>140 g m(-2) h(-1)). Histologically, tape-stripped skin with detectable eATP had a viable epidermis and a subepidermal neutrophil infiltrate, and administration of apyrase reduced the inflammatory infiltrate. Neither a viable epidermis nor an upper dermal neutrophil infiltrate was observed after excessive tape stripping. These results suggest that tape stripping prompts ATP release from viable keratinocytes, which facilitates inflammatory cell migration. Eluc-agarose may be useful in the in vivo detection of eATP in murine models of skin diseases.

  2. Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

    Science.gov (United States)

    Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

    2010-01-01

    Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

  3. Strain background modifies phenotypes in the ATP8B1-deficient mouse.

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    Sohela Shah

    Full Text Available BACKGROUND: Mutations in ATP8B1 (FIC1 underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis to intermittent (benign recurrent intrahepatic cholestasis. The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6, 129, and (B6-129 F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.

  4. Glibenclamide decreases ATP-induced intracellular calcium transient elevation via inhibiting reactive oxygen species and mitochondrial activity in macrophages.

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    Duo-ling Li

    Full Text Available Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP-induced intracellular calcium ([Ca(2+]i handling in Raw 264.7 macrophages. In the present study, [Ca(2+]i transient, reactive oxygen species (ROS and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+ channel blockers had no effect on the resting [Ca(2+]i of Raw 264.7 cells. Extracellular ATP (100 µM induced [Ca(2+]i transient elevation independent of extracellular Ca(2+. The transient elevation was inhibited by an ROS scavenger (tiron and mitochondria inhibitor (rotenone. Glibenclamide and 5-hydroxydecanoate (5-HD also decreased ATP-induced [Ca(2+]i transient elevation, but pinacidil and other unselective K(+ channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+]i transient induced by extracellular thapsigargin (Tg, 1 µM. Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

  5. ATP sensitizes H460 lung carcinoma cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Swennen, Els L R; Ummels, Vanessa; Buss, Irina; Jaehde, Ulrich; Bast, Aalt; Dagnelie, Pieter C

    2010-03-30

    Platinum resistance of cancer cells may evolve due to a decrease in intracellular drug accumulation, decreased cell permeability or by an increased deactivation of the drug by glutathione (GSH). The aim of this study was (1) to investigate the effect of adenosine 5'-triphosphate (ATP) on the cytotoxicity of cisplatin in a large cell lung carcinoma cell line (H460), and (2) to examine the potential involvement of increased cisplatin uptake, GSH depletion and pyrimidine starvation by ATP in this effect. H460 cells were harvested and seeded (5% CO(2); 37 degrees C). Subsequently, cells were incubated with medium or ATP followed by an incubation with cisplatin. Cytotoxicity screening was analyzed by the sulforhodamine B (SRB) colorimetric assay, lactate dehydrogenase and caspase-3/7 activity. Pre-incubation for 72h with 0.3 and 3mM ATP strongly enhanced the anti-proliferative potency of cisplatin 2.9- and 7.6-fold, respectively. Moreover, after incubation of H460 cells with 0.3mM ATP the intracellular platinum concentration increased, indicating increased cisplatin uptake by ATP. ATP, despite lowering the LD(50) of cisplatin, did not modulate GSH levels in H460 cells. ATP itself showed a biphasic effect on H460 cell growth: 0.3mM inhibited H460 cell growth via the pyrimidine starvation effect, activation of caspase-3/7 and LDH leakage, while 3mM ATP showed no effect on cell growth. In conclusion, ATP sensitizes the H460 cells to cisplatin-induced apoptosis. The effect of 0.3mM ATP is not due to GSH depletion but involves increased cisplatin uptake and pyrimidine starvation due to ATP conversion to adenosine followed by cellular uptake.

  6. The ATP-P2X7 signalling axis is dispensable for obesity-associated inflammasome activation in adipose tissue

    NARCIS (Netherlands)

    Sun, S.; Xia, S.; Ji, Y.; Kersten, A.H.; Qi, L.

    2012-01-01

    Inflammasome activation in adipose tissue has been implicated in obesity-associated insulin resistance and type 2 diabetes. However, when and how inflammasome is activated in adipose tissue remains speculative. Here we test the hypothesis that extracellular ATP, a potent stimulus of inflammasome in

  7. The ATP-P2X7 signalling axis is dispensable for obesity-associated inflammasome activation in adipose tissue

    NARCIS (Netherlands)

    Sun, S.; Xia, S.; Ji, Y.; Kersten, A.H.; Qi, L.

    2012-01-01

    Inflammasome activation in adipose tissue has been implicated in obesity-associated insulin resistance and type 2 diabetes. However, when and how inflammasome is activated in adipose tissue remains speculative. Here we test the hypothesis that extracellular ATP, a potent stimulus of inflammasome in

  8. ATP as a peripheral mediator of pain.

    Science.gov (United States)

    Hamilton, S G; McMahon, S B

    2000-07-01

    This article reviews the extent to which recent studies substantiate the hypothesis that ATP functions as a peripheral pain mediator. The discovery of the P2X family of ion channels (for which ATP is a ligand) and, in particular, the highly selective distribution of the P2X(3) receptor within the rat nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. ATP elicits excitatory inward currents in small diameter sensory ganglion cells. These currents resemble those elicited by ATP on recombinantly expressed heteromeric P2X(2/3) channels as well as homomultimers consisting of P2X(2) and P2X(3). In vivo behavioural models have characterised the algogenic properties of ATP in normal conditions and in models of peripheral sensitisation. In humans, iontophoresis of ATP induces modest pain. In rats and humans the response is dependent on capsaicin sensitive neurons and is augmented in the presence of inflammatory mediators. Since ATP can be released in the vicinity of peripheral nociceptive terminals under a variety of conditions, there exists a purinergic chain of biological processes linking tissue damage to pain perception. The challenge remains to prove a physiological role for endogenous ATP in activating this chain of events.

  9. Red blood cell ATP/ADP & nitric oxide: The best vasodilators in diabetic patients

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    Bakhtiari Nuredin

    2012-08-01

    Full Text Available Abstract Background Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose levels that result from defects in insulin secretion, or action, or both. Inspired by previous report the release of ATP from RBCs, which may participate in vessel dilation by stimulating NO production in the endothelium through purinergic receptor signaling and so, the aim of this study is to clearly determined relationship between RBC ATP/ADP ratio with nitric oxide. Methods The ATP/ADP ratio of erythrocytes among four groups of normal individuals (young & middle age, athletes’ subjects and diabetic patients were compared and the relationship between ATP/ADP ratio and NO level of plasma was determined with AVOVA test and bioluminescence method. Results ATP/ADP level in four groups normal (young & middle age, athletes, diabetes] are measured and analyzed with ANOVA test that show a significant difference between groups (P-value Conclusion In this study, a positive relationship between RBC ATP/ADP ratio and NO was found. Based on the obtained result, higher RBC ATP/ADP content may control the ratio of plasma NO in different individuals, also this results show that ATP can activate endothelial cells in NO production and is a main factor in releasing of NO from endothelial cells.

  10. Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP.

    Science.gov (United States)

    Karmakar, Mausita; Katsnelson, Michael A; Dubyak, George R; Pearlman, Eric

    2016-02-15

    Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K(+), NLRP3 inflammasome activation and IL-1β secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca(2+)], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca(2+)]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1β secretion by neutrophils likely has a significant, wide ranging clinical impact.

  11. Effects of acute and chronic administration of venlafaxine and desipramine on extracellular monoamine levels in the mouse prefrontal cortex and striatum.

    Science.gov (United States)

    Higashino, Kosuke; Ago, Yukio; Umehara, Masato; Kita, Yuki; Fujita, Kazumi; Takuma, Kazuhiro; Matsuda, Toshio

    2014-04-15

    Prefrontal catecholamine neurotransmission plays a key role in the therapeutic actions of drugs for attention-deficit/hyperactivity disorder (ADHD). We have recently shown that serotonin/noradrenaline reuptake inhibitors and the noradrenaline reuptake inhibitor desipramine attenuated horizontal hyperactivity in spontaneously hypertensive rats, an animal model of ADHD, and that these drugs are potential pharmacotherapeutics for ADHD. In this study, we used in vivo microdialysis to study the effects of acute and chronic (once daily for 3 weeks) administration of the serotonin/noradrenaline reuptake inhibitor venlafaxine and the noradrenaline reuptake inhibitor desipramine on noradrenaline, dopamine, and serotonin levels, and the expression of the neuronal activity marker c-Fos in the mouse prefrontal cortex and striatum. Both acute and chronic venlafaxine administration increased prefrontal noradrenaline, dopamine, and serotonin levels and striatal noradrenaline and serotonin levels. Both acute and chronic desipramine administration increased prefrontal noradrenaline and dopamine levels and striatal noradrenaline levels, with chronic administration yielding stronger increase. Chronic desipramine did not affect striatal dopamine and serotonin levels. Both acute and chronic venlafaxine administration increased the expression of c-Fos in the prefrontal cortex, whereas chronic, but not acute, desipramine administration increased the expression of c-Fos in the prefrontal cortex. Both acute and chronic venlafaxine administration increased the striatal c-Fos expression to some degree, whereas desipramine administration did not. These results suggest that acute and chronic venlafaxine and chronic desipramine administration maximally activate the prefrontal adrenergic and dopaminergic systems without affecting striatal dopaminergic systems in mice.

  12. Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells.

    Science.gov (United States)

    Toma, Ildikó; Bansal, Eric; Meer, Elliott J; Kang, Jung Julie; Vargas, Sarah L; Peti-Peterdi, János

    2008-06-01

    Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.

  13. Urinary ATP may be a dynamic biomarker of detrusor overactivity in women with overactive bladder syndrome.

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    Miguel Silva-Ramos

    Full Text Available BACKGROUND: Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB release more ATP than controls. Here, we compared the urinary ATP concentration in samples collected non-invasively from OAB women with detrusor overactivity and age-matched controls. METHODS: Patients with neurologic diseases, history of malignancy, urinary tract infections or renal impairment (creatinine clearance <70 ml/min were excluded. All patients completed a 3-day voiding diary, a 24 h urine collection and blood sampling to evaluate creatinine clearance. Urine samples collected during voluntary voids were immediately freeze-preserved for ATP determination by the luciferin-luciferase bioluminescence assay; for comparison purposes, samples were also tested for urinary nerve growth factor (NGF by ELISA. RESULTS: The urinary content of ATP, but not of NGF, normalized to patients' urine creatinine levels (ATP/Cr or urinary volume (ATP.Vol were significantly (P<0.05 higher in OAB women with detrusor overactivity (n = 34 than in healthy controls (n = 30. Significant differences between the two groups were still observed by boosting urinary ATP/Cr content after water intake, but these were not detected for NGF/Cr. In OAB patients, urinary ATP/Cr levels correlated inversely with mean voided volumes determined in a 3-day voiding diary. CONCLUSION: A high area under the receiver operator characteristics (ROC curve (0.741; 95% CI 0.62-0.86; P<0.001 is consistent with urinary ATP/Cr being a highly sensitive dynamic biomarker for assessing detrusor overactivity in women with OAB syndrome.

  14. Genetic variation in ATP5O is associated with skeletal muscle ATP50 mRNA expression and glucose uptake in young twins.

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    Tina Rönn

    Full Text Available BACKGROUND: Impaired oxidative capacity of skeletal muscle mitochondria contribute to insulin resistance and type 2 diabetes (T2D. Furthermore, mRNA expression of genes involved in oxidative phosphorylation, including ATP5O, is reduced in skeletal muscle from T2D patients. Our aims were to investigate mechanisms regulating ATP5O expression in skeletal muscle and association with glucose metabolism, and the relationship between ATP5O single nucleotide polymorphisms (SNPs and risk of T2D. METHODOLOGY/PRINCIPAL FINDINGS: ATP5O mRNA expression was analyzed in skeletal muscle from young (n = 86 and elderly (n = 68 non-diabetic twins before and after a hyperinsulinemic euglycemic clamp. 11 SNPs from the ATP5O locus were genotyped in the twins and a T2D case-control cohort (n = 1466. DNA methylation of the ATP5O promoter was analyzed in twins (n = 22 using bisulfite sequencing. The mRNA level of ATP5O in skeletal muscle was reduced in elderly compared with young twins, both during basal and insulin-stimulated conditions (p<0.0005. The degree of DNA methylation around the transcription start of ATP5O was <1% in both young and elderly twins and not associated with mRNA expression (p = 0.32. The mRNA level of ATP5O in skeletal muscle was positively related to insulin-stimulated glucose uptake (regression coefficient = 6.6; p = 0.02. Furthermore, two SNPs were associated with both ATP5O mRNA expression (rs12482697: T/T versus T/G; p = 0.02 and rs11088262: A/A versus A/G; p = 0.004 and glucose uptake (rs11088262: A/A versus A/G; p = 0.002 and rs12482697: T/T versus T/G; p = 0.005 in the young twins. However, we could not detect any genetic association with T2D. CONCLUSIONS/SIGNIFICANCE: Genetic variation and age are associated with skeletal muscle ATP5O mRNA expression and glucose disposal rate, suggesting that combinations of genetic and non-genetic factors may cause the reduced expression of ATP5O in T2D muscle. These findings propose a role for ATP5O, in

  15. CB-1 receptors modulate the effect of the selective serotonin reuptake inhibitor, citalopram on extracellular serotonin levels in the rat prefrontal cortex

    NARCIS (Netherlands)

    Kleijn, Jelle; Cremers, Thomas I. F. H.; Hofland, Corry M.; Westerink, Ben H. C.

    2011-01-01

    A large percentage of depressed individuals use drugs of abuse, like cannabis. This study investigates the impact of cannabis on the pharmacological effects of the antidepressant citalopram. Using microdialysis in the prefrontal cortex of rats we monitored serotonin levels before and after cannabino

  16. CB-1 receptors modulate the effect of the selective serotonin reuptake inhibitor, citalopram on extracellular serotonin levels in the rat prefrontal cortex

    NARCIS (Netherlands)

    Kleijn, Jelle; Cremers, Thomas I. F. H.; Hofland, Corry M.; Westerink, Ben H. C.

    2011-01-01

    A large percentage of depressed individuals use drugs of abuse, like cannabis. This study investigates the impact of cannabis on the pharmacological effects of the antidepressant citalopram. Using microdialysis in the prefrontal cortex of rats we monitored serotonin levels before and after cannabino

  17. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

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    Kimberly L. James

    2016-08-01

    Full Text Available Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1 for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4 in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.

  18. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

    Science.gov (United States)

    Xing, Shu; Grol, Matthew W; Grutter, Peter H; Dixon, S Jeffrey; Komarova, Svetlana V

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

  19. (31)P-MRS of healthy human brain: ATP synthesis, metabolite concentrations, pH, and T1 relaxation times.

    Science.gov (United States)

    Ren, Jimin; Sherry, A Dean; Malloy, Craig R

    2015-11-01

    The conventional method for measuring brain ATP synthesis is (31)P saturation transfer (ST), a technique typically dependent on prolonged pre-saturation with γ-ATP. In this study, ATP synthesis rate in resting human brain is evaluated using EBIT (exchange kinetics by band inversion transfer), a technique based on slow recovery of γ-ATP magnetization in the absence of B1 field following co-inversion of PCr and ATP resonances with a short adiabatic pulse. The unidirectional rate constant for the Pi → γ-ATP reaction is 0.21 ± 0.04 s(-1) and the ATP synthesis rate is 9.9 ± 2.1 mmol min(-1)  kg(-1) in human brain (n = 12 subjects), consistent with the results by ST. Therefore, EBIT could be a useful alternative to ST in studying brain energy metabolism in normal physiology and under pathological conditions. In addition to ATP synthesis, all detectable (31)P signals are analyzed to determine the brain concentration of phosphorus metabolites, including UDPG at around 10 ppm, a previously reported resonance in liver tissues and now confirmed in human brain. Inversion recovery measurements indicate that UDPG, like its diphosphate analogue NAD, has apparent T1 shorter than that of monophosphates (Pi, PMEs, and PDEs) but longer than that of triphosphate ATP, highlighting the significance of the (31)P-(31)P dipolar mechanism in T1 relaxation of polyphosphates. Another interesting finding is the observation of approximately 40% shorter T1 for intracellular Pi relative to extracellular Pi, attributed to the modulation by the intracellular phosphoryl exchange reaction Pi ↔ γ-ATP. The sufficiently separated intra- and extracellular Pi signals also permit the distinction of pH between intra- and extracellular environments (pH 7.0 versus pH 7.4). In summary, quantitative (31)P MRS in combination with ATP synthesis, pH, and T1 relaxation measurements may offer a promising tool to detect biochemical alterations at early stages of brain dysfunctions and diseases.

  20. Glioactive ATP controls BDNF recycling in cortical astrocytes

    Science.gov (United States)

    Vignoli, Beatrice; Canossa, Marco

    2017-01-01

    ABSTRACT We have recently reported that long-term memory retention requires synaptic glia for proBDNF uptake and recycling. Through the recycling course, glial cells release endocytic BDNF, a mechanism that is activated in response to glutamate via AMPA and mGluRI/II receptors. Cortical astrocytes express receptors for many different transmitters suggesting for a complex signaling controlling endocytic BDNF secretion. Here, we demonstrated that the extracellular nucleotide ATP, activating P2X and P2Y receptors, regulates endocytic BDNF secretion in cultured astrocytes. Our data indicate that distinct glioactive molecules can participate in BDNF glial recycling and suggest that cortical astrocytes contributing to neuronal plasticity can be influenced by neurotransmitters in tune with synaptic needs.

  1. Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion Current Rectification at Polyimidazolium-Modified Micropipettes.

    Science.gov (United States)

    Zhang, Kailin; He, Xiulan; Liu, Yang; Yu, Ping; Fei, Junjie; Mao, Lanqun

    2017-06-20

    Development of new principles and methods for cerebral ATP assay is highly imperative not only for determining ATP dynamics in brain but also for understanding physiological and pathological processes related to ATP. Herein, we for the first time demonstrate that micrometer scale ion current rectification (MICR) at a polyimidazolium brush-modified micropipette can be used as the signal transduction output for the cerebral ATP assay with a high selectivity. The rationale for ATP assay is essentially based on the competitive binding ability between positively charged polyimidazolium and ATP toward negatively charged ATP aptamer. The method is well responsive to ATP with a good linearity within a concentration range from 5 nM to 100 nM, and high selectivity toward ATP. These properties essentially enable the method to determine the cerebral ATP by combining in vivo microdialysis. The basal dialysate level of ATP in rat brain cortex is determined to be 11.32 ± 2.36 nM (n = 3). This study demonstrates that the MICR-based sensors could be potentially used for monitoring neurochemicals in cerebral systems.

  2. Extracellular Gd-CA

    DEFF Research Database (Denmark)

    Thomsen, Henrik S; Marckmann, Peter

    2008-01-01

    Until recently it was believed that extracellular gadolinium-based contrast agents were safe for both the kidneys and all other organs within the dose range up to 0.3 mmol/kg body weight. However, in 2006, it was demonstrated that some gadolinium-based contrast agents may trig the development of ...

  3. No effect of aluminium upon the hydrolysis of ATP in the coronary circulation of the isolated working rat heart.

    Science.gov (United States)

    Korchazhkina, O; Wright, G; Exley, C

    1999-08-30

    Adenosine 5'-triphosphate (ATP) is now recognised as an important extracellular signalling molecule. Its action at a number of specific receptors is mediated by the activity of ectonucleotidases. We have optimised a high performance liquid chromatography (HPLC) method to allow the simultaneous determination of ATP, and the products of its hydrolysis, in the coronary effluent of an isolated working rat heart. The method is extremely sensitive allowing picomolar quantities of product to be determined. We have used this method to investigate the influence of aluminium on the hydrolysis of ATP by an ecto-ATPase located in the luminal surface of the coronary endothelium of the rat heart. Aluminium did not influence the hydrolysis of ATP by this enzyme.

  4. Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer's disease patients.

    Science.gov (United States)

    Esteras, Noemí; Alquézar, Carolina; Bermejo-Pareja, Félix; Bialopiotrowicz, Emilia; Wojda, Urszula; Martín-Requero, Angeles

    2013-04-01

    Previously, we reported a Ca(2+)/calmodulin (CaM)-dependent impairment of apoptosis induced by serum deprivation in Alzheimer's disease (AD) lymphoblasts. These cell lines showed downregulation of extracellular signal-regulated kinase (ERK)1/2 activity and elevated content of p21 compared with control cells. The aim of this study was to delineate the molecular mechanism underlying the distinct regulation of p21 content in AD cells. Quantitative reverse transcription polymerase chain reaction analysis demonstrated increased p21 messenger RNA (mRNA) levels in AD cells. The ERK1/2 inhibitor, PD98059, prevented death of control cells and enhanced p21 mRNA and protein levels. The CaM antagonist, calmidazolium, and the CaMKII inhibitor, KN-62, normalized the survival pattern of AD lymphoblasts by augmenting ERK1/2 activation and reducing p21 mRNA and protein levels. Upregulation of p21 transcription in AD cells appears to be the consequence of increased activity of forkhead box O3a (FOXO3a) as the result of diminished ERK1/2-mediated phosphorylation of this transcription factor, which in turn facilitates its nuclear accumulation. Murine double minute 2 (MDM2) protein levels were decreased in AD cells relative to control lymphoblasts, suggesting an impairment of FOXO3a degradation.

  5. ATP and Presentation Service for Mizar Formalizations

    CERN Document Server

    Urban, Josef; Sitcliffe, Geoff

    2011-01-01

    This paper describes the Automated Reasoning for Mizar (MizAR) service, which integrates several automated reasoning, artificial intelligence, and presentation tools with Mizar and its authoring environment. The service provides ATP assistance to Mizar authors in finding and explaining proofs, and offers generation of Mizar problems as challenges to ATP systems. The service is based on a sound translation from the Mizar language to that of first-order ATP systems, and relies on the recent progress in application of ATP systems in large theories containing tens of thousands of available facts. We present the main features of MizAR services, followed by an account of initial experiments in finding proofs with the ATP assistance. Our initial experience indicates that the tool offers substantial help in exploring the Mizar library and in preparing new Mizar articles.

  6. [ATP in the metabolism of ruminants].

    Science.gov (United States)

    Bergner, H

    1991-10-01

    The ATP yield from the carbohydrates of anaerobically living microorganisms in the rumen amounts to only 5-10% of the ATP yield of the intermediary metabolism in the presence of oxygen. Vital functions and thus microbial protein synthesis are due to protein degradation in the rumen. The ATP yield in the intermediary metabolism of ruminants is mainly achieved from propionate and microbial protein by means of gluconeogenesis because the absorption of glucose from digested starch is very low. The relationships between ATP yield in the rumen and the processes of glucose provision for the production of lactose as well as the protein content of the milk are shown. As important processes of ATP production in microorganisms from easily soluble carbohydrates take place in silage preparations before feed intake, the corresponding consequences for the metabolism of high-performance cows fed with silage are shown.

  7. HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels.

    Science.gov (United States)

    Zhang, Hai Xia; Akrouh, Alejandro; Kurata, Harley T; Remedi, Maria Sara; Lawton, Jennifer S; Nichols, Colin G

    2011-03-01

    Murine ventricular and atrial ATP-sensitive potassium (K(ATP)) channels contain different sulfonylurea receptors (ventricular K(ATP) channels are Kir6.2/SUR2A complexes, while atrial K(ATP) channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K(ATP) channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K(ATP) channels over atrial K(ATP) channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as (86)Rb(+) efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K(ATP) currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K(ATP) currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits (86)Rb(+) effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K(ATP) channels.

  8. Top-down control analysis of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes.

    Science.gov (United States)

    Ainscow, E K; Brand, M D

    1999-08-01

    Control analysis was used to analyse the internal control of rat hepatocyte metabolism. The reactions of the cell were grouped into nine metabolic blocks linked by five key intermediates. The blocks were glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption. The linking intermediates were intracellular glucose-6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential. The steady-state fluxes through the blocks and the levels of the intermediates were measured in the absence and presence of specific effectors of hepatocyte metabolism. Application of the multiple modulation approach gave the kinetic responses of each block to each intermediate (the elasticities). These were then used to calculate all of the control coefficients, which describe the degree of control each block had over the level of each intermediate, and over the rate of each process. Within this full description of control, many different interactions could be identified. One key finding was that the processes that consumed ATP had only 35% of the control over the rate of ATP consumption. Instead, the reactions that produced ATP exerted the most control over ATP consumption rate; particularly important were mitochondrial phosphorylation (30% of control) and glycolysis (19%). The rate of glycolysis was positively controlled by the glycolytic enzymes themselves (66% of control) and by ATP consumption (47%). Mitochondrial production of ATP, including oxidative, proton leak and phosphorylation processes, had negative control over glycolysis (-26%; the Pasteur effect). In contrast, glycolysis had little control over the rate of ATP production by the mitochondria (-10%; the Crabtree effect). Control over the flux through the mitochondrial phosphorylation block was shared between pyruvate oxidation (23%), ATP consumption (28%) and the

  9. Thermodynamics of proton transport coupled ATP synthesis.

    Science.gov (United States)

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase.

  10. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

    Directory of Open Access Journals (Sweden)

    Joseph E Ippolito

    Full Text Available Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5 on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.

  11. A futile cycle, formed between two ATP-dependant -glutamyl cycle enzymes, -glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

    Indian Academy of Sciences (India)

    Akhilesh Kumar; Anand Kumar Bachhawat

    2010-03-01

    Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the -glutamyl cycle leads to ATP depletion. The enzyme -glutamyl cysteine synthetase (-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, -glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

  12. RNAi-mediated silencing of Atp6i and Atp6i haploinsufficiency prevents both bone loss and inflammation in a mouse model of periodontal disease.

    Directory of Open Access Journals (Sweden)

    Hongbing Jiang

    Full Text Available Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i(+/- mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.

  13. Feasibility study of B16 melanoma therapy using oxidized ATP to target purinergic receptor P2X7.

    Science.gov (United States)

    Hattori, Fumie; Ohshima, Yasuhiro; Seki, Shizuka; Tsukimoto, Mitsutoshi; Sato, Mitsuru; Takenouchi, Takato; Suzuki, Akina; Takai, Erina; Kitani, Hiroshi; Harada, Hitoshi; Kojima, Shuji

    2012-11-15

    The P2X7 receptor is not only involved in cell proliferation, but also acts as an adenosine 5'-triphosphate (ATP)-gated non-selective channel, and its expression is increased in human melanoma. An irreversible antagonist of P2X7, such as oxidized ATP (oxATP), might block P2X7 receptor-mediated ATP release and proliferative signaling. Therefore, we carried out basic studies to test this idea and to examine the feasibility of using oxATP to treat B16 melanoma. We first found that low-pH conditions (mimicking the hypoxia and acidosis commonly seen in solid tumors) induced P2X7 receptor-mediated ATP release from B16 melanoma cells. Then, we compared the proliferation rates of B16 melanoma wild-type cells and B16 P2X7 receptor-knockdown clone (P2X7-KDC) cells in the presence of P2X7 agonists. The proliferation rate, as well as the ATP release, of agonist-treated P2X7-KDC cells was lower than that of agonist-treated wild-type cells. Next, the effect of P2X7 antagonist oxATP on B16 melanoma cell growth was examined in vitro and in vivo. oxATP significantly decreased B16 melanoma cell proliferation in vitro, and also significantly inhibited tumor growth in B16 melanoma-bearing mice. These data indicate that extracellularly released ATP may serve as an intercellular signaling molecule. We propose that the P2X7 receptor is a promising target for treatment of solid tumors.

  14. Towards the development of an automated ATP measuring platform to monitor microbial quality of drinking water

    DEFF Research Database (Denmark)

    Tatari, Karolina; Hansen, C. B.; Rasmussen, A.

    clogged the microfluidic channels. An alternative thermal lysis step was implemented, by adding a flow-though heating/cooling step to the system. Thermal lysis showed efficient release of ATP from an E. coli dilution, but the releasing efficiency varied according to the type of water. Overall......This work aimed to develop an automated and nearly on-line method to monitor ATP levels in drinking water as an indicator of microbial contamination. The system consists of a microfluidic cartridge installed in a light tight box, where the sample is mixed with the reagents and the emitted light...... is detected by a photomultiplier. Temperature in the assay box is controlled and set to 25°C. Calibration of the system using ATP standard solutions was successful, both for free and for total ATP. Chemical release of ATP by reagent addition however resulted in the formation of particles that ultimately...

  15. IV ATP potentiates midazolam sedation as assessed by bispectral index.

    Science.gov (United States)

    Sakurai, Satoru; Fukunaga, Atsuo; Ichinohe, Tatsuya; Kaneko, Yuzuru

    2014-01-01

    In this study, by measuring bispectral index (BIS), we tested the hypothesis that intravenous adenosine 5'-triphosphate (ATP) infusion would deepen the level of midazolam-induced sedation. Ten healthy volunteers underwent 2 experiments with at least 2 weeks' interval: immediately after intravenous bolus administration of midazolam (0.04 mg/kg), they received continuous infusion of either ATP infusion (100 μg/kg/min) or placebo (saline) for 40 minutes in a double-blind, randomized, crossover manner. Changes in BIS values and responsiveness to verbal command as well as cardiorespiratory variables were observed throughout the study periods. Administration of midazolam alone reduced BIS value from control: 97 ± 1 to 68 ± 18 at 25 minutes, which was accompanied by significant cardiopulmonary depressant effects, while maintaining responsiveness to verbal command (consciousness) throughout the study period. Coadministration of ATP with midazolam further reduced BIS value to 51 ± 13, associated with complete loss of consciousness without adverse effect on the cardiorespiratory systems. We conclude that the addition of ATP infusion to midazolam significantly enhances midazolam sedation without disturbing cardiorespiratory functions.

  16. ATP content and biomass activity in sequential anaerobic/aerobic reactors

    Institute of Scientific and Technical Information of China (English)

    陈红

    2004-01-01

    Specific ATP content of volatile solids was measured to characterize the sludge activity in a sequential anaerobic/aerobic wastewater treatment system, with an upflow anaerobic sludge blanket (UASB) reactor and a three-phase aerobic fluidized bed (AFB) reactor. The wastewater COD level was 2000(3000 mg/L in simulation of real textile wastewater. The ATP content and the specific ATP contents of volatile solids at different heights of the UASB reactor and those of the suspended and immobilized biomass in the AFB reactor were measured. In the UASB reactor, the maximum value of specific ATP (0.85 mg ATP/g VS) was obtained at a hydraulic retention time (HRT) 7.14 h in the blanket solution. In the AFB reactor, the specific ATP content of suspended biomass was higher than that of immobilized biomass and increased with hydraulic retention time reaching a maximum value of 1.6 mg ATP/g VS at hydraulic retention time 4.35 h. The ATP content of anaerobes in the UASB effluent declined rapidly under aerobic conditions following a 2nd-order kinetic model.

  17. ATP content and biomass activity in sequential anaerobic/aerobic reactors

    Institute of Scientific and Technical Information of China (English)

    陈红

    2004-01-01

    Specific ATP content of volatile solids was measured to characterize the sludge activity in a sequential anaerobic/aerobic wastewater treatment system, with an upflow anaerobic sludge blanket (UASB) reactor and a three-phase aerobic fluidized bed (AFB) reactor. The wastewater COD level was 2000-3000 mg/L in simulation of real textile wastewater. The ATP content and the specific ATP contents of volatile solids at different heights of the UASB reactor and those of the suspended and immobilized biomass in the AFB reactor were measured. In the UASB reactor, the maximum value of specific ATP (0.85 mg ATP/g VS) was obtained at a hydraulic retention time (HRT) 7.14 h in the blanket solution. In the AFB reactor, the specific ATP content of suspended biomass was higher than that of immobilized biomass and increased with hydraulic retention time reaching a maximum value of 1.6 mg ATP/g VS at hydraulic retention time 4.35 h. The ATP content of anaerobes in the UASB effluent declined rapidly under aerobic conditions following a 2nd-order kinetic model.

  18. Structural, biochemical and genetic characterization of dissimilatory ATP sulfurylase from Allochromatium vinosum.

    Directory of Open Access Journals (Sweden)

    Kristian Parey

    Full Text Available ATP sulfurylase (ATPS catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2- with ATP to adenosine 5'-phosphosulfate (APS and pyrophosphate (PPi. In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.

  19. [A review of recent researches on correlation between ATP and acupuncture efficacies].

    Science.gov (United States)

    Chen, Bo; Guo, Yi; Zhao, Xue; Liu, Yang-Yang; Li, Zhong-Zheng; Li, Ying-Hong; Guo, Yong-Ming

    2012-08-01

    It has been documented that adenosine triphosphate (ATP) is a multifunctional nucleoside triphosphate used in cells, including chemical energy transportation, extra- and intracellular signaling, cell structure maintaining, DNA and RNA synthesis, etc. In the present paper, the authors reviewed studies on the involvement of ATP in different efficacies of acupuncture intervention from the following four aspects. 1) ATP release in the stimulated acupoint area is one of the key factors for producing acupuncture analgesia; 2) Acupuncture induced suppression of ATP activity in the central nervous system results in pain relief; 3) ATP application on the human body surface may strengthen the sensation propagation along the meridian; 4) Favorable regulation of acupuncture intervention on the abnormal functional activities of some viscera often accompanies with an increase of ATP content and ATPase activity in the related internal organs. It has been proposed that ATP, Ca2+ and reactive oxygen species (ROS) are closely related each other in the life activities of the organism. Hence, a reasonable regulation on ATP levels in the related organs of the body may be a new approach for raising clinical therapeutic effects of acupuncture therapy.

  20. Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling.

    Science.gov (United States)

    Plattner, Helmut; Verkhratsky, Alexei

    2016-08-05

    From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally

  1. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Low-level laser therapy stimulates tissue repair and reduces the extracellular matrix degradation in rats with induced arthritis in the temporomandibular joint.

    Science.gov (United States)

    Lemos, George Azevedo; Rissi, Renato; de Souza Pires, Ivan Luiz; de Oliveira, Letícia Prado; de Aro, Andrea Aparecida; Pimentel, Edson Rosa; Palomari, Evanisi Teresa

    2016-08-01

    The objective of this study was to characterize morphological and biochemistry action of low-level laser therapy (LLLT) on induced arthritis in the temporomandibular joint (TMJ) of rats. Twenty-four male Wistar rats were randomly divided into groups with 12 animals each: (AG) group with arthritis induced in the left TMJ and (LG) group with arthritis induced in the left TMJ and treated with LLLT (830 nm, 30 mW, 3 J/cm(2)). Right TMJs in the AG group were used as noninjected control group (CG). Arthritis was induced by intra-articular injection of 50 μl Complete Freund's Adjuvant (CFA) and LLLT began 1 week after arthritis induction. Histopathological analysis was performed using sections stained with hematoxylin-eosin, Toluidine Blue, and picrosirius. Biochemical analysis was determined by the total concentration of sulfated glycosaminoglycans (GAGs) and evaluation of matrix metalloproteinases (MMP-2 and MMP-9). Statistical analysis was performed using paired and unpaired t tests, with p arthritis.

  3. Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus.

    Science.gov (United States)

    de Paula Soares, Cleiton; Rodrigues, Elisete Pains; de Paula Ferreira, Jéssica; Simões Araújo, Jean Luiz; Rouws, Luc Felicianus Marie; Baldani, José Ivo; Vidal, Marcia Soares

    2015-03-01

    TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

  4. Receptor-like activity evoked by extracellular ADP in Arabidopsis root epidermal plasma membrane.

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M

    2011-07-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca(2+) influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca(2+) in root epidermal protoplasts. An inward Ca(2+)-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca(2+) efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

  5. Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis Root Epidermal Plasma Membrane1

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M.

    2011-01-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermal protoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM. PMID:21562328

  6. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2012-01-01

    , but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production...... satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential...... in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging....

  7. The two-hit hypothesis for neuroinflammation: role of exogenous ATP in modulating inflammation in the brain

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    Bernd L. Fiebich

    2014-09-01

    Full Text Available Brain inflammation is a common occurrence following responses to varied insults such as bacterial infections, stroke, traumatic brain injury and neurodegenerative disorders. A common mediator for these varied inflammatory responses is prostaglandin E2 (PGE2, produced by the enzymatic activity of cyclooxygenases (COX 1 and 2. Previous attempts to reduce neuronal inflammation through COX inhibition, by use of nonsteroidal anti-inflammatory drugs (NSAIDs, have met with limited success. We are proposing the two-hit model for neuronal injury – an initial localized inflammation mediated by PGE2 (first hit and the simultaneous release of adenosine triphosphate (ATP by injured cells (second hit, which significantly enhances the inflammatory response through increased synthesis of PGE2. Several evidences on the role of exogenous ATP in inflammation have been reported, including contrary instances where extracellular ATP reduces inflammatory events. In this review, we will examine the current literature on the role of P2 receptors, to which ATP binds, in modulating inflammatory reactions during neurodegeneration. Targeting the P2 receptors, therefore, provides a therapeutic alternative to reduce inflammation in the brain. P2 receptor-based anti-inflammatory drugs (PBAIDs will retain the activities of essential COX enzymes, yet will significantly reduce neuroinflammation by decreasing the enhanced production of PGE2 by extracellular ATP.

  8. Species and agonist dependent zinc modulation of endogenous and recombinant ATP-gated P2X7 receptors.

    Science.gov (United States)

    Moore, Samantha F; Mackenzie, Amanda B

    2008-12-15

    Zinc (Zn2+) and copper (Cu2+) are key signalling molecules in the immune system and regulate the activity of many ion channels. Both Zn2+ and Cu2+ potently inhibit rat P2X7 receptors via a binding site identified by mutagenesis. Here we show that extracellular Cu2+ also potently inhibits mouse P2X7 receptors. By contrast, the receptor expression system and agonist strongly influence the action of extracellular Zn2+ at mouse P2X7 receptors. Consistent with previous reports, Zn2+ inhibits recombinant rat P2X7 receptors. However, recombinant mouse P2X7 receptors are potentiated by Zn2+ when activated by ATP4- but inhibited when stimulated with the ATP analogue BzATP4-. Endogenous murine macrophage P2X7 receptors are not modulated by Zn2+ when stimulated by ATP4- however Zn2+ inhibits BzATP4- mediated responses. In summary, these findings provide a fundamental insight into the differential actions of Zn2+ and Cu2+ between different P2X7 receptor species.

  9. P2X receptor-mediated ATP purinergic signaling in health and disease

    Directory of Open Access Journals (Sweden)

    Jiang LH

    2012-09-01

    Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

  10. Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

    Energy Technology Data Exchange (ETDEWEB)

    Gikas, P.; Livingston, A.G. (Imperial Coll., London (United Kingdom). Dept. of Chemical Engineering)

    1993-12-01

    This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bio-reactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h[sup [minus]1] in the CST bioreactor and between 0.111 and 0.500h[sup [minus]1] in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g[sup [minus]1] dry weight (dw) as dilution rate increases from 0.027 to 0.115 h[sup [minus]1]. At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g[sup [minus]1] dw, which is assumed to be the quantity of ATP in 100% viable biomass, In the TPAL bioreactor, the ATP level increased with dilution rat in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g[sup [minus]1] dw at dilution rates between 0.111 and 0.200 h[sup [minus]1] to approximately 0.119 mg ATP g[sup [minus]1] dw at dilution rates between 0.300 and 0.500 h[sup [minus]1].

  11. Cardiac specific ATP-sensitive K+ channel (KATP) overexpression results in embryonic lethality.

    Science.gov (United States)

    Toib, Amir; Zhang, Hai Xia; Broekelmann, Thomas J; Hyrc, Krzysztof L; Guo, Qiusha; Chen, Feng; Remedi, Maria S; Nichols, Colin G

    2012-09-01

    Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.

  12. Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular noradrenaline, serotonin, and dopamine levels in Wistar-Kyoto and spontaneously hypertensive rats.

    Science.gov (United States)

    Tchekalarova, Jana; Loyens, Ellen; Smolders, Ilse

    2015-05-01

    In the management of epilepsy, AT1 receptor antagonists have been suggested as an additional treatment strategy. A hyperactive brain angiotensin (Ang) II system and upregulated AT1 receptors are implicated in the cerebrovascular alterations in a genetic form of hypertension. Uncontrolled hypertension could also, in turn, be a risk factor for a seizure threshold decrease and development of epileptogenesis. The present study aimed to assess the effects of the selective AT1 receptor antagonist ZD7155 on kainic acid (KA)-induced status epilepticus (SE) development and accompanying changes in the hippocampal extracellular (EC) neurotransmitter levels of noradrenaline (NAD), serotonin (5-HT), and dopamine (DA) in spontaneously hypertensive rats (SHRs) and their parent strain Wistar-Kyoto (WKY) rats, since monoamines are well-known neurotransmitters involved in mechanisms of both epilepsy and hypertension. Status epilepticus was evoked in freely moving rats by a repetitive intraperitoneal (i.p.) administration of KA in subconvulsant doses. In the treatment group, ZD7155 (5mg/kg i.p.) was coadministered with the first KA injection. Spontaneously hypertensive rats exhibited higher susceptibility to SE than WKY rats, but the AT1 receptor antagonist did not alter the development of SE in SHRs or in WKY rats. In vivo microdialysis demonstrated significant KA-induced increases of the hippocampal NAD and DA levels in SHRs and of NAD, 5-HT, and DA in WKY rats. Although SHRs developed more severe seizures while receiving a lower dose of KA compared to WKY rats, AT1 receptor antagonism completely prevented all KA-induced increases of hippocampal monoamine levels in both rat strains without affecting seizure development per se. These results suggest a lack of direct relationship between KA-induced seizure susceptibility and adaptive changes of hippocampal NAD, 5-HT, and DA levels in the effects of ZD7155 in WKY rats and SHRs.

  13. Customized ATP towpreg. [Automated Tow Placement

    Science.gov (United States)

    Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

    1992-01-01

    Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placement from the robot head to the tool. This study examined the key towpreg ribbon properties and dimensions which play a significant role in ATP. Analysis of the heat transfer process window indicates that adequate heating can be achieved at lay down rates as high as 1 m/sec. While heat transfer did not appear to be the limiting factor, resin flow and fiber movement into tow lap gaps could be. Accordingly, consideration was given to towpreg ribbon having uniform yet non-rectangular cross sections. Dimensional integrity of the towpreg ribbon combined with customized ribbon architecture offer great promise for processing advances in ATP of high performance composites.

  14. An RNA motif that binds ATP

    Science.gov (United States)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  15. Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

    DEFF Research Database (Denmark)

    Mortensen, Stefan P.; Gonzalez-Alonso, Jose; Nielsen, Jens Jung

    2009-01-01

    ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial...... ATP and NE concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and norepinephrine (NE) concentrations were measured during: 1) femoral arterial ATP infusion (0.......42+/-0.04 and 2.26+/-0.52 mumol/min; mean+/-SEM) and 2) one-leg knee-extensor exercise (18+/-0 and 37+/-2W) in 10 healthy, male subjects. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from ~0.3 L/min at baseline to 4.2+/-0.3 and 4.6+/-0.5 L/min, respectively, whereas...

  16. The role of individual domains and the significance of shedding of ATP6AP2/(prorenin receptor in vacuolar H(+-ATPase biogenesis.

    Directory of Open Access Journals (Sweden)

    Kenichiro Kinouchi

    Full Text Available The ATPase 6 accessory protein 2 (ATP6AP2/(prorenin receptor (PRR is essential for the biogenesis of active vacuolar H(+-ATPase (V-ATPase. Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD and transmembrane domain (TM of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M and causes X-linked mental retardation Hedera type (MRXSH and X-linked parkinsonism with spasticity (XPDS in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  17. The role of individual domains and the significance of shedding of ATP6AP2/(pro)renin receptor in vacuolar H(+)-ATPase biogenesis.

    Science.gov (United States)

    Kinouchi, Kenichiro; Ichihara, Atsuhiro; Sano, Motoaki; Sun-Wada, Ge-Hong; Wada, Yoh; Ochi, Hiroki; Fukuda, Toru; Bokuda, Kanako; Kurosawa, Hideaki; Yoshida, Naohiro; Takeda, Shu; Fukuda, Keiichi; Itoh, Hiroshi

    2013-01-01

    The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H(+)-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  18. Role of ATP in the sensitivity to heat and the induction of apoptosis in mammalian cells.

    Science.gov (United States)

    Miyazaki, N; Kurihara, K; Nakano, H; Shinohara, K

    2002-01-01

    Heat-induced cell death and apoptosis were studied with respect to intracellular ATP. Studies on the relationship between hyperthermic cell-killing at 44 degrees C and cellular ATP levels in four cell lines grown as monolayers and six cell lines grown in suspension showed good correlations between cellular ATP levels and the sensitivity to heat. D(0) values (the dose required to reduce survival in the linear portion of the response by 63%) linearly increased with an increase in cellular ATP levels. No such changes in sensitivity to heat were observed between the cells cultured at different cell densities, regardless of the change in the cellular ATP level. These results suggest that cellular intrinsic ability to supply ATP rather than the level of pooled ATP per se is responsible for the thermal response. Heat-induced apoptosis in L5178Y cells was observed following treatment at 42 degrees C for 70 min, 44 degrees C for 20 min or 47 degrees C for 3 min, which corresponded to surviving fractions of 25, 0.6 and 0.8%, respectively, but not at 47 degrees C for 20 min, indicating that mild heat shock induced apoptosis. 2-deoxyglucose (2DG) and 2,4-dinitrophenol (DNP) increased the sensitivity to heat and affected the mode of cell death. Cells treated with 2DG and DNP (2DG/DNP) were heated at 42 degrees C for 20 min, and then incubated at 37 degrees C for up to 2h in the presence or absence of 2DG/DNP. In the absence of 2DG/DNP, the cellular ATP level recovered to 76% of the control level and DNA ladder formation was observed, whereas in the presence of 2DG/DNP, the cellular ATP level was further decreased (3-7% of the control) and no DNA fragmentation was detected. These results suggest that the inhibition of ATP synthesis is closely associated with the enhancement of sensitivity to heat and that ATP is required for the induction of apoptosis.

  19. Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells.

    Science.gov (United States)

    Aromolaran, Ademuyiwa S; Zima, Aleksey V; Blatter, Lothar A

    2007-07-01

    The role of glycolytically generated ATP in Ca(2+)/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca(2+) signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca(2+) concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + beta-hydroxybutyrate (beta-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca(2+)](i). The rise of [Ca(2+)](i) was characterized by a transient spike followed by a small sustained plateau of elevated [Ca(2+)](i). In the absence of extracellular Ca(2+) 2-DG caused an increase in [Ca(2+)](i), suggesting that inhibition of glycolysis directly triggered release of Ca(2+) from intracellular endoplasmic reticulum (ER) Ca(2+) stores. The inositol-1,4,5-trisphosphate receptor (IP(3)R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca(2+) response. Ca(2+) release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca(2+)](i) wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca(2+)](i). Propagating [Ca(2+)](i) waves were preceded by [Ca(2+)](i) oscillations and small, highly localized elevations of [Ca(2+)](i) (Ca(2+) puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca(2+) response, and vice versa during inhibition of CaMKII 2-DG-induced Ca(2+) release was attenuated. Similar results were obtained with pyr + beta-HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg(2+) concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca(2+) release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP(3)R.

  20. Electrophysiology of autonomic neuromuscular transmission involving ATP.

    Science.gov (United States)

    Sneddon, P

    2000-07-01

    Electrophysiological investigations of autonomic neuromuscular transmission have provided great insights into the role of ATP as a neurotransmitter. Burnstock and Holman made the first recordings of excitatory junction potentials (e.j.p.s) produced by sympathetic nerves innervating the smooth muscle of the guinea-pig vas deferens. This led to the identification of ATP as the mediator of e.j.p.s in this tissue, where ATP acts as a cotransmitter with noradrenaline. The e.j.p.s are mediated solely by ATP acting on P2X(1) receptors leading to action potentials and a rapid phasic contraction, whilst noradrenaline mediates a slower, tonic contraction which is not dependent on membrane depolarisation. Subsequent electrophysiological studies of the autonomic innervation of smooth muscles of the urogenital, gastrointestinal and cardiovascular systems have revealed a similar pattern of response, where ATP mediates a fast electrical and mechanical response, whilst another transmitter such as noradrenaline, acetylcholine, nitric oxide or a peptide mediates a slower response. The modulation of junction potentials by a variety of pre-junctional receptors and the mechanism of inactivation of ATP as a neurotransmitter will also be described.

  1. Adenosine-5'-triphosphate (ATP supplementation improves low peak muscle torque and torque fatigue during repeated high intensity exercise sets

    Directory of Open Access Journals (Sweden)

    Rathmacher John A

    2012-10-01

    Full Text Available Abstract Background Intracellular concentrations of adenosine-5’-triphosphate (ATP are many times greater than extracellular concentrations (1–10 mM versus 10–100 nM, respectively and cellular release of ATP is tightly controlled. Transient rises in extracellular ATP and its metabolite adenosine have important signaling roles; and acting through purinergic receptors, can increase blood flow and oxygenation of tissues; and act as neurotransmitters. Increased blood flow not only increases substrate availability but may also aid in recovery through removal of metabolic waste products allowing muscles to accomplish more work with less fatigue. The objective of the present study was to determine if supplemental ATP would improve muscle torque, power, work, or fatigue during repeated bouts of high intensity resistance exercise. Methods Sixteen participants (8 male and 8 female; ages: 21–34 years were enrolled in a double-blinded, placebo-controlled study using a crossover design. The participants received either supplemental ATP (400 mg/d divided into 2 daily doses or placebo for 15 d. After an overnight fast, participants underwent strength and fatigue testing, consisting of 3 sets of 50 maximal knee extensions performed on a Biodex® leg dynamometer. Results No differences were detected in high peak torque, power, or total work with ATP supplementation; however, low peak torque in set 2 was significantly improved (p Conclusions Supplementation with 400 mg ATP/d for 15 days tended to reduce muscle fatigue and improved a participant’s ability to maintain a higher force output at the end of an exhaustive exercise bout.

  2. Modeling interactions among individual P2 receptors to explain complex response patterns over a wide range of ATP concentrations

    Directory of Open Access Journals (Sweden)

    Shu Xing

    2016-07-01

    Full Text Available EExtracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6. In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1/2 in that range (e.g. P2Y4 or P2X5 exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

  3. Effect of Controlled Freezing and Open-Pulled Straw (OPS) Vitrification on ATP Content and Reactive Oxygen Species (ROS) Level of Bovine In Vitro Produced Blastocysts%不同冷冻方法对牛体外胚胎ATP含量与ROS水平的影响

    Institute of Scientific and Technical Information of China (English)

    赵学明; 杜卫华; 王栋; 郝海生; 朱化彬

    2012-01-01

    [Objective] The experiment was designed to investigate the effect of cryopreservation on ATP content and ROS level of bovine blastocysts produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). [Method] Bovine in vitro produced blastocysts were cryopreserved by controlled freezing and OPS vitrification. ATP Bioluminescence Assay Kit HS II and GENMED ROS Assay Kit were used to analyze ATP content and ROS level in cryopreserved blastocysts. [Result] (1) For each kind of blastocysts (IVF or SCNT), significant difference was observed between survival rates of controlled freezing group ((81.25±4.98)% or (49.41±2.24)%) and OPS vitrification group ((93.25±5.17)% or (77.56±3.52)%) (P<0.05). (2) For IVF blastocysts, ATP content was significantly decreased from (0.76±0.04) pmol (fresh group) to (0.45±0.03) pmol (controlled freezing group) or (0.63±0.05) pmol (vitrification group) (P<0.05), and the same was found for SCNT blastocysts ((0.40±0.02) pmol to (0.22±0.01) pmol or (0.33±0.02) pmol) (P<0.05). ATP content of each kind of blastocysts (IVF or SCNT) in controlled freezing group ((0.45±0.03) pmol or (0.22±0.01) pmol) was significantly lower than that in OPS vitrification group ((0.63±0.05) pmol or (0.33±0.02) pmol) (P<0.05). (3) ROS level of fresh IVF blastocysts ((48.52±2.65) cps) or SCNT blastocysts ((27.36±2.23) cps) was significantly lower than that of OPS vitrification group ((74.34±4.24) cps or (43.21±3.35) cps) (P<0.05), but higher than that of controlled freezing group ((35.61±4.32) cps or (16.56±2.52) cps) (P<0.05). [Conclusion] The present work indicated that vitrification was more efficient in the cryopreservation of bovine blastocysts derived from FVF or SCNT with comparison of controlled freezing. Meanwhile, both vitrification and controlled freezing significantly altered ATP content and ROS level in those blastocysts.%[目的]探讨冷冻保存对牛体外生产胚胎能量代谢、有氧代谢造成的影响.[

  4. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  5. Local radiotherapy increases the level of autoantibodies to ribosomal P0 protein but not to heat shock proteins, extracellular matrix molecules and EGFR/ErbB2 receptors in prostate cancer patients.

    Science.gov (United States)

    Ingrosso, Gianluca; Fantini, Massimo; Nardi, Alessandra; Benvenuto, Monica; Sacchetti, Pamela; Masuelli, Laura; Ponti, Elisabetta; Frajese, Giovanni Vanni; Lista, Florigio; Schillaci, Orazio; Santoni, Riccardo; Modesti, Andrea; Bei, Roberto

    2013-03-01

    Prostate cancer is a common cancer among men in developed countries. Although hormonotherapy and radiotherapy (RT) represent valid therapies for prostate cancer treatment, novel immunological approaches have been explored. The development of clinical trials employing cancer vaccines has indicated that immune response to tumor antigens can be boosted and that vaccine administration can improve patient survival. Immune response to tumor antigens could also be enhanced after standard therapies. In the present study, we determined the occurrence of antibodies to extracellular matrix (ECM) molecules, heat shock protein (HSP), ribosomal P0 protein, EGFR, ErbB2 and prostate-specific antigen (PSA) in 35 prostate cancer patients prior to and following local RT and hormonotherapy. We demonstrated that immunity to P0, ECM molecules [collagens (C) CI, CIII, CV, fibronectin (FN) and laminin (LM)] and to HSP90 was associated with malignancy in untreated patients. None of the patient sera showed antibodies to EGFR, while 2 and 1 patients showed reactivity to ErbB2 and PSA, respectively. We also demonstrated that 8 months after therapy the IgG serum levels to CI, CIII, FN and HSP90 significantly decreased. Conversely, the level of P0 autoantibodies increased after therapy in 10 patients. Five of the 10 patients with increased levels of P0 autoantibodies were treated with RT plus hormonotherapy. Treatment of patients did not change the levels of antibodies against EGFR, ErbB2 and PSA. Our results indicated that the modification of antibody level to self molecules after standard treatment of prostate cancer patients is influenced by the type of antigen. Ribosomal P0 protein appears to be a high immunogenic antigen and its immunogenicity increases following RT. In addition, 10 patients with increased levels of autoantibodies to P0 showed PSA mean levels lower than the remaining 25 patients at 18 months. This study may contribute to a better understanding of the

  6. 冬虫夏草对肾衰患者细胞氨基酸代谢的影响%The Influence of Cordyceps Sinensis on the Extracellular and Intracellular Free Amino Acid Levels in Patients with CRF

    Institute of Scientific and Technical Information of China (English)

    朱淳; 左静南; 朱汉威

    2000-01-01

    Objective To study the Cordyceps Sinensis acting on the metabolism of intracellular and extracellular amino acids in patients with CRF. Methods We observed ten patients with CRF, compared the free amino acid concentrations in plasma, erthrocytes and skeletal muscle before and after Cordyceps Sinensis treatment by the amino acid automatic analytical instrument. Results After the CRF group took Cordyceps Sinensis, the Leu, Ile, Thr, Lys, Cys, Tyr concentrations in plasma and Tyr, Glu in erythrocytes approached the normal levels. In one sample of skeletal muscle, the Thr, Lys concentrations approached the normal, whereas both the intracellular and extracellular Val concentrations were still decreased remarkably as compared with the normal controls. Conclusion In patinets with CRF, Cordyceps Sinensis can improve the metabolic disorder notably, increase the dropped free amino acids in varying degrees, but couldn't correct completely the disturbance.%目的 研究冬虫夏草(虫草)对慢性肾功能衰竭(CRF)患者细胞内外氨基酸代谢的影响。 方法 采用LKB-4400型氨基酸自动分析仪测定10例CRF患者服用虫草前后的血浆、红细胞、骨骼肌细胞内的游离氨基酸浓度。结果 CRF患者服用虫草后血浆中亮氨酸、异亮氨酸、苏氨酸、赖氨酸、胱氨酸、酪氨酸浓度和红细胞中酪氨酸、谷氨酸浓度接近正常,1例骨骼肌标本苏氨酸、赖氨酸浓度接近正常;而细胞内外缬氨酸浓度仍显著降低。结论 虫草能显著改善CRF患者的氨基酸代谢紊乱,使下降的游离氨基酸水平均有不同程度的提高,但不能使其完全恢复正常。

  7. Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

    Science.gov (United States)

    Ah-Seng, Yoan; Rech, Jérôme; Lane, David; Bouet, Jean-Yves

    2013-01-01

    Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. PMID:24367270

  8. Defining the role of ATP hydrolysis in mitotic segregation of bacterial plasmids.

    Directory of Open Access Journals (Sweden)

    Yoan Ah-Seng

    Full Text Available Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP, together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.

  9. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels

    Directory of Open Access Journals (Sweden)

    Go Kasuya

    2016-02-01

    Full Text Available P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

  10. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  11. Extracellular Matrix Proteins

    Directory of Open Access Journals (Sweden)

    Linda Christian Carrijo-Carvalho

    2012-01-01

    Full Text Available Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair.

  12. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

    2016-05-16

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

  13. ATP as an indicator of biomass activity in thermophilic upflow anaerobic sludge blanket reactor

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    This work investigated the biomass activity in a thermophilic upflow anaerobic sludge blanket (UASB) reactor of wastewater treatment. Synthetic textile wastewater with pH 10-11, COD level of 2000-3000 mg/L was tested. Cellular adenosine triphosphate (ATP) in volatile solids (VS; mg ATP/gVS) was measured and expressed as specific ATP content to compare the biomass activity in up zone and lower zone in UASB reactor. The result shows that the specific ATP content based on total volatile solids (VS)in lower zone (0. 046 mgATP/gVS average) is much lower than that in up zone (0.62 mgATP/gVS average) due to high content of inactive biomass and high pH in lower zone. The SATP in up zone increases as HRT increases and approaches to a maximum value of 0.85 mgATP/gVS at HRT of 7h, then decreases. It shows most of the total VS in up zone represent active bacterial biomass at HRT of 7h. Rate of subtract utilization is directly related to the activity of microorganisms in the reactor. The effect of HRT on SATP in lower zone is not as significant as on SATP in up zone. The buffer capacity of the thermophilic UASB reactor is very good. It is the activity of sludge granules in lower zone that give the UASB reactor such a good buffer capacity to the inlet high pH.

  14. ATP monitoring technology for microbial growth control in potable water systems

    Science.gov (United States)

    Whalen, Patrick A.; Whalen, Philip J.; Cairns, James E.

    2006-05-01

    ATP (Adenosine Triphosphate) is the primary energy transfer molecule present in all living biological cells on Earth. ATP cannot be produced or maintained by anything but a living organism, and as such, its measurement is a direct indication of biological activity. The main advantage of ATP as a biological indicator is the speed of the analysis - from collecting the sample to obtaining the result, only minutes are required. The technology to measure ATP is already widely utilized to verify disinfection efficacy in the food industry and is also commonly applied in industrial water processes such as cooling water systems to monitor microbial growth and biocide applications. Research has indicated that ATP measurement technology can also play a key role in such important industries as potable water distribution and biological wastewater treatment. As will be detailed in this paper, LuminUltra Technologies has developed and applied ATP measurement technologies designed for any water type, and as such can provide a method to rapidly and accurately determine the level of biological activity in drinking water supplies. Because of its speed and specificity to biological activity, ATP measurement can play a key role in defending against failing drinking water quality, including those encountered during routine operation and also bioterrorism.

  15. Macula densa cell signaling involves ATP release through a maxi anion channel.

    Science.gov (United States)

    Bell, Phillip Darwin; Lapointe, Jean-Yves; Sabirov, Ravshan; Hayashi, Seiji; Peti-Peterdi, Janos; Manabe, Ken-Ichi; Kovacs, Gergely; Okada, Yasunobu

    2003-04-01

    Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl](L)) and transmit signals to the mesangial cellafferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch-clamp studies of macula densa cells, we identified an [NaCl](L)-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 microM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl](L), by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca(2+) concentration to extracellular application of ATP (EC(50) 0.8 microM). Importantly, we also found increases in cytosolic Ca(2+) concentration with elevations in [NaCl](L), when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y(2) receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

  16. Ultrasensitive bioluminescent determinations of adenosine triphosphate (ATP) for investigating the energetics of host-grown microbes

    Science.gov (United States)

    Hanks, J. H.; Dhople, A. M.

    1975-01-01

    Stability and optimal concentrations of reagents were studied in bioluminescence assay of ATP levels. Luciferase enzyme was prepared and purified using Sephadex G-100. Interdependencies between enzyme and luciferin concentrations in presence of optimal Mg are illustrated. Optimal ionic strength was confirmed to be 0.05 M for the four buffers tested. Adapted features of the R- and H-systems are summarized, as well as the percentages of ATP pools released from representative microbes by heat and chloroform.

  17. Synthetic peptides target ATP translocase of ‘Candidatus Liberibacter asiaticus’ to block ATP uptake

    Science.gov (United States)

    As an obligate intracellular pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) may act as an “energy parasite” by importing ATP from its host’s cells. We previously demonstrated that the Las translocase NttA (gb|ACX71867.1) is functional in Escherichia coli and enables the direct import of ATP/ADP...

  18. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... release of both ATP-consuming and ATP-generating enzymes into pancreatic juice. This newly discovered richness of secreted enzymes underscores the importance of purine signaling between acini and pancreatic ducts lumen and implies regulation of the purine-converting enzymes release....

  19. Relationship between L-glutamate-regulated intracellular Na+ dynamics and ATP hydrolysis in astrocytes.

    Science.gov (United States)

    Magistretti, P J; Chatton, J-Y

    2005-01-01

    Glutamate uptake into astrocytes and the resulting increase in intracellular Na+ (Na+(i)) have been identified as a key signal coupling excitatory neuronal activity to increased glucose utilization. Arguments based mostly on mathematical modeling led to the conclusion that physiological concentrations of glutamate more than double astrocytic Na+/K+-ATPase activity, which should proportionally increase its ATP hydrolysis rate. This hypothesis was tested in the present study by fluorescence monitoring of free Mg2+ (Mg2+(i)), a parameter that inversely correlates with ATP levels. Glutamate application measurably increased Mg2+(i) (i.e. decreased ATP), which was reversible after glutamate washout. Na+(i) and ATP changes were then directly compared by simultaneous Na+(i) and Mg2+ imaging. Glutamate increased both parameters with different rates and blocking the Na+/K+-ATPase during the glutamate-evoked Na+(i) response, resulted in a drop of Mg2+(i) levels (i.e. increased ATP). Taken together, this study demonstrates the tight correlation between glutamate transport, Na+ homeostasis and ATP levels in astrocytes.

  20. Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth

    Energy Technology Data Exchange (ETDEWEB)

    Ashino, T.; Varadarajan, S.; Urao, N.; Oshikawa, J.; Chen, G. -F.; Wang, H.; Huo, Y.; Finney, L.; Vogt, S.; McKinney, R. D.; Maryon, E. B.; Kaplan, J. H.; Ushio-Fukai, M.; Fukai, T. (Biosciences Division); ( XSD); ( PSC-USR); (Univ. of Illinois at Chicago); (Univ. of Minnesota)

    2010-09-09

    Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

  1. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  2. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  3. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.

  4. Altered ATP7A expression and other compensatory responses in a murine model of Menkes disease.

    Science.gov (United States)

    Niciu, Mark J; Ma, Xin-Ming; El Meskini, Rajaâ; Pachter, Joel S; Mains, Richard E; Eipper, Betty A

    2007-09-01

    Mutations in the copper-transporter ATP7A lead to severe neurodegeneration in the mottled brindled hemizygous male (MoBr/y) mouse and human patients with Menkes disease. Our earlier studies demonstrated cell-type- and -stage-specific changes in ATP7A protein expression during postnatal neurodevelopment. Here we examined copper and cuproenzyme levels in MoBr/y mice to search for compensatory responses. While all MoBr/y neocortical subcellular fractions had decreased copper levels, the greatest decrease (8-fold) was observed in cytosol. Immunostaining for ATP7A revealed decreased levels in MoBr/y hippocampal pyramidal and cerebellar Purkinje neurons. In contrast, an upregulation of ATP7A protein occurred in MoBr/y endothelial cells, perhaps to compensate for a lack of copper in the neuropil. MoBr/y astrocytes and microglia increased their physical association with the blood-brain barrier. No alterations in ATP7A levels were observed in ependymal cells, arguing for specificity in the alteration observed at the blood-brain barrier. The decreased expression of ATP7A protein in MoBr/y Purkinje cells was associated with impaired synaptogenesis and dramatic cytoskeletal dysfunction. Immunoblotting failed to reveal any compensatory increase in levels of ATP7B. While total levels of several cuproenzymes (peptide-amidating monooxygenase, SOD1, and SOD3) were unaltered in the MoBr/y brain, levels of amidated cholecystokinin (CCK8) and amidated pituitary adenylate cyclase-activating polypeptide (PACAP38) were reduced in a tissue-specific fashion. The compensatory changes observed in the neurovascular unit provide insight into the success of copper injections within a defined neurodevelopmental period.

  5. Expression of ATP-insensitive KATP channels in pancreatic beta-cells underlies a spectrum of diabetic phenotypes.

    Science.gov (United States)

    Koster, Joseph C; Remedi, Maria S; Masia, Ricard; Patton, Brian; Tong, Ailing; Nichols, Colin G

    2006-11-01

    Glucose metabolism in pancreatic beta-cells elevates cytoplasmic [ATP]/[ADP], causing closure of ATP-sensitive K(+) channels (K(ATP) channels), Ca(2+) entry through voltage-dependent Ca(2+) channels, and insulin release. Decreased responsiveness of K(ATP) channels to the [ATP]/[ADP] ratio should lead to decreased insulin secretion and diabetes. We generated mice expressing K(ATP) channels with reduced ATP sensitivity in their beta-cells. Previously, we described a severe diabetes, with nearly complete neonatal lethality, in four lines (A-C and E) of these mice. We have now analyzed an additional three lines (D, F, and G) in which the transgene is expressed at relatively low levels. These animals survive past weaning but are glucose intolerant and can develop severe diabetes. Despite normal islet morphology and insulin content, islets from glucose-intolerant animals exhibit reduced glucose-stimulated insulin secretion. The data demonstrate that a range of phenotypes can be expected for a reduction in ATP sensitivity of beta-cell K(ATP) channels and provide models for the corollary neonatal diabetes in humans.

  6. Honing in on the ATP Release Channel in Taste Cells.

    Science.gov (United States)

    Medler, Kathryn F

    2015-09-01

    Studies over the last 8 years have identified 3 potential channels that appear to release ATP from Type II cells in response to taste stimuli. These studies have taken different methodological approaches but have all provided data supporting their candidate channel as the ATP release channel. These potential channels include Pannexin 1, Connexins (30 and/or 43), and most recently, the Calhm1 channel. Two papers in this issue of Chemical Senses provide compelling new evidence that Pannexin 1 is not the ATP release channel. Tordoff et al. did a thorough behavioral analysis of the Pannexin1 knock out mouse and found that these animals have the same behavioral responses as wild type mice for 7 different taste stimuli that were tested. Vandenbeuch et al. presented an equally thorough analysis of the gustatory nerve responses in the Pannexin1 knock out mouse and found no differences compared with controls. Thus when the role of Pannexin 1 is analyzed at the systems level, it is not required for normal taste perception. Further studies are needed to determine the role of this hemichannel in taste cells.

  7. ATP, P2X receptors and pain pathways.

    Science.gov (United States)

    Ding, Y; Cesare, P; Drew, L; Nikitaki, D; Wood, J N

    2000-07-01

    A role for ATP in nociception and pain induction was proposed on the basis of human psychophysical experiments shortly after the formulation of the purinergic hypothesis. Following the pharmacological definition of distinct P2X and P2Y purinergic receptor subtypes by Burnstock and his collaborators, molecular cloning studies have identified the gene products that underlie the effects of ATP on peripheral sensory neurons. One particular receptor, P2X(3), is of particular interest in the context of pain pathways, because it is relatively selectively expressed at high levels by nociceptive sensory neurons. Evidence that this receptor may play a role in the excitation of sensory neurons has recently been complemented by studies that suggest an additional presynaptic role in the regulation of glutamate release from primary afferent neurons in the dorsal horn of the spinal cord. In this brief review, we discuss the present state of knowledge of the role of ATP in pain induction through its action on peripheral P2X receptors.

  8. ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation.

    Science.gov (United States)

    Riteau, N; Baron, L; Villeret, B; Guillou, N; Savigny, F; Ryffel, B; Rassendren, F; Le Bert, M; Gombault, A; Couillin, I

    2012-10-11

    Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.

  9. Extracellular adenosine 5'-triphosphate and lipopolysaccharide induce interleukin-1β release in canine blood.

    Science.gov (United States)

    Spildrejorde, Mari; Curtis, Stephen J; Curtis, Belinda L; Sluyter, Ronald

    2014-01-15

    Binding of extracellular adenosine 5'-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30 min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs.

  10. Studies on luciferin-luciferase ATP assay in plants (etiolated wheat germs, and bean leaves

    Directory of Open Access Journals (Sweden)

    A. Barbaro

    2015-01-01

    Full Text Available For ATP determination by the method of bioluminescence apparatus of home production was adapted from the equipment available in any isotope laboratory. The measurement error did not exceed 1.5 per cent. Methodical experiments concerned the choice of the extraction, fixation and storage methods of plant material for determination at the given moment of the amount of ATP in the tissues, unchanged by the analytical procedure. The highest ATP amounts were recovered by extraction with perchloric acid at high (25% concentrations of the tissue in the homogenate. The best way of fixation of the material for later analyses was found to be freezing of ready extracts. Lyophilization and freezing of the plant material caused a several-fold decrease of the ATP level in the tissues. These results suggest the necessity of working in conditions of low temperature during the entire analytical procedure and strict observation of time limitation.

  11. P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells.

    Science.gov (United States)

    Eun, So Young; Ko, Young Shin; Park, Sang Won; Chang, Ki Churl; Kim, Hye Jung

    2015-07-01

    Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

  12. High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition

    Directory of Open Access Journals (Sweden)

    Schwarz Elisabeth

    2007-11-01

    Full Text Available Abstract Background Smad4 is a tumour suppressor frequently inactivated in pancreatic and colorectal cancers. We have recently reported loss of Smad4 in every fourth carcinoma of the uterine cervix. Smad4 transmits signals from the TGF-β superfamily of cytokines and functions as a versatile transcriptional co-modulator. The prevailing view suggests that the tumour suppressor function of Smad4 primarily resides in its capability to mediate TGF-β growth inhibitory responses. However, accumulating evidence indicates, that the acquisition of TGF-β resistance and loss of Smad4 may be independent events in the carcinogenic process. Through inducible reexpression of Smad4 in cervical cancer cells we wished to shed more light on this issue and to identify target genes implicated in Smad4 dependent tumor suppression. Methods Smad4-deficient human C4-II cervical carcinoma cells were used to establish inducible Smad4 reexpression using the commercial Tet-on™ system (Clontech. The impact of Smad4 reexpression on cell growth was analysed in vitro and in vivo. Transcriptional responses were assessed through profiling on cDNA macroarrays (Clontech and validated through Northern blotting. Results Clones were obtained that express Smad4 at widely varying levels from approximately physiological to 50-fold overexpression. Smad4-mediated tumour suppression in vivo was apparent at physiological expression levels as well as in Smad4 overexpressing clones. Smad4 reexpression in a dose-dependent manner was associated with transcriptional induction of the extracellular matrix-associated genes, BigH3, fibronectin and PAI-1, in response to TGF-β. Smad4-dependent regulation of these secreted Smad4 targets is not restricted to cervical carcinoma cells and was confirmed in pancreatic carcinoma cells reexpressing Smad4 after retroviral transduction and in a stable Smad4 knockdown model. On the other hand, the classical cell cycle-associated TGF-β target genes, c-myc, p

  13. Interference sources in ATP bioluminescence assay of silica nanoparticle toxicity to activated sludge.

    Science.gov (United States)

    Sibag, Mark; Kim, Seung Hwan; Kim, Choah; Kim, Hee Jun; Cho, Jinwoo

    2015-06-01

    ATP measurement provides an overview of the general state of microbial activity, and thus it has proven useful for the evaluation of nanoparticle toxicity in activated sludge. ATP bioluminescence assay, however, is susceptible to interference by the components of activated sludge other than biomass. This paper presents the interference identified specific to the use of this assay after activated sludge respiration inhibition test of silica nanoparticles (OECD 209). We observed a high degree of interference (90%) in the presence of 100 mg/L silica nanoparticles and a low level of ATP being measured (0.01 μM); and 30% interference by the synthetic medium regardless of silica nanoparticle concentration and ATP level in the samples. ATP measurement in activated sludge with different MLSS concentrations revealed interference of high biomass content. In conclusion, silica nanoparticles, synthetic medium and activated sludge samples themselves interfere with ATP bioluminescence; this will need to be considered in the evaluation of silica nanoparticle toxicity to activated sludge when this type of assay is used.

  14. The uses and abuses of rapid bioluminescence-based ATP assays.

    Science.gov (United States)

    Shama, G; Malik, D J

    2013-03-01

    Bioluminescence-based ATP testing of solid surfaces has become well established in the food processing industry as part of general hazard analysis and critical control points (HACCP) measures. The rise in healthcare associated infections (HAIs) at the turn of the century focussed attention on the environment as a potential reservoir of the agents responsible for such infections. In response to the need for objective methods of assessing the efficiency of cleaning in healthcare establishments and for rapid methods for detecting the presence of the pathogens responsible for HAIs, it was proposed that ATP testing of environmental surfaces be introduced. We examine the basis behind the assumptions inherent in these proposals. Intracellular ATP levels are shown to vary between microbial taxa and according to environmental conditions. Good correlations between microbial numbers and ATP levels have been obtained under certain specific conditions, but never within healthcare settings. Notwithstanding, ATP testing may still have a role in providing reassurance that cleaning regimes are being carried out satisfactorily. However, ATP results should not be interpreted as surrogate indicators for the presence of microbial pathogens.

  15. Gene Expression and Activity Profiling Reveal a Significant Contribution of Exo-Phosphotransferases to the Extracellular Nucleotides Metabolism in HUVEC Endothelial Cells.

    Science.gov (United States)

    Wujak, Magdalena; Hetmann, Anna; Porowińska, Dorota; Roszek, Katarzyna

    2017-06-01

    Purinergic signaling maintains local tissue homeostasis in blood vessels via the regulation of vascular tone, blood platelet aggregation, cell proliferation, and differentiation as well as inflammatory responses. Extracellular purines are important signaling molecules in the vasculature, and both purine-hydrolysing as well as -phosphorylating enzymes are considered to selectively govern extracellular nucleotide/nucleoside metabolism. Recent studies have provided some evidence for the existence of these enzymes in a soluble form in human blood and their secretion into the extracellular space under physiological and pathological conditions. However, the comprehensive analysis of endothelium-derived enzymes involved in purine metabolic pathways has received no attention so far. In the presented study, in vitro cultured human umbilical vein endothelial cells (HUVEC) are shown to be an abundant source of exo-nucleotidases comprising 5'-nucleotidase (exo-5'-NT), and nucleoside triphosphate diphosphohydrolases (exo-NTPDase) as well as phosphotransferases, represented by nucleoside diphosphate kinase (exo-NDPK) and adenylate kinase (exo-AK). An attempt is also made to demonstrate that, in contrast to the metabolic pattern determined on the endothelial cell surface, exo-phosphorylating activities markedly predominate over exo-hydrolytic ones. We present for the first time the expression profiles of genes encoding isoenzymes belonging to distinct nucleotide kinase and nucleotidase families. The genes encoding NDPK1, NDPK2, AK1, and AK2 phosphotransferases have been shown to be expressed at the highest level in HUVEC cells. The data indicate the coexistence of secreted and cell-associated factors of endothelial origin mediating ATP-consuming and ATP-generating pathways with the predominance of exo-phosphotransferases activity. The described enzymes contribute to the regulation of purinergic signal duration and extent in the venous vasculature. J. Cell. Biochem. 118: 1341

  16. Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes.

    Science.gov (United States)

    Kamidate, Tamio; Yanashita, Kenji; Tani, Hirofumi; Ishida, Akihiko; Notani, Mizuyo

    2006-01-01

    Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an ATP extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.

  17. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    Science.gov (United States)

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  18. Electrophysiological effects of ATP on brain neurones.

    Science.gov (United States)

    Illes, P; Nieber, K; Nörenberg, W

    1996-12-01

    1. The electrophysiological effects of ATP on brain neurones are either due to the direct activation of P2 purinoceptors by the unmetabolized nucleotide or to the indirect activation of P1. purinoceptors by the degradation product adenosine. 2. Two subtypes of P2 purinoceptors are involved, a ligand-activated ion channel (P2X) and a G protein-coupled receptor (P2Y). Hence, the stimulation of P2X purinoceptors leads to a cationic conductance increase, while the stimulation of P2Y purinoceptors leads to a G protein-mediated opening or closure of potassium channels. 3. ATP may induce a calcium-dependent potassium current by increasing the intracellular Ca2+ concentration. This is due either to the entry of Ca2+ via P2X purinoceptors or to the activation of metabotropic P2Y purinoceptors followed by signaling via the G protein/phospholipase C/inositol 1,4,5-trisphosphate (IP3) cascade. Eventually, IP3 releases Ca2+ from its intracellular pools. 4. There is no convincing evidence for the presence of P2U purinoceptors sensitive to both ATP and UTP, or pyrimidinoceptors sensitive to UTP only, in the central nervous system (CNS). 5. ATP-sensitive P2X and P2Y purinoceptors show a wide distribution in the CNS and appear to regulate important neuronal functions.

  19. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  20. Calcium and ATP control multiple vital functions

    Science.gov (United States)

    Verkhratsky, Alexei

    2016-01-01

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca2+ concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca2+ concentration (which in all life forms is kept around 50–100 nM) forms the basis for a universal intracellular signalling system in which Ca2+ acts as a second messenger. Maintenance of transmembrane Ca2+ gradients, in turn, requires ATP-dependent Ca2+ transport, thus further emphasizing the inseparable links between these two substances. Ca2+ signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca2+ signalling relies on cell specific Ca2+ signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca2+ signalling toolkits lead to aberrant Ca2+ signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377728

  1. S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

    Science.gov (United States)

    Módis, Katalin; Ju, YoungJun; Ahmad, Akbar; Untereiner, Ashley A; Altaany, Zaid; Wu, Lingyun; Szabo, Csaba; Wang, Rui

    2016-11-01

    levels decrease) S-sulfhydration of ATP5A1 decreased as well. In conclusion, H2S induces S-sulfhydration of ATP5A1 at C244 and C294. This post-translational modification may be a physiological mechanism to maintain ATP synthase in a physiologically activated state, thereby supporting mitochondrial bioenergetics. The sulfhydration of ATP synthase may be a dynamic process, which may be regulated by endogenous H2S levels under various pathophysiological conditions.

  2. Integrating printed microfluidics with silicon photomultipliers for miniaturised and highly sensitive ATP bioluminescence detection.

    Science.gov (United States)

    Santangelo, M F; Libertino, S; Turner, A P F; Filippini, D; Mak, W C

    2018-01-15

    Bioluminescence has been widely used for important biosensing applications such as the measurement of adenosine triphosphate (ATP), the energy unit in biological systems and an indicator of vital processes. The current technology for detection is mainly based on large equipment such as readers and imaging systems, which require intensive and time-consuming procedures. A miniaturised bioluminescence sensing system, which would allow sensitive and continuous monitoring of ATP, with an integrated and low-cost disposable microfluidic chamber for handling of biological samples, is highly desirable. Here, we report the design, fabrication and testing of 3D printed microfluidics chips coupled with silicon photomultipliers (SiPMs) for high sensitive real-time ATP detection. The 3D microfluidic chip reduces reactant consumption and facilitates solution delivery close to the SiPM to increase the detection efficiency. Our system detects ATP with a limit of detection (LoD) of 8nM and an analytical dynamic range between 15nM and 1µM, showing a stability error of 3%, and a reproducibility error below of 20%. We demonstrate the dynamic monitoring of ATP in a continuous-flow system exhibiting a fast response time, ~4s, and a full recovery to the baseline level within 17s. Moreover, the SiPM-based bioluminescence sensing system shows a similar analytical dynamic range for ATP detection to that of a full-size PerkinElmer laboratory luminescence reader. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations.

    Science.gov (United States)

    Furukawa-Hagiya, Tomoka; Furuta, Tadaomi; Chiba, Shuntaro; Sohma, Yoshiro; Sakurai, Minoru

    2013-01-10

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions.

  4. Effects and mechanism of acid rain on plant chloroplast ATP synthase.

    Science.gov (United States)

    Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2016-09-01

    Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

  5. Assessment of the role in protection and pathogenesis of the Chlamydia muridarum V-type ATP synthase subunit A (AtpA) (TC0582).

    Science.gov (United States)

    Cheng, Chunmei; Jain, Pooja; Pal, Sukumar; Tifrea, Delia; Sun, Guifeng; Teng, Andy A; Liang, Xiaowu; Felgner, Philip L; de la Maza, Luis M

    2014-02-01

    A novel Chlamydia muridarum antigen (TC0582) was used to vaccinate BALB/c mice. Mice were also immunized with other components of the ATP synthase complex (TC0580, TC0581, and TC0584), or with the major outer membrane protein (MOMP). TC0582 was also formulated in combination with TC0580, TC0581 or MOMP. TC0582 alone, or in combination with the other antigens, elicited strong Chlamydia-specific humoral and cellular immune responses. Vaccinated animals were challenged intranasally and the course of the infection was followed for 10 days. Based on percentage change in body weight, lung weight, and number of Chlamydia inclusion forming units recovered from the lungs, mice immunized with TC0582, TC0581 or MOMP, as single antigens, showed significant protection. Mice immunized with combinations of two antigens were also protected but the level of protection was not additive. TC0582 has sequence homology with the eukaryotic ATP synthase subunit A (AtpA). Therefore, to determine if immunization with TC0582, or with Chlamydia, elicited antibodies that cross-reacted with the mouse AtpA, the two proteins were printed on a microarray. Sera from mice immunized with TC0582 and/or live Chlamydia, strongly reacted with TC0582 but did not recognize the mouse AtpA. In conclusion, TC0582 may be considered as a Chlamydia vaccine candidate.

  6. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased.

    Science.gov (United States)

    Minet, Ariane D; Gaster, Michael

    2012-06-01

    The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content, but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging.

  7. Seahorse Xfe 24 Extracellular Flux Analyzer-Based Analysis of Cellular Respiration in Caenorhabditis elegans.

    Science.gov (United States)

    Luz, Anthony L; Smith, Latasha L; Rooney, John P; Meyer, Joel N

    2015-11-02

    Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans.

  8. Autocrine regulation of TGF-β1-induced cell migration by exocytosis of ATP and activation of P2 receptors in human lung cancer cells.

    Science.gov (United States)

    Takai, Erina; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Sawada, Keisuke; Moriyama, Yoshinori; Kojima, Shuji

    2012-11-01

    TGF-β1 plays a key role in cancer progression through induction of various biological effects, including cell migration. Extracellular nucleotides, such as ATP, released from cells play a role in signaling through activation of P2 receptors. We show here that exocytosis of ATP followed by activation of P2 receptors play a key role in TGF-β1-induced actin remodeling associated with cell migration. Treatment with TGF-β1 facilitated migration of human lung cancer A549 cells, which was blocked by pretreatment with ecto-nucleotidase and P2 receptor antagonists. ATP and P2 agonists facilitated cell migration. TGF-β1-induced actin remodeling, which contributes to cell migration, was also suppressed by pretreatment with ecto-nucleotidase and P2 receptor antagonists. Knockdown of P2X7 receptor suppressed TGF-β1-induced migration and actin remodeling. These results indicate the involvement of TGF-β1-induced ATP release in cell migration, at least in part, through activation of P2X7 receptors. TGF-β1 caused release of ATP from A549 cells within 10 minutes. Both ATP-enriched vesicles and expression of a vesicular nucleotide transporter (VNUT) SLC17A9, which is responsible for exocytosis of ATP, were found in cytosol of A549 cells. TGF-β1 failed to induce release of ATP from SLC17A9-knockdown cells. TGF-β1-induced cell migration and actin remodeling were also decreased in SLC17A9-knockdown cells. These results suggest the importance of exocytosis of ATP in cell migration. We conclude that autocrine signaling through exocytosis of ATP and activation of P2 receptors is required for the amplification of TGF-β1-induced migration of lung cancer cells.

  9. ATP measurements for monitoring microbial drinking water quality

    DEFF Research Database (Denmark)

    Vang, Óluva Karin

    methods are vital for an improved surveillance and distribution of clean and safe drinking water. One of these rapid methods is the ATP assay. This thesis encompasses various methodological aspects of the ATP assay describing the principal and theory of the ATP assay measurement. ATP is the main energy...... carrying molecule in living cells, thus ATP can be used as a parameter for microbial activity. ATP is extracted from cells through cell lysis and subsequently assayed with the luciferase enzyme and its substrate luciferin, resulting in bioluminescence, i.e. light emission which can be quantified....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP...

  10. Extracellular vesicles for drug delivery

    NARCIS (Netherlands)

    Vader, Pieter; Mol, Emma A; Pasterkamp, Gerard; Schiffelers, Raymond M

    Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained

  11. Calcium signals in the nucleus accumbens: Activation of astrocytes by ATP and succinate

    Directory of Open Access Journals (Sweden)

    Emri Zsuzsa

    2011-10-01

    Full Text Available Abstract Background Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc, and thereby to explore the action of citric acid cycle intermediate succinate (SUC. Results We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43 and the glial fibrillary acidic protein (GFAP indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthioadenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 μM in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. Conclusions We

  12. NS5ATP6 modulates intracellular triglyceride content through FGF21 and independently of SIRT1 and SREBP1.

    Science.gov (United States)

    Li, Zhongshu; Feng, Shenghu; Zhou, Li; Liu, Shunai; Cheng, Jun

    2016-06-17

    The prevalence of nonalcoholic fatty liver disease (NAFLD) is rising strikingly in Western countries and China. The molecular biological mechanism of NAFLD remains unclear, with no effective therapies developed so far. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone, with safe lipid lowering effects. FGF21 analogs are being developed for clinical application. Here we demonstrated that a novel gene, NS5ATP6, modulated intracellular triglyceride (TG) content independently of sirtuin1 (SIRT1) and sterol regulatory element binding protein 1 (SREBP1) in HepG2 cells. Interestingly, NS5ATP6 regulated FGF21 expression both at the mRNA and protein levels. The modulatory effects of NS5ATP6 on intracellular TG content depended upon FGF21. Further studies revealed that NS5ATP6 decreased the promoter activity of FGF21. In addition, NS5ATP6 regulated the expression of miR-577, which directly targeted and regulated FGF21. Therefore, miR-577 might be involved in NS5ATP6 regulation of FGF21 at the post-transcriptional level. In conclusion, NS5ATP6 regulates the intracellular TG level via FGF21, and independently of SIRT1 and SREBP1.

  13. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highl

  14. An ATP synthase harboring an atypical γ–subunit is involved in ATP synthesis in tomato fruit chromoplasts

    National Research Council Canada - National Science Library

    Pateraki, Irini; Renato, Marta; Azcón‐Bieto, Joaquín; Boronat, Albert

    2013-01-01

    ... and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP , and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources...

  15. Extracellular DNA: the tip of root defenses?

    Science.gov (United States)

    Hawes, Martha C; Curlango-Rivera, Gilberto; Wen, Fushi; White, Gerard J; Vanetten, Hans D; Xiong, Zhongguo

    2011-06-01

    This review discusses how extracellular DNA (exDNA) might function in plant defense, and at what level(s) of innate immunity this process might operate. A new role for extracellular factors in mammalian defense has been described in a series of studies. These studies reveal that cells including neutrophils, eosinophils, and mast cells produce 'extracellular traps' (ETs) consisting of histone-linked exDNA. When pathogens are attracted to such ETs, they are trapped and killed. When the exDNA component of ETs is degraded, trapping is impaired and resistance against invasion is reduced. Conversely, mutation of microbial genes encoding exDNases that degrade exDNA results in loss of virulence. This discovery that exDNases are virulence factors opens new avenues for disease control. In plants, exDNA is required for defense of the root tip. Innate immunity-related proteins are among a group of >100 proteins secreted from the root cap and root border cell populations. Direct tests revealed that exDNA also is rapidly synthesized and exported from the root tip. When this exDNA is degraded by the endonuclease DNase 1, root tip resistance to fungal infection is lost; when the polymeric structure is degraded more slowly, by the exonuclease BAL31, loss of resistance to fungal infection is delayed accordingly. The results suggest that root border cells may function in a manner analogous to that which occurs in mammalian cells.

  16. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  17. Yeast ADP/ATP Carrier Isoform 2

    Science.gov (United States)

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  18. Altered ATP7A expression and other compensatory responses in a murine model of Menkes disease

    OpenAIRE

    Niciu, Mark J.; Ma, Xin-Ming; Meskini, Rajaâ El; Pachter, Joel S.; Mains, Richard E.; Eipper, Betty A.

    2007-01-01

    Mutations in the copper-transporter ATP7A lead to severe neurodegeneration in the mottled brindled hemizygous male (MoBr/y) mouse and human patients with Menkes disease. Our earlier studies demonstrated cell-type and stage-specific changes in ATP7A protein expression during postnatal neurodevelopment. Here we examined copper and cuproenzyme levels in MoBr/y mice to search for compensatory responses. While all MoBr/y neocortical subcellular fractions had decreased copper levels, the greatest d...

  19. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    Science.gov (United States)

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.

  20. Activation of volume-regulated Cl− channels by ACh and ATP in Xenopus follicles

    Science.gov (United States)

    Pérez-Samartín, Alberto L; Miledi, Ricardo; Arellano, Rogelio O

    2000-01-01

    Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application.In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−.Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0.Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm.Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to −20 mV. This single-channel activity was increased by application of ACh or ATP.The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling.All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity

  1. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and

  2. Human endomembrane H sup + pump strongly resembles the ATP-synthetase of Archaebacteria

    Energy Technology Data Exchange (ETDEWEB)

    Suedhof, T.C.; Stone, D.K.; Johnston, P.A.; Xie, Xiaosong (Univ. of Texas Southwestern Medical Center, Dallas (USA)); Fried, V.A. (Saint Jude Children' s Research Hospital, Memphis, TN (USA))

    1989-08-01

    Preparations of mammalian H{sup +} pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H{sup +} pump are unknown from animal cells. The authors report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H{sup +} pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H{sup +} gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H{sup +} pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

  3. Shockwaves increase T-cell proliferation and IL-2 expression through ATP release, P2X7 receptors, and FAK activation.

    Science.gov (United States)

    Yu, Tiecheng; Junger, Wolfgang G; Yuan, Changji; Jin, An; Zhao, Yi; Zheng, Xueqing; Zeng, Yanjun; Liu, Jianguo

    2010-03-01

    Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm(2) induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  4. Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors.

    Directory of Open Access Journals (Sweden)

    Ayumi Kawano

    Full Text Available Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2, and ATP release and interleukin (IL-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors.

  5. The distribution of ATP within tomato (Lycopersicon esculentum Mill.) embryos correlates with germination whee as total ATP concentration does not

    NARCIS (Netherlands)

    Spoelstra, P.; Joosen, R.V.L.; Hilhorst, H.W.M.

    2002-01-01

    The distribution of ATP in tomato seeds was visualized by monitoring the luminescence of frozen sections on top of a gel containing all the components of the luciferase reaction, but excluding ATP. ATP was imaged in germinating tomato seeds at intervals of 3, 6, 17, 24 and 48 h and in seeds with pri

  6. The distribution of ATP within tomato (Lycopersicon esculentum Mill.) embryos correlates with germination whee as total ATP concentration does not

    NARCIS (Netherlands)

    Spoelstra, P.; Joosen, R.V.L.; Hilhorst, H.W.M.

    2002-01-01

    The distribution of ATP in tomato seeds was visualized by monitoring the luminescence of frozen sections on top of a gel containing all the components of the luciferase reaction, but excluding ATP. ATP was imaged in germinating tomato seeds at intervals of 3, 6, 17, 24 and 48 h and in seeds with

  7. In Situ ATP Bioluminescent Measurements in Subglacial Environments - The Engabreen Glacier in the Norwegian Arctic

    Science.gov (United States)

    Cullen, D. C.; Wadham, J. L.; Pancost, R.; Kelly, S.; Barnett, M. J.; Jackson, M.

    2007-12-01

    Engabreen is a northern outlet glacier from the western Svartisen ice cap on the Nordland coast of Norway just inside the Arctic Circle. A unique feature of the glacier is a man-made tunnel system within the bedrock beneath the glacier that offers scientists direct access to the glacier-bedrock interface. This unique facility - called the Engabreen Subglacial Laboratory - is ideal to test developments of new in situ analytical techniques. We have used the facility to perform the first in situ detection of microbial life in a subglacial environment using standard off-the-shelf ATP bioluminescence detection technology and therefore using ATP levels as a proxy of microbial life. Measurements were performed both in melt-waters in the tunnels and from melted ice samples directly from the glacier-bedrock interface. Levels of ATP above background were detected and appeared to be associated with suspended sediment particles rather than in the water or ice component. This indicated the presence of microbial life. Development of protocols for in situ sample processing and use of in situ ATP measurements in the directing and choice of sampling points for other techniques was explored. This study has shown that off-the-shelf portable ATP bioluminescence can be used to perform in situ measurements within sub-glacial environments but that further development work is required to optimize experimental protocols and to correlate findings with other life detection and enumeration techniques.

  8. Increased ATP generation in the host cell is required for efficient vaccinia virus production

    Directory of Open Access Journals (Sweden)

    Hsu Che-Fang

    2009-09-01

    Full Text Available Abstract To search for cellular genes up-regulated by vaccinia virus (VV infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

  9. Fluoride decreased the sperm ATP of mice through inhabiting mitochondrial respiration.

    Science.gov (United States)

    Sun, Zilong; Zhang, Wen; Xue, Xingchen; Zhang, Yuliang; Niu, Ruiyan; Li, Xuying; Li, Baojun; Wang, Xiaowen; Wang, Jundong

    2016-02-01

    Fluoride-induced low sperm motility was observed in accumulated investigations. However, the effect of fluoride exposure on ATP generation which is essential to sperm motility remains to be elucidated. In this study, 120 healthy male mice were orally administrated with 0, 25, 50, and 100 mg L(-1) NaF for 90 d. Results showed that compared with controls, fluoride ingestion significantly reduced sperm count, survival, as well as mobility and total ATP level in sperm untreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or pyruvate, which was used to establish glycolysis or mitochondrial respiration model, respectively. Data further revealed that sperm mobility and ATP level under mitochondrial respiration condition were significantly suppressed, while no statistical difference occurred in the model of glycolysis, indicating ATP derived from mitochondria was affected. Moreover, mRNA expressions of mitochondrial cytochrome b (mt-Cytb) and cytochrome c oxidase subunit 2 (mt-COX2), two important molecules in mitochondrial electron transport chain (ETC), were down-regulated in all fluoride treatment groups. Mitochondria in sperm of mice exposed to 100 mg L(-1) NaF appeared to be irregular and vacuolated. These findings suggested that decreased sperm motility induced by fluoride may result from low ATP generation due to the disturbed ETC in sperm mitochondrial. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Regulation of Synaptic Transmission by Ambient Extracellular Glutamate

    OpenAIRE

    Featherstone, David E.; Scott A. Shippy

    2007-01-01

    Many neuroscientists assume that ambient extracellular glutamate concentrations in the nervous system are biologically negligible under nonpathological conditions. This assumption is false. Hundreds of studies over several decades suggest that ambient extracellular glutamate levels in the intact mammalian brain are ~0.5 to ~5 μM. This has important implications. Glutamate receptors are desensitized by glutamate concentrations significantly lower than needed for receptor activation; 0.5 to 5 μ...

  11. Evidence that acute taurine treatment alters extracellular AMP hydrolysis and adenosine deaminase activity in zebrafish brain membranes.

    Science.gov (United States)

    Rosemberg, Denis Broock; Kist, Luiza Wilges; Etchart, Renata Jardim; Rico, Eduardo Pacheco; Langoni, Andrei Silveira; Dias, Renato Dutra; Bogo, Maurício Reis; Bonan, Carla Denise; Souza, Diogo Onofre

    2010-09-06

    Taurine is one of the most abundant free amino acids in excitable tissues. In the brain, extracellular taurine may act as an inhibitory neurotransmitter, neuromodulator, and neuroprotector. Nucleotides are ubiquitous signaling molecules that play crucial roles for brain function. The inactivation of nucleotide-mediated signaling is controlled by ectonucleotidases, which include the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. These enzymes hydrolyze ATP/GTP to adenosine/guanosine, which exert a modulatory role controlling several neurotransmitter systems. The nucleoside adenosine can be inactivated in extracellular or intracellular milieu by adenosine deaminase (ADA). In this report, we tested whether acute taurine treatment at supra-physiological concentrations alters NTPDase, ecto-5'-nucleotidase, and ADA activities in zebrafish brain. Fish were treated with 42, 150, and 400 mg L(-1) taurine for 1h, the brains were dissected and the enzyme assays were performed. Although the NTPDase activities were not altered, 150 and 400 mg L(-1) taurine increased AMP hydrolysis (128 and 153%, respectively) in zebrafish brain membranes and significantly decreased ecto-ADA activity (29 and 38%, respectively). In vitro assays demonstrated that taurine did not change AMP hydrolysis, whereas it promoted a significant decrease in ecto-ADA activity at 150 and 400 mg L(-1) (24 and 26%, respectively). Altogether, our data provide the first evidence that taurine exposure modulates the ecto-enzymes responsible for controlling extracellular adenosine levels in zebrafish brain. These findings could be relevant to evaluate potential beneficial effects promoted by acute taurine treatment in the central nervous system (CNS) of this species. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Mapping the interactions between ATP and the sarcoplasmic reticulum Ca 2 + -ATPase with ATP and ATP analogs studied by Fourier transform infrared spectroscopy

    OpenAIRE

    Liu, Man

    2004-01-01

    Die Infrarotspektroskopie in Verbindung mit photoaktivierbaren Substraten wurde zur Untersuchung von Substrat-Protein-Wechselwirkungen eingesetzt. Dabei wurden Konformationsänderungen der Ca2+-ATPase des Sarkoplasmatischen Retikulums bei Bindung des Nukleotids, der Phosphorylierung der ATPase und der Hydrolyse des Phosphoenzyms beobachtet. Verwender wurden das native Substrat ATP und seine Analoga ADP, AMPPNP, 2'-deoxyATP, 3'-deoxyATP, ITP, AMP, Pyrophosphat, Ribosetriphosphat und TNP-AMP beo...

  13. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    Science.gov (United States)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  14. Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

    Directory of Open Access Journals (Sweden)

    Deanna M Navaroli

    Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

  15. Expression of ATP6V1C1 during oral carcinogenesis.

    Science.gov (United States)

    Oliveira Alves, M G; Carta, C F L; Padín-Iruegas, M-E; Pérez-Sayáns, M; Suarez-Peñaranda, J M; Issa, J S; García-García, A; Almeida, J D

    2016-01-01

    We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.

  16. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

    Science.gov (United States)

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P; Yi, Ling; Kaler, Stephen G; Lutsenko, Svetlana; Ralle, Martina

    2016-08-05

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Ratiometric detection of adenosine triphosphate (ATP) in water and real-time monitoring of apyrase activity with a tripodal zinc complex.

    Science.gov (United States)

    Butler, Stephen J

    2014-11-24

    Two tripodal fluorescent probes Zn⋅L(1,2) have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn⋅L(1) allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of several competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.

  18. Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop.

    Science.gov (United States)

    Michalski, Kevin; Kawate, Toshimitsu

    2016-02-01

    Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.

  19. Clostridium pasteurianum F1Fo ATP Synthase: Operon, Composition, and Some Properties

    OpenAIRE

    2003-01-01

    The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ɛ), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of C. pasteurianum was low compared to what ha...

  20. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

    Directory of Open Access Journals (Sweden)

    A. V. Oberemko

    2014-12-01

    Full Text Available This review presents a generalized definition of vesicles as bilayer extracellular organelles of all celular forms of life: not only eu-, but also prokaryotic. The structure and composition of extracellular vesicles, history of research, nomenclature, their impact on life processes in health and disease are discussed. Moreover, vesicles may be useful as clinical instruments for biomarkers, and they are promising as biotechnological drug. However, many questions in this area are still unresolved and need to be addressed in the future. The most interesting from the point of view of practical health care represents a direction to study the effect of exosomes and microvesicles in the development and progression of a particular disease, the possibility of adjusting the pathological process by means of extracellular vesicles of a particular type, acting as an active ingredient. Relevant is the further elucidation of the role and importance of exosomes to the surrounding cells, tissues and organs at the molecular level, the prospects for the use of non-cellular vesicles as biomarkers of disease.

  1. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites.

    Science.gov (United States)

    Coutinho-Silva, Robson; Ojcius, David M

    2012-11-01

    Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.

  2. Metabolic requirements for neutrophil extracellular traps formation

    Science.gov (United States)

    Rodríguez-Espinosa, Oscar; Rojas-Espinosa, Oscar; Moreno-Altamirano, María Maximina Bertha; López-Villegas, Edgar Oliver; Sánchez-García, Francisco Javier

    2015-01-01

    As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 1010 to 1011 cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis. PMID:25545227

  3. Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1β Release.

    Science.gov (United States)

    Hecker, Andreas; Küllmar, Mira; Wilker, Sigrid; Richter, Katrin; Zakrzewicz, Anna; Atanasova, Srebrena; Mathes, Verena; Timm, Thomas; Lerner, Sabrina; Klein, Jochen; Kaufmann, Andreas; Bauer, Stefan; Padberg, Winfried; Kummer, Wolfgang; Janciauskiene, Sabina; Fronius, Martin; Schweda, Elke K H; Lochnit, Günter; Grau, Veronika

    2015-09-01

    IL-1β is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1β plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1β release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1β synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1β by caspase-1, and release of mature IL-1β. Mechanisms controlling IL-1β release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1β release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1β and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

  4. Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

    Science.gov (United States)

    del Puerto, Ana; Díaz-Hernández, Juan-Ignacio; Tapia, Mónica; Gomez-Villafuertes, Rosa; Benitez, María José; Zhang, Jin; Miras-Portugal, María Teresa; Wandosell, Francisco; Díaz-Hernández, Miguel; Garrido, Juan José

    2012-01-01

    In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

  5. Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.

    Science.gov (United States)

    Habermacher, Chloé; Martz, Adeline; Calimet, Nicolas; Lemoine, Damien; Peverini, Laurie; Specht, Alexandre; Cecchini, Marco; Grutter, Thomas

    2016-01-25

    P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as 'molecular tweezers' to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

  6. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

    Science.gov (United States)

    Birket, Matthew J; Orr, Adam L; Gerencser, Akos A; Madden, David T; Vitelli, Cathy; Swistowski, Andrzej; Brand, Martin D; Zeng, Xianmin

    2011-02-01

    Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1β and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

  7. Red blood cells (RBCs), epoxyeicosatrienoic acids (EETs) and adenosine triphosphate (ATP).

    Science.gov (United States)

    Jiang, Houli; Anderson, Gail D; McGiff, John C

    2010-01-01

    In addition to serving as carriers of O(2), red blood cells (RBCs) regulate vascular resistance and the distribution of microvascular perfusion by liberating adenosine triphosphate (ATP) and epoxyeicosatrienoic acids (EETs) upon exposure to a low O(2) environment. Therefore, RBCs act as sensors that respond to low pO(2) by releasing millimolar amounts of ATP, a signaling molecule, and lipid mediators (EETs). The release of EETs occurs by a mechanism that is activated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, which should greatly amplify the circulatory response to ATP. RBCs are reservoirs of EETs and the primary sources of plasma EETs, which are esterified to the phospholipids of lipoproteins. Levels of free EETs in plasma are low, about 3% of circulating EETs. RBC EETs are produced by direct oxidation of arachidonic acid (AA) esterified to glycerophospholipids and the monooxygenase-like activity of hemoglobin. On release, EETs affect vascular tone, produce profibrinolysis and dampen inflammation. A soluble epoxide hydrolase (sEH) regulates the concentrations of RBC and vascular EETs by metabolizing both cis- and trans-EETs to form dihydroxyeicosatrienoic acids (DHETs). The function and pathophysiological roles of trans-EETs and erythro-DHETs has yet to be integrated into a physiological and pathophysiological context.

  8. Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system

    Energy Technology Data Exchange (ETDEWEB)

    Ciechanover, A.; Wolin, S.L.. Steitz, J.A.; Lodish, H.F.

    1985-03-01

    Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Here the authors show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of /sup 125/I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNA/sup His/, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.

  9. Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

    Science.gov (United States)

    Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

    2016-04-01

    Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.

  10. ATP Cofactor energético

    OpenAIRE

    Granados Moreno, Jairo Enrique

    2014-01-01

    El documento explica la importancia de la adenosín trifosfato ó trifosfato de adenosina (ATP). Presenta los flujos y tipos de energía en animales. Estos conceptos son muy útiles a la hora de entender el flujo energético en la naturaleza pues permite comprender que en el paso de los compuestos por todos los procesos metabólicos, por ejemplo de la glucosa al CO2 hay unas 21 reacciones, se va liberando energía en forma de calor e incrementando la entropía, en definitiva

  11. Mammary gland copper transport is stimulated by prolactin through alterations in Ctr1 and Atp7A localization.

    Science.gov (United States)

    Kelleher, Shannon L; Lönnerdal, Bo

    2006-10-01

    Milk copper (Cu) concentration declines and directly reflects the stage of lactation. Three Cu-specific transporters (Ctr1, Atp7A, Atp7B) have been identified in the mammary gland; however, the integrated role they play in milk Cu secretion is not understood. Whereas the regulation of milk composition by the lactogenic hormone prolactin (PRL) has been documented, the specific contribution of PRL to this process is largely unknown. Using the lactating rat as a model, we determined that the normal decline in milk Cu concentration parallels declining Cu availability to the mammary gland and is associated with decreased Atp7B protein levels. Mammary gland Cu transport was highest during early lactation and was stimulated by suckling and hyperprolactinemia, which was associated with Ctr1 and Atp7A localization at the plasma membrane. Using cultured mammary epithelial cells (HC11), we demonstrated that Ctr1 stains in association with intracellular vesicles that partially colocalize with transferrin receptor (recycling endosome marker). Atp7A was primarily colocalized with mannose 6-phosphate receptor (M6PR; late endosome marker), whereas Atp7B was partially colocalized with protein disulfide isomerase (endoplasmic reticulum marker), TGN38 (trans-Golgi network marker) and M6PR. Prolactin stimulated Cu transport as a result of increased Ctr1 and Atp7A abundance at the plasma membrane. Although the molecular mechanisms responsible for these posttranslational changes are not understood, transient changes in prolactin signaling play a role in the regulation of mammary gland Cu secretion during lactation.

  12. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

    Science.gov (United States)

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

    2013-01-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  13. Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

    Science.gov (United States)

    Blodgett, David M.; De Zutter, Julie K.; Levine, Kara B.; Karim, Pusha; Carruthers, Anthony

    2007-01-01

    Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-