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Sample records for carbenoxolone

  1. Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis.

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    Teshima, Takahiro; Matsumoto, Hirotaka; Okusa, Tomoko; Nakamura, Yumi; Koyama, Hidekazu

    2015-01-01

    Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to suppressive negative feedback by glucocorticoids. The abnormal expression of 11beta-hydroxysteroid dehydrogenase (11HSD), which is a cortisol metabolic enzyme, is found in human and murine corticotroph adenomas. Our recent studies demonstrated that canine corticotroph adenomas also have abnormal expression of 11HSD. 11HSD has two isoforms in dogs, 11HSD type1 (HSD11B1), which converts cortisone into active cortisol, and 11HSD type2 (HSD11B2), which converts cortisol into inactive cortisone. It has been suggested that glucocorticoid resistance in corticotroph tumors is related to the overexpression of HSD11B2. Therefore it was our aim to investigate the effects of carbenoxolone (CBX), an 11HSD inhibitor, on the healthy dog's pituitary-adrenal axis. Dogs were administered 50 mg/kg of CBX twice each day for 15 days. During CBX administration, no adverse effects were observed in any dogs. The plasma adrenocorticotropic hormone (ACTH), and serum cortisol and cortisone concentrations were significantly lower at day 7 and 15 following corticotropin releasing hormone stimulation. After completion of CBX administration, the HSD11B1 mRNA expression was higher, and HSD11B2 mRNA expression was significantly lower in the pituitaries. Moreover, proopiomelanocortin mRNA expression was lower, and the ratio of ACTH-positive cells in the anterior pituitary was also significantly lower after CBX treatment. In adrenal glands treated with CBX, HSD11B1 and HSD11B2 mRNA expression were both lower compared to normal canine adrenal glands. The results of this study suggested that CBX inhibits ACTH secretion from pituitary due to altered 11HSD expressions, and is potentially useful for the treatment of canine Cushing's disease.

  2. Carbenoxolone as a novel therapy for attenuation of cancer-induced bone pain

    DEFF Research Database (Denmark)

    Falk, Sarah

    2018-01-01

    to elucidate the underlying mechanisms using the two specific blockers 37,43Gap27 and 43Gap26. Compared with vehicle treatment, chronic systemic administration of 20 mg/kg or 40 mg/kg carbenoxolone caused a significantly later onset and attenuation of movement-evoked and on-going pain, assessed with limb use...... and weight-bearing respectively. In addition, the carbenoxolone-treated groups demonstrated a significant delay in time to reach the humane endpoint. Acute intrathecal administration of 37,43Gap27 significantly attenuated both limb use and weight-bearing, whereas 43Gap26 had a less pronounced effect...

  3. Trauma-induced reactive gliosis is reduced after treatment with octanol and carbenoxolone.

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    Andersson, Heléne C; Anderson, Michelle F; Porritt, Michelle J; Nodin, Christina; Blomstrand, Fredrik; Nilsson, Michael

    2011-07-01

    Reactive gliosis and scar formation after brain injury can inhibit the recovery process. As many glial cells utilize gap junctions for intercellular signaling, this study investigated whether two commonly used gap junction blockers, octanol and carbenoxolone, could attenuate reactive gliosis following a minor traumatic brain injury. Octanol (710 mg/kg) or carbenoxolone (90 mg/kg) was administered 30 minutes before or after a needle track injury in adult male Sprague-Dawley rats. To mark dividing cells, animals were injected with bromodeoxyuridine (BrdU; 150 mg/kg) intraperitoneally two times per day, 8 hours apart and killed 2 days later. Immunohistochemistry for BrdU and markers for reactive glial cells [glial fibrillary acidic protein (GFAP), ED1, and NG2] were investigated using immunohistochemistry and western blot techniques. Two days after injury, increased cellular proliferation, activated astrocytes and microglia, and upregulation of NG2 expression were observed surrounding the injury site. Octanol and carbenoxolone administrated prior to injury significantly decreased cell proliferation by 60 and 70% respectively. The distance of GFAP immunoreactive astrocytes from the wound margin was decreased by 32 and 18% when octanol was administrated prior to or post injury respectively. Treatment with octanol also decreased the number of reactive microglia by 55% and, when administrated prior to injury, octanol reduced the distance of NG2 expression from the wound by 48%. The present study demonstrates that two important components of reactive gliosis, cellular activation and proliferation, can be attenuated by octanol and carbenoxolone.

  4. TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice

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    Sara Crespo Yanguas

    2018-03-01

    Full Text Available Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5′-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

  5. Protective effects of carbenoxolone, an 11β-HSD1 inhibitor, against chemical induced dry eye syndrome.

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    Na, Yoon-Ju; Choi, Kyoung-Jin; Park, Sung Bum; Sung, Hye-Rim; Jung, Won Hoon; Kim, Hee Youn; Rhee, Sang Dal; Kim, Ki Young

    2017-11-01

    Dry eye syndrome (DES) is a disorder of the eye due to tear deficiency or excessive evaporation that causes damage to the eye and is associated with discomfort and dryness. 11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is an enzyme that converts inactive cortisone to active cortisol. Recently, 11β-HSD1 has been expressed in human and rodent eyes and has been recognized as a target of glaucoma. In this study, the therapeutic effects and underlying mechanisms of topical carbenoxolone, an 11β-HSD1 inhibitor, were investigated in benzalkonium chloride (BAC)-treated human conjunctival epithelial cells and a rat DES model. In the in vitro study, carbenoxolone dose-dependently inhibited cell death and 11β-HSD1 activity in BAC-treated human conjunctival epithelial cells. For the in vivo study, carbenoxolone or a solvent was administered to the BAC-induced DES model twice daily. BAC-treated rat eyes showed significant increases in ocular surface damage, a reduction of tears, decrease corneal thickness, corneal basement membrane destruction, apoptosis in the conjunctival epithelium, and expression of pro-inflammatory cytokines (TNF-α and IL-6) and 11β-HSD1. These effects of BAC were reversed by topical carbenoxolone treatment. These results demonstrate that carbenoxolone can prevent DES by inhibiting pro-inflammatory cytokine expression and cell death of the corneal and conjunctival epithelium via inhibition of both 11β-HSD1 activity and expression in the eyes of BAC-treated rats. It is suggested that topical 11β-HSD1 inhibitors may provide a new therapeutic window in the prevention and/or treatment of DES.

  6. Carbenoxolone does not cross the blood brain barrier: an HPLC study

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    Burnham William M

    2006-01-01

    Full Text Available Abstract Background Carbenoxolone (CBX is a widely used gap junctional blocker. Considering several reports indicating that transient gap junctional blockade could be a favourable intervention following injuries to central nervous tissue, and some current enthusiasm in studies using systemic injections of CBX, it is imperative to consider the penetration of CBX into central nervous tissue after systemic administrations. So far, only very indirect evidence suggests that CBX penetrates into the central nervous system after systemic administrations. We thus determined the amounts of CBX present in the blood and the cerebrospinal fluid of rats after intraperitoneal administration, using high performance liquid chromatography Results CBX was found in the blood of the animals, up to 90 minutes post-injection. However, the cerebrospinal fluid concentration of CBX was negligible. Conclusion Thus, we conclude that, most likely, CBX does not penetrate the blood brain barrier and therefore recommend careful consideration in the manner of administration, when a central effect is desired.

  7. Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson's disease.

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    Thakur, Poonam; Nehru, Bimla

    2015-02-01

    α-Synuclein aggregation contributes to the Parkinson's disease (PD) pathology in multiple ways-the two most important being the activation of neuroinflammation and mitochondrial dysfunction. Our recent studies have shown the beneficial effects of a heat shock protein (HSP) inducer, carbenoxolone (Cbx), in reducing the aggregation of α-synuclein in a rotenone-based rat model of PD. The present study was designed to explore its ability to attenuate the α-synuclein-mediated alterations in neuroinflammation and mitochondrial functions. The PD model was generated by the rotenone administration (2 mg/kg b.wt.) to the male SD rats for a period of 5 weeks. Cbx (20 mg/kg b.wt.) co-administration was seen to reduce the activation of astrocytes incited by rotenone. Subsequently, the release of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β was inhibited. Further, the expression level of various inflammatory mediators such as COX-2, iNOS, and NF-κB was also reduced following Cbx co-treatment. Cbx was also shown to reduce the rotenone-induced decline in activity of mitochondrial complexes-I, -II, and -IV. Protection of mitochondrial functions and reduction in neuroinflammation lead to the lesser production of ROS and subsequently reduced oxidative stress. This was reflected by the increase in both the cytosolic and mitochondrial GSH levels as well as SOD activity during Cbx co-treatment. Thus, Cbx reduces the inflammatory response and improves the mitochondrial dysfunctions by reducing α-synuclein aggregation. In addition, it also reduces the associated oxidative stress. Due to its ability to target the multiple pathways implicated in the PD, Cbx can serve as a highly beneficial prophylactic agent.

  8. Carbenoxolone induced depression of rhythmogenesis in the pre-Bötzinger Complex

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    VanDam Richard J

    2008-05-01

    Full Text Available Abstract Background Carbenoxolone (CBX, a gap junction uncoupler, alters the functioning of the pre-Bötzinger Complex (preBötC, a central pattern generating neuronal network important for the production of respiratory rhythm in mammals. Even when isolated in a 1/2 mm-thick slice of medulla oblongata from neonatal mouse the preBötC continues producing periodic bursts of action potentials, termed population bursts that are thought to be important in generating various patterns of inspiration, in vivo. Whether gap junction communication contributes to preBötC rhythmogenesis remains unresolved, largely because existing gap junction uncouplers exert numerous non-specific effects (e.g., inhibition of active transport, alteration of membrane conductances. Here, we determined whether CBX alters preBötC rhythmogenesis by altering membrane properties including input resistance (Rin, voltage-gated Na+ current (INa, and/or voltage-gated K+ current (IK, rather than by blocking gap junction communication. To do so we used a medullary slice preparation, network-level recordings, whole-cell voltage clamp, and glycyrrhizic acid (GZA; a substance used as a control for CBX, since it is similar in structure and does not block gap junctions. Results Whereas neither of the control treatments [artificial cerebrospinal fluid (aCSF or GZA (50 μM] noticeably affected preBötC rhythmogenesis, CBX (50 μM decreased the frequency, area and amplitude of population bursts, eventually terminating population burst production after 45–60 min. Both CBX and GZA decreased neuronal Rin and induced an outward holding current. Although neither agent altered the steady state component of IK evoked by depolarizing voltage steps, CBX, but not GZA, increased peak INa. Conclusion The data presented herein are consistent with the notion that gap junction communication is important for preBötC rhythmogenesis. By comparing the effects of CBX and GZA on membrane properties our data a

  9. 15-PGDH inhibitors: the antiulcer effects of carbenoxolone, pioglitazone and verapamil in indomethacin induced peptic ulcer rats.

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    Moustafa, Y M; El-Azab, M F; Fouda, A

    2013-01-01

    15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the enzyme responsible for prostaglandins (PGs) metabolism. PGs have an important role in the protection of stomach mucosa against destructive stimuli. The aim of the present study is to investigate the inhibitory effect of carbenoxolone, pioglitazone and verapamil on 15-PGDH enzyme. The experiments were carried out in the Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt from May 2011 to August 2011. Adult male albino rats were fasted for 18 hours before administration of high dose of indomethacin (30 mg/kg, p.o.), except for the negative control group which received saline only, followed by pyloric ligation to induce acute gastric ulcers. The rats were pretreated orally with saline, pioglitazone (20 mg/kg), verapamil (25 mg/kg), carbenoxolone (30 mg/kg) or their combinations 30 minutes before indomethacin. The rats were sacrificed after four hours of pyloric ligation. The effects of the previous treatments on the ulcer index (Ui), the microscopic appearance of gastric mucosa, the gastric acid output, the gastric barrier mucus content, and 15-PGDH enzyme activity were determined. Indomethacin resulted in severe ulceration and increased gastric acid output (p ulcer index, gastric acid output and 15-PGDH activity (p ulcer index, gastric acid output and 15-PGDH activity (p stomach mucosa.

  10. Carbenoxolone treatment ameliorated metabolic syndrome in WNIN/Ob obese rats, but induced severe fat loss and glucose intolerance in lean rats.

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    Siva Sankara Vara Prasad Sakamuri

    Full Text Available BACKGROUND: 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1 regulates local glucocorticoid action in tissues by catalysing conversion of inactive glucocorticoids to active glucocorticoids. 11β-HSD1 inhibition ameliorates obesity and associated co-morbidities. Here, we tested the effect of 11β-HSD inhibitor, carbenoxolone (CBX on obesity and associated comorbidities in obese rats of WNIN/Ob strain, a new animal model for genetic obesity. METHODOLOGY/PRINCIPAL FINDINGS: Subcutaneous injection of CBX (50 mg/kg body weight or volume-matched vehicle was given once daily for four weeks to three month-old WNIN/Ob lean and obese rats (n = 6 for each phenotype and for each treatment. Body composition, plasma lipids and hormones were assayed. Hepatic steatosis, adipose tissue morphology, inflammation and fibrosis were also studied. Insulin resistance and glucose intolerance were determined along with tissue glycogen content. Gene expressions were determined in liver and adipose tissue. CBX significantly inhibited 11β-HSD1 activity in liver and adipose tissue of WNIN/Ob lean and obese rats. CBX significantly decreased body fat percentage, hypertriglyceridemia, hypercholesterolemia, insulin resistance in obese rats. CBX ameliorated hepatic steatosis, adipocyte hypertrophy, adipose tissue inflammation and fibrosis in obese rats. Tissue glycogen content was significantly decreased by CBX in liver and adipose tissue of obese rats. Severe fat loss and glucose- intolerance were observed in lean rats after CBX treatment. CONCLUSIONS/SIGNIFICANCE: We conclude that 11β-HSD1 inhibition by CBX decreases obesity and associated co-morbidities in WNIN/Ob obese rats. Our study supports the hypothesis that inhibition of 11β-HSD1 is a key strategy to treat metabolic syndrome. Severe fat loss and glucose -intolerance by CBX treatment in lean rats suggest that chronic 11β-HSD1 inhibition may lead to insulin resistance in normal conditions.

  11. Conduction slowing by the gap junctional uncoupler carbenoxolone

    NARCIS (Netherlands)

    de Groot, [No Value; Veenstra, T; Verkerk, AO; Wilders, R; Smits, JPP; Wilms-Schopman, FJG; Wiegerinck, RF; Bourier, J; Belterman, CNW; Coronel, R; Verheijck, EE

    2003-01-01

    Background: Cellular electrical coupling is essential for normal propagation of the cardiac action potential, whereas reduced electrical coupling is associated with arrhythrmas. Known cellular uncoupling agents have severe side effects on membrane ionic currents. We investigated the effect of

  12. Activation, Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

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    Hansen, Daniel Bloch; Ye, Zu-Cheng; Calloe, Kirstine

    2014-01-01

    overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized...

  13. Role of gap junctional coupling in astrocytic networks in the determination of global ischaemia-induced oxidative stress and hippocampal damage.

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    Perez Velazquez, Jose L; Kokarovtseva, Larisa; Sarbaziha, Raheleh; Jeyapalan, Zina; Leshchenko, Yevgen

    2006-01-01

    While there is evidence that gap junctions play important roles in the determination of cell injuries, there is not much known about mechanisms by which gap junctional communication may exert these functions. Using a global model of transient ischaemia in rats, we found that pretreatment with the gap junctional blockers carbenoxolone, 18alpha-glycyrrhetinic acid and endothelin, applied via cannulae implanted into the hippocampus in one hemisphere, resulted in decreased numbers of TUNEL-positive neurons, as compared with the contralateral hippocampus that received saline injection. Post-treatment with carbenoxolone for up to 30 min after the stroke injury still resulted in decreased cell death, but post-treatment at 90 min after the ischaemic insult did not result in differences in cell death. However, quinine, an inhibitor of Cx36-mediated gap junctional coupling, did not result in appreciable neuroprotection. Searching for a possible mechanism for the observed protective effects, possible actions of the gap junctional blockers in the electrical activity of the hippocampus during the ischaemic insult were assessed using intracerebral recordings, with no differences observed between the saline-injected and the contralateral drug-injected hippocampus. However, a significant reduction in lipid peroxides, a measure of free radical formation, in the hippocampus treated with carbenoxolone, revealed that the actions of gap junctional coupling during injuries may be causally related to oxidative stress. These observations suggest that coupling in glial networks may be functionally important in determining neuronal vulnerability to oxidative injuries.

  14. Effects of gap junction blockers on human neocortical synchronization.

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    Gigout, S; Louvel, J; Kawasaki, H; D'Antuono, M; Armand, V; Kurcewicz, I; Olivier, A; Laschet, J; Turak, B; Devaux, B; Pumain, R; Avoli, M

    2006-06-01

    Field potentials and intracellular recordings were obtained from human neocortical slices to study the role of gap junctions (GJ) in neuronal network synchronization. First, we examined the effects of GJ blockers (i.e., carbenoxolone, octanol, quinine, and quinidine) on the spontaneous synchronous events (duration = 0.2-1.1 s; intervals of occurrence = 3-27 s) generated by neocortical slices obtained from temporal lobe epileptic patients during application of 4-aminopyridine (4AP, 50 muM) and glutamatergic receptor antagonists. The synchronicity of these potentials (recorded at distances up to 5 mm) was decreased by GJ blockers within 20 min of application, while prolonged GJ blockers treatment at higher doses made them disappear with different time courses. Second, we found that slices from patients with focal cortical dysplasia (FCD) could generate in normal medium spontaneous synchronous discharges (duration = 0.4-8 s; intervals of occurrence = 0.5-90 s) that were (i) abolished by NMDA receptor antagonists and (ii) slowed down by carbenoxolone. Finally, octanol or carbenoxolone blocked 4AP-induced ictal-like discharges (duration = up to 35 s) in FCD slices. These data indicate that GJ play a role in synchronizing human neocortical networks and may implement epileptiform activity in FCD.

  15. Point correlation dimension can reveal functional changes caused by gap junction blockers in the 4-aminopyridine in vivo rat epilepsy model

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    Jardanhazy, Anett [Department of Neurology, University of Szeged, Semmelweis u. 6, Szeged H-6725 (Hungary); Molnar, Mark [Department of Psychophysiology, Institute for Psychology of the Hungarian Academy of Sciences, P.O. Box 398, Budapest H-1394 (Hungary)], E-mail: molnar@cogpsyphy.hu; Jardanhazy, Tamas [Department of Neurology, University of Szeged, Semmelweis u. 6, Szeged H-6725 (Hungary)], E-mail: jt@nepsy.szote.u-szeged.hu

    2009-04-15

    The contribution of gap junction (GJ) blockers to seizure initiation was reexamined by means of an analysis on nonlinear dynamics with point correlation dimension (PD2i) at as well as around the primary focus, and mirror focus in an already active 4-aminopyridine-induced in vivo epilepsy model. From the data base of the ECoGs of anesthetized adult rats treated with quinine, a selective blocker of Cx36, and in combination with an additional broad-spectrum GJ blocker, carbenoxolone, 14 cases of each condition were reexamined with a stationarity insensitive nonlinear PD2i method. The blockade of the Cx36 channels decreased the usual drop of the point correlation dimension at the beginning of the seizures, and this was enhanced by the additional use of the global blocker carbenoxolone. The so-called characteristic DC shift just prior to seizure onset denotes a low dimensional seizure event and the recognizable seizures display very variable, rapidly changing dynamics, as revealed by the PD2i analysis. This nonlinear PD2i analysis demonstrated that the different GJ blockers in the already active epileptic model helped seizure initiation, but exerted inhibitory effects on the seizure onset itself, acting differently on the local components of the network organization generating seizure discharges, possibly changing the coupling strengths and time delays in the GJ-s.

  16. Point correlation dimension can reveal functional changes caused by gap junction blockers in the 4-aminopyridine in vivo rat epilepsy model

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    Jardanhazy, Anett; Molnar, Mark; Jardanhazy, Tamas

    2009-01-01

    The contribution of gap junction (GJ) blockers to seizure initiation was reexamined by means of an analysis on nonlinear dynamics with point correlation dimension (PD2i) at as well as around the primary focus, and mirror focus in an already active 4-aminopyridine-induced in vivo epilepsy model. From the data base of the ECoGs of anesthetized adult rats treated with quinine, a selective blocker of Cx36, and in combination with an additional broad-spectrum GJ blocker, carbenoxolone, 14 cases of each condition were reexamined with a stationarity insensitive nonlinear PD2i method. The blockade of the Cx36 channels decreased the usual drop of the point correlation dimension at the beginning of the seizures, and this was enhanced by the additional use of the global blocker carbenoxolone. The so-called characteristic DC shift just prior to seizure onset denotes a low dimensional seizure event and the recognizable seizures display very variable, rapidly changing dynamics, as revealed by the PD2i analysis. This nonlinear PD2i analysis demonstrated that the different GJ blockers in the already active epileptic model helped seizure initiation, but exerted inhibitory effects on the seizure onset itself, acting differently on the local components of the network organization generating seizure discharges, possibly changing the coupling strengths and time delays in the GJ-s.

  17. Gap junction protein connexin43 exacerbates lung vascular permeability.

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    James J O'Donnell

    Full Text Available Increased vascular permeability causes pulmonary edema that impairs arterial oxygenation and thus contributes to morbidity and mortality associated with Acute Respiratory Distress Syndrome and sepsis. Although components of intercellular adhesive and tight junctions are critical for maintaining the endothelial barrier, there has been limited study of the roles of gap junctions and their component proteins (connexins. Since connexins can modulate inflammatory signaling in other systems, we hypothesized that connexins may also regulate pulmonary endothelial permeability. The relationships between connexins and the permeability response to inflammatory stimuli were studied in cultured human pulmonary endothelial cells. Prolonged treatment with thrombin, lipopolysaccharide, or pathological cyclic stretch increased levels of mRNA and protein for the major connexin, connexin43 (Cx43. Thrombin and lipopolysaccharide both increased intercellular communication assayed by transfer of microinjected Lucifer yellow. Although thrombin decreased transendothelial resistance in these cells, the response was attenuated by pretreatment with the connexin inhibitor carbenoxolone. Additionally, the decreases of transendothelial resistance produced by either thrombin or lipopolysaccharide were attenuated by reducing Cx43 expression by siRNA knockdown. Both carbenoxolone and Cx43 knockdown also abrogated thrombin-induced phosphorylation of myosin light chain. Taken together, these data suggest that increased lung vascular permeability induced by inflammatory conditions may be amplified via increased expression of Cx43 and intercellular communication among pulmonary endothelial cells.

  18. An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

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    Droguett, Karla; Rios, Mariana; Carreño, Daniela V; Navarrete, Camilo; Fuentes, Christian; Villalón, Manuel; Barrera, Nelson P

    2017-07-15

    Extracellular ATP, in association with [Ca 2+ ] i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca 2+ ] i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca 2+ ] i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml -1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an

  19. Role of connexin 32 hemichannels in the release of ATP from peripheral nerves.

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    Nualart-Marti, Anna; del Molino, Ezequiel Mas; Grandes, Xènia; Bahima, Laia; Martin-Satué, Mireia; Puchal, Rafel; Fasciani, Ilaria; González-Nieto, Daniel; Ziganshin, Bulat; Llobet, Artur; Barrio, Luis C; Solsona, Carles

    2013-12-01

    Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy. Copyright © 2013 Wiley Periodicals, Inc.

  20. Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes.

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    Hansen, Fernanda; Galland, Fabiana; Lirio, Franciane; de Souza, Daniela Fraga; Da Ré, Carollina; Pacheco, Rafaela Ferreira; Vizuete, Adriana Fernanda; Quincozes-Santos, André; Leite, Marina Concli; Gonçalves, Carlos-Alberto

    2017-01-01

    The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance.

  1. Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

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    Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

    2013-01-01

    Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

  2. L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

    KAUST Repository

    Jourdain, P.

    2016-02-19

    Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

  3. Active sulforhodamine 101 uptake into hippocampal astrocytes.

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    Christian Schnell

    Full Text Available Sulforhodamine 101 (SR101 is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrolateral medulla. Although carbenoxolone is able to decrease the SR101-labeling of astrocytes in the hippocampus, it is unlikely that SR101 is taken up via gap-junction hemichannels because mefloquine, a blocker for pannexin and connexin hemichannels, was unable to prevent SR101-labeling of hippocampal astrocytes. However, SR101-labeling of the hippocampal astrocytes was significantly reduced by substrates of organic anion transport polypeptides, including estron-3-sulfate and dehydroepiandrosterone sulfate, suggesting that SR101 is actively transported into hippocampal astrocytes.

  4. Connexins and M3 Muscarinic Receptors Contribute to Heterogeneous Ca2+ Signaling in Mouse Aortic Endothelium

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    François-Xavier Boittin

    2013-02-01

    Full Text Available Background/Aims: Smooth muscle tone is controlled by Ca2+ signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40 and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca2+ signaling of the mouse aorta. Methods: Ca2+ imaging was performed on intact aortic endothelium from both wild type (Cx40+/+ and Connexin40-deficient (Cx40 -/- mice. Results: Acetylcholine (ACh induced early fast and high amplitude Ca2+ transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca2+ transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca2+ waves, indicating that Cx40 contributes to the spreading of Ca2+ signals. The propagation of those Ca2+ responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca2+ waves. Conclusions: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca2+ signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

  5. Electrical coupling between hippocampal astrocytes in rat brain slices.

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    Meme, William; Vandecasteele, Marie; Giaume, Christian; Venance, Laurent

    2009-04-01

    Gap junctions in astrocytes play a crucial role in intercellular communication by supporting both biochemical and electrical coupling between adjacent cells. Despite the critical role of electrical coupling in the network organization of these glial cells, the electrophysiological properties of gap junctions have been characterized in cultures while no direct evidence has been sought in situ. In the present study, gap-junctional currents were investigated using simultaneous dual whole-cell patch-clamp recordings between astrocytes from rat hippocampal slices. Bidirectional electrotonic coupling was observed in 82% of the cell pairs with an average coupling coefficient of 5.1%. Double patch-clamp analysis indicated that junctional currents were independent of the transjunctional voltage over a range from -100 to +110 mV. Interestingly, astrocytic electrical coupling displayed weak low-pass filtering properties compared to neuronal electrical synapses. Finally, during uncoupling processes triggered by either the gap-junction inhibitor carbenoxolone or endothelin-1, an increase in the input resistance in the injected cell paralleled the decrease in the coupling coefficient. Altogether, these results demonstrate that hippocampal astrocytes are electrically coupled through gap-junction channels characterized by properties that are distinct from those of electrical synapses between neurons. In addition, gap-junctional communication is efficiently regulated by endogenous compounds. This is taken to represent a mode of communication that may have important implications for the functional role of astrocyte networks in situ.

  6. Mechanism of Mitochondrial Connexin43′s Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury

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    Shuai Hou

    2016-05-01

    Full Text Available We observed mitochondrial connexin43 (mtCx43 expression under cerebral ischemia-reperfusion (I/R injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO. Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD activity and malondialdehyde (MDA content. MtCx43, p-mtCx43, protein kinase C (PKC, and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX and diazoxide (DZX groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists.

  7. Spondias purpurea L. (Anacardiaceae: Antioxidant and Antiulcer Activities of the Leaf Hexane Extract

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    Cynthia Layse Ferreira de Almeida

    2017-01-01

    Full Text Available Spondias purpurea is used in folk medicine to treat diarrhea and diuresis. The objective of this study was to evaluate the phytochemical profile and antioxidant and antiulcer activities of the hexane extract of the leaves of S. purpurea (SpHE. Phytochemical profile was evaluated via thin layer chromatography (TLC and HPLC. SpHE was screened for antioxidant activities using DPPH, ABTS, FRAP, and phosphomolybdenum assays. To determine its antiulcer properties, animals were pretreated with injured control, lansoprazole, ranitidine, carbenoxolone, or SpHE (12.5, 25, and 50 mg/kg and were screened; acute ulcers were induced by HCl/ethanol, absolute ethanol, and nonsteroidal anti-inflammatory drug (NSAID. TLC revealed the presence of flavonoids, whereas HPLC analysis showed the presence of caffeic acid and epigallocatechin. The phenolic compounds and in vitro assays showed antioxidant activity. After gastric ulcer induction by using HCl/ethanol, SpHE reduced the area of ulcerative lesions by 82, 91, and 88%, respectively. In ethanol, SpHE reduced the area of ulcerative lesions by 77, 93, and 92%, respectively. In the NSAID, the percentages of protection were 70, 76, and 78%, respectively. SpHE promoted the minimization of ulcers, increased the levels of reduced glutathione, and decreased tumor necrosis factor. S. purpurea has antioxidant and antiulcer properties.

  8. L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

    KAUST Repository

    Jourdain, P.; Allaman, I.; Rothenfusser, K.; Fiumelli, Hubert; Marquet, P.; Magistretti, Pierre J.

    2016-01-01

    Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

  9. Gap junctions and inhibitory synapses modulate inspiratory motoneuron synchronization.

    Science.gov (United States)

    Bou-Flores, C; Berger, A J

    2001-04-01

    Interneuronal electrical coupling via gap junctions and chemical synaptic inhibitory transmission are known to have roles in the generation and synchronization of activity in neuronal networks. Uncertainty exists regarding the roles of these two modes of interneuronal communication in the central respiratory rhythm-generating system. To assess their roles, we performed studies on both the neonatal mouse medullary slice and en bloc brain stem-spinal cord preparations where rhythmic inspiratory motor activity can readily be recorded from both hypoglossal and phrenic nerve roots. The rhythmic inspiratory activity observed had two temporal characteristics: the basic respiratory frequency occurring on a long time scale and the synchronous neuronal discharge within the inspiratory burst occurring on a short time scale. In both preparations, we observed that bath application of gap-junction blockers, including 18 alpha-glycyrrhetinic acid, 18 beta-glycyrrhetinic acid, and carbenoxolone, all caused a reduction in respiratory frequency. In contrast, peak integrated phrenic and hypoglossal inspiratory activity was not significantly changed by gap-junction blockade. On a short-time-scale, gap-junction blockade increased the degree of synchronization within an inspiratory burst observed in both nerves. In contrast, opposite results were observed with blockade of GABA(A) and glycine receptors. We found that respiratory frequency increased with receptor blockade, and simultaneous blockade of both receptors consistently resulted in a reduction in short-time-scale synchronized activity observed in phrenic and hypoglossal inspiratory bursts. These results support the concept that the central respiratory system has two components: a rhythm generator responsible for the production of respiratory cycle timing and an inspiratory pattern generator that is involved in short-time-scale synchronization. In the neonatal rodent, properties of both components can be regulated by interneuronal

  10. Expression and roles of pannexins in ATP release in the pituitary gland.

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    Li, Shuo; Bjelobaba, Ivana; Yan, Zonghe; Kucka, Marek; Tomic, Melanija; Stojilkovic, Stanko S

    2011-06-01

    Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland.

  11. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Hui [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Zhuo, Liling [College of Life Science, Zaozhuang University, Zaozhuang, Shandong, 277160 (China); Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China)

    2015-04-17

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

  12. Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

    Science.gov (United States)

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Mel'nikov, P A; Cherepanov, S A; Levinsky, A B; Chehonin, V P

    2016-02-01

    The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

  13. Neuroinflammatory contributions to pain after SCI: roles for central glial mechanisms and nociceptor-mediated host defense.

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    Walters, Edgar T

    2014-08-01

    Neuropathic pain after spinal cord injury (SCI) is common, often intractable, and can be severely debilitating. A number of mechanisms have been proposed for this pain, which are discussed briefly, along with methods for revealing SCI pain in animal models, such as the recently applied conditioned place preference test. During the last decade, studies of animal models have shown that both central neuroinflammation and behavioral hypersensitivity (indirect reflex measures of pain) persist chronically after SCI. Interventions that reduce neuroinflammation have been found to ameliorate pain-related behavior, such as treatment with agents that inhibit the activation states of microglia and/or astroglia (including IL-10, minocycline, etanercept, propentofylline, ibudilast, licofelone, SP600125, carbenoxolone). Reversal of pain-related behavior has also been shown with disruption by an inhibitor (CR8) and/or genetic deletion of cell cycle-related proteins, deletion of a truncated receptor (trkB.T1) for brain-derived neurotrophic factor (BDNF), or reduction by antisense knockdown or an inhibitor (AMG9810) of the activity of channels (TRPV1 or Nav1.8) important for electrical activity in primary nociceptors. Nociceptor activity is known to drive central neuroinflammation in peripheral injury models, and nociceptors appear to be an integral component of host defense. Thus, emerging results suggest that spinal and systemic effects of SCI can activate nociceptor-mediated host defense responses that interact via neuroinflammatory signaling with complex central consequences of SCI to drive chronic pain. This broader view of SCI-induced neuroinflammation suggests new targets, and additional complications, for efforts to develop effective treatments for neuropathic SCI pain. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Dual functional extracellular recording using a light-addressable potentiometric sensor for bitter signal transduction.

    Science.gov (United States)

    Du, Liping; Wang, Jian; Chen, Wei; Zhao, Luhang; Wu, Chunsheng; Wang, Ping

    2018-08-31

    This paper presents a dual functional extracellular recording biosensor based on a light-addressable potentiometric sensor (LAPS). The design and fabrication of this biosensor make it possible to record both extracellular membrane potential changes and ATP release from a single taste bud cell for the first time. For detecting ATP release, LAPS chip was functionalized with ATP-sensitive DNA aptamer by covalent immobilization. Taste bud cells isolated from rat were cultured on LAPS surface. When the desired single taste bud cell was illuminated by modulated light, ATP release from single taste bud cells can be measured by recording the shifts of bias voltage-photocurrent curves (I-V curves) when the LAPS chip is working in discrete mode. On the other hand, extracellular membrane potential changes can be monitored by recording the fluctuation of LAPS photocurrent when the LAPS chip is working in continuous mode. The results show this biosensor can effectively record the enhancive effect of the bitter substance and inhibitory effect of the carbenoxolone (CBX) on the extracellular membrane potential changes and ATP release of single taste bud cells. In addition, the inhibitory effect of CBX also confirms LAPS extracellular recordings are originated from bitter signal transduction. It is proved this biosensor is suitable for extracellular recording of ATP release and membrane potential changes of single taste bud cells. It is suggested this biosensor could be applied to investigating taste signal transduction at the single-cell level as well as applied to other types of cells which have similar functions to taste bud cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Pannexin 1 channels: new actors in the regulation of catecholamine release from adrenal chromaffin cells

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    Fanny eMomboisse

    2014-09-01

    Full Text Available Chromaffin cells of the adrenal gland medulla synthesize and store hormones and peptides, which are released into the blood circulation in response to stress. Among them, adrenaline is critical for the fight-or-flight response. This neurosecretory process is highly regulated and depends on cytosolic [Ca2+]. By forming channels at the plasma membrane, pannexin-1 (Panx1 is a protein involved in many physiological and pathological processes amplifying ATP release and/or Ca2+ signals. Here, we show that Panx1 is expressed in the adrenal gland where it plays a role by regulating the release of catecholamines. In fact, inhibitors of Panx1 channels, such as carbenoxolone (Cbx and probenecid, reduced the secretory activity induced with the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 50 µM in whole adrenal glands. A similar inhibitory effect was observed in single chromaffin cells using Cbx or 10Panx1 peptide, another Panx1 channel inhibitors. Given that the secretory response depends on cytosolic [Ca2+] and Panx1 channels are permeable to Ca2+, we studied the possible implication of Panx1 channels in the Ca2+ signaling occurring during the secretory process. In support of this possibility, Panx1 channel inhibitors significantly reduced the Ca2+ signals evoked by DMPP in single chromaffin cells. However, the Ca2+ signals induced by caffeine in the absence of extracellular Ca2+ was not affected by Panx1 channel inhibitors, suggesting that this mechanism does not involve Ca2+ release from the endoplasmic reticulum. Conversely, Panx1 inhibitors significantly blocked the DMPP-induce dye uptake, supporting the idea that Panx1 forms functional channels at the plasma membrane. These findings indicate that Panx1 channels participate in the control the Ca2+ signal that triggers the secretory response of adrenal chromaffin cells. This mechanism could have physiological implications during the response to stress.

  16. Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling.

    Science.gov (United States)

    Begandt, Daniela; Bader, Almke; Antonopoulos, Georgios C; Schomaker, Markus; Kalies, Stefan; Meyer, Heiko; Ripken, Tammo; Ngezahayo, Anaclet

    2015-10-01

    The present report evaluates the advantages of using the gold nanoparticle-mediated laser perforation (GNOME LP) technique as a computer-controlled cell optoperforation to introduce Lucifer yellow (LY) into cells in order to analyze the gap junction coupling in cell monolayers. To permeabilize GM-7373 endothelial cells grown in a 24 multiwell plate with GNOME LP, a laser beam of 88 μm in diameter was applied in the presence of gold nanoparticles and LY. After 10 min to allow dye uptake and diffusion through gap junctions, we observed a LY-positive cell band of 179 ± 8 μm width. The presence of the gap junction channel blocker carbenoxolone during the optoperforation reduced the LY-positive band to 95 ± 6 μm. Additionally, a forskolin-related enhancement of gap junction coupling, recently found using the scrape loading technique, was also observed using GNOME LP. Further, an automatic cell imaging and a subsequent semi-automatic quantification of the images using a java-based ImageJ-plugin were performed in a high-throughput sequence. Moreover, the GNOME LP was used on cells such as RBE4 rat brain endothelial cells, which cannot be mechanically scraped as well as on three-dimensionally cultivated cells, opening the possibility to implement the GNOME LP technique for analysis of gap junction coupling in tissues. We conclude that the GNOME LP technique allows a high-throughput automated analysis of gap junction coupling in cells. Moreover this non-invasive technique could be used on monolayers that do not support mechanical scraping as well as on cells in tissue allowing an in vivo/ex vivo analysis of gap junction coupling.

  17. 1,2,3,4,6 Penta-O-galloyl-β-d-glucose, a bioactivity guided isolated compound from Mangifera indica inhibits 11β-HSD-1 and ameliorates high fat diet-induced diabetes in C57BL/6 mice.

    Science.gov (United States)

    Mohan, C G; Viswanatha, G L; Savinay, G; Rajendra, C E; Halemani, Praveen D

    2013-03-15

    Methanolic leaf extract of Mangifera indica (MEMI) was subjected to bioactivity guided fractionation in order to identify the active antidiabetic constituent. 32 fractions were evaluated for possible 11β-HSD-1 inhibition activity under in vitro conditions. The EA-7/8-9/10-4 fraction was evolved as a most potent fraction among all the fractions and it was identified as well known gallotannin compound 1,2,3,4,6 penta-O-galloyl-β-d-glucose (PGG) by spectral analysis. Based on these results the PGG was further evaluated in ex vivo 11β-HSD-1 inhibition assay and high fat diet (HFD)-induced diabetes in male C57BL/6 mice. Single dose (10, 25, 50 and 100mg/kg) of PGG and carbenoxolone (CBX) have dose dependently inhibited the 11β-HSD-1 activity in liver and adipose tissue. Furthermore, HFD appraisal to male C57BL/6 mice caused severe hyperglycemia, hypertriglyceridemia, elevated levels of plasma corticosterone and insulin, increased liver and white adipose mass with increase in body weight was observed compare to normal control. Also, oral glucose tolerance was significantly impaired compare to normal control. Interestingly, post-treatment with PGG for 21 days had alleviated the HFD-induced biochemical alterations and improved oral glucose tolerance compare to HFD-control. In conclusion, the PGG isolated from MEMI inhibits 11β-HSD-1 activity and ameliorates HFD-induced diabetes in male C57BL/6 mice. Copyright © 2013 Elsevier GmbH. All rights reserved.

  18. Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

    Science.gov (United States)

    Yamamoto, Y.; Yoshizaki, G.; Takeuchi, T.; Soyano, K.; Patino, R.

    2008-01-01

    Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18??-glycyrrhetinic acid (??-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20??-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption. ?? 2007 Elsevier Inc. All rights reserved.

  19. Thalamic gap junctions control local neuronal synchrony and influence macroscopic oscillation amplitude during EEG alpha rhythms

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    Stuart eHughes

    2011-08-01

    Full Text Available Although EEG alpha ( (8-13 Hz rhythms are often considered to reflect an ‘idling’ brain state, numerous studies indicate that they are also related to many aspects of perception. Recently, we outlined a potential cellular substrate by which such aspects of perception might be linked to basic  rhythm mechanisms. This scheme relies on a specialized subset of rhythmically bursting thalamocortical (TC neurons (high-threshold bursting cells in the lateral geniculate nucleus (LGN which are interconnected by gap junctions (GJs. By engaging GABAergic interneurons, that in turn inhibit conventional relay-mode TC neurons, these cells can lead to an effective temporal framing of thalamic relay-mode output. Although the role of GJs is pivotal in this scheme, evidence for their involvement in thalamic  rhythms has thus far mainly derived from experiments in in vitro slice preparations. In addition, direct anatomical evidence of neuronal GJs in the LGN is currently lacking. To address the first of these issues we tested the effects of the GJ inhibitors, carbenoxolone (CBX and 18-glycyrrhetinic acid (18-GA, given directly to the LGN via reverse microdialysis, on spontaneous LGN and EEG  rhythms in behaving cats. We also examined the effect of CBX on  rhythm-related LGN unit activity. Indicative of a role for thalamic GJs in these activities, 18-GA and CBX reversibly suppressed both LGN and EEG  rhythms, with CBX also decreasing neuronal synchrony. To address the second point, we used electron microscopy to obtain definitive ultrastructural evidence for the presence of GJs between neurons in the cat LGN. As interneurons show no phenotypic evidence of GJ coupling (i.e. dye-coupling and spikelets we conclude that these GJs must belong to TC neurons. The potential significance of these findings for relating macroscopic changes in  rhythms to basic cellular processes is discussed.

  20. Contribution of microglia and astrocytes to the central sensitization, inflammatory and neuropathic pain in the juvenile rat

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    Ikeda Hiroshi

    2012-06-01

    Full Text Available Abstract Background The development of pain after peripheral nerve and tissue injury involves not only neuronal pathways but also immune cells and glia. Central sensitization is thought to be a mechanism for such persistent pain, and ATP involves in the process. We examined the contribution of glia to neuronal excitation in the juvenile rat spinal dorsal horn which is subjected to neuropathic and inflammatory pain. Results In rats subjected to neuropathic pain, immunoreactivity for the microglial marker OX42 was markedly increased. In contrast, in rats subjected to inflammatory pain, immunoreactivity for the astrocyte marker glial fibrillary acidic protein was increased slightly. Optically-recorded neuronal excitation induced by single-pulse stimulation to the dorsal root was augmented in rats subjected to neuropathic and inflammatory pain compared to control rats. The bath application of a glial inhibitor minocycline and a p38 mitogen-activated protein kinase inhibitor SB203580 inhibited the neuronal excitation in rats subjected to neuropathic pain. A specific P2X1,2,3,4 antagonist TNP-ATP largely inhibited the neuronal excitation only in rats subjected to neuropathic pain rats. In contrast, an astroglial toxin L-alpha-aminoadipate, a gap junction blocker carbenoxolone and c-Jun N-terminal kinase inhibitor SP600125 inhibited the neuronal excitation only in rats subjected to inflammatory pain. A greater number of cells in spinal cord slices from rats subjected to neuropathic pain showed Ca2+ signaling in response to puff application of ATP. This Ca2+ signaling was inhibited by minocycline and TNP-ATP. Conclusions These results directly support the notion that microglia is more involved in neuropathic pain and astrocyte in inflammatory pain.

  1. Gap junctions and hydrogen peroxide are involved in endothelium-derived hyperpolarising responses to bradykinin in omental arteries and veins isolated from pregnant women.

    Science.gov (United States)

    Hammond, Stephanie; Mathewson, Alastair M; Baker, Philip N; Mayhew, Terry M; Dunn, William R

    2011-10-01

    Altered endothelial function may underlie human cardiovascular diseases, including hypertension, diabetes and pre-eclampsia. While much is known about endothelial function in small arteries, very little is known about endothelial responses in small veins isolated from humans. Therefore, we assessed endothelium-dependent responses in omental arteries and veins isolated from healthy pregnant women, focussing on endothelium-dependent hyperpolarising (EDH) mechanisms. Human omental arteries and veins were obtained from women undergoing elective caesarean sections and examined using pressure myography. In pressurised vessels, the effects of proposed inhibitors of EDH production/function were examined on responses to bradykinin. The expression of connexins Cx37, 40 and 43 was assessed using immunohistochemistry. Bradykinin caused vasodilatation in human pressurised omental arteries and veins. In both vessels, responses to bradykinin were partially blocked in the presence of the gap junction uncoupler, carbenoxolone, and reduced further with the addition of catalase, which acts to degrade H(2)O(2). The effect of catalase alone was more pronounced in venous preparations. All three connexins were expressed in both arteries and veins, with a similar distribution pattern, where Cx37 and Cx40 were located mainly in the endothelium and Cx43 located mostly in the media. These data show that, in human omental vessels, an EDH mechanism is produced in response to bradykinin that involves gap junction communication and the production of H(2)O(2). These mechanisms may be involved in the haemodynamic alterations that take place during pregnancy, and any aberration in their function could contribute to raised blood pressure in hypertensive disorders of pregnancy, such as pre-eclampsia. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Localization of connexin 43 gap junctions and hemichannels in tanycytes of adult mice.

    Science.gov (United States)

    Szilvásy-Szabó, Anett; Varga, Edina; Beliczai, Zsuzsa; Lechan, Ronald M; Fekete, Csaba

    2017-10-15

    Tanycytes are specialized glial cells lining the lateral walls and the floor of the third ventricle behind the optic chiasm. In addition to functioning as barrier cells, they also have an important role in the regulation of neuroendocrine axes and energy homeostasis. To determine whether tanycytes communicate with each other via Connexin 43 (Cx43) gap junctions, individual tanycytes were loaded with Lucifer yellow (LY) through a patch pipette. In all cases, LY filled a larger group of tanycytes as well as blood vessels adjacent to tanycyte processes. The Cx43-blocker, carbenoxolone, inhibited spreading of LY. The greatest density of Cx43-immunoreactive spots was observed in the cell membrane of α-tanycyte cell bodies. Cx43-immunoreactivity was also present in the membrane of β-tanycyte cell bodies, but in lower density. Processes of both types of tanycytes also contained Cx43-immunoreactivity. At the ultrastructural level, Cx43-immunoreactivity was present in the cell membrane of all types of tanycytes including their ventricular surface, but gap junctions were more frequent among α-tanycytes. Cx43-immunoreactivity was also observed in the cell membrane between contacting tanycyte endfeet processes, and between tanycyte endfeet process and axon varicosities in the external zone of the median eminence and capillaries in the arcuate nucleus and median eminence. These results suggest that gap junctions are present not only among tanycytes, but also between tanycytes and the axons of hypophysiotropic neurons. Cx43 hemichannels may also facilitate the transport between tanycytes and extracellular fluids, including the cerebrospinal fluid, extracellular space of the median eminence and bloodstream. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Astrocyte sigma-1 receptors modulate connexin 43 expression leading to the induction of below-level mechanical allodynia in spinal cord injured mice.

    Science.gov (United States)

    Choi, Sheu-Ran; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kwon, Soon-Gu; Choi, Hoon-Seong; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2016-12-01

    We have previously shown using a spinal cord injury (SCI) model that gap junctions contribute to the early spread of astrocyte activation in the lumbar spinal cord and that this astrocyte communication plays critical role in the induction of central neuropathic pain. Sigma-1 receptors (Sig-1Rs) have been implicated in spinal astrocyte activation and the development of peripheral neuropathic pain, yet their contribution to central neuropathic pain remains unknown. Thus, we investigated whether SCI upregulates spinal Sig-1Rs, which in turn increase the expression of the astrocytic gap junction protein, connexin 43 (Cx43) leading to the induction of central neuropathic pain. A thoracic spinal cord hemisection significantly increased both astrocyte activation and Cx43 expression in lumbar dorsal horn. Sig-1Rs were also increased in lumbar dorsal horn astrocytes, but not neurons or microglia. Intrathecal injection of an astrocyte metabolic inhibitor (fluorocitrate); a gap junction/hemichannel blocker (carbenoxolone); or a Cx43 mimetic peptide ( 43 Gap26) significantly reduced SCI-induced bilateral below-level mechanical allodynia. Blockade of Sig-1Rs with BD1047 during the induction phase of pain significantly suppressed the SCI-induced development of mechanical allodynia, astrocyte activation, increased expression of Cx43 in both total and membrane levels, and increased association of Cx43 with Sig-1R. However, SCI did not change the expression of oligodendrocyte (Cx32) or neuronal (Cx36) gap junction proteins. These findings demonstrate that SCI activates astrocyte Sig-1Rs leading to increases in the expression of the gap junction protein, Cx43 and astrocyte activation in the lumbar dorsal horn, and ultimately contribute to the induction of bilateral below-level mechanical allodynia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity.

    Science.gov (United States)

    Karpuk, Nikolay; Burkovetskaya, Maria; Fritz, Teresa; Angle, Amanda; Kielian, Tammy

    2011-01-12

    Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

  5. Endoplasmic reticulum stress is increased in adipose tissue of women with gestational diabetes.

    Directory of Open Access Journals (Sweden)

    Stella Liong

    Full Text Available Maternal obesity and gestational diabetes mellitus (GDM are two increasingly common and important obstetric complications that are associated with severe long-term health risks to mothers and babies. IL-1β, which is increased in obese and GDM pregnancies, plays an important role in the pathophysiology of these two pregnancy complications. In non-pregnant tissues, endoplasmic (ER stress is increased in diabetes and can induce IL-1β via inflammasome activation. The aim of this study was to determine whether ER stress is increased in omental adipose tissue of women with GDM, and if ER stress can also upregulate inflammasome-dependent secretion of IL-1β. ER stress markers IRE1α, GRP78 and XBP-1s were significantly increased in adipose tissue of obese compared to lean pregnant women. ER stress was also increased in adipose tissue of women with GDM compared to BMI-matched normal glucose tolerant (NGT women. Thapsigargin, an ER stress activator, induced upregulated secretion of mature IL-1α and IL-1β in human omental adipose tissue explants primed with bacterial endotoxin LPS, the viral dsRNA analogue poly(I:C or the pro-inflammatory cytokine TNF-α. Inhibition of capase-1 with Ac-YVAD-CHO resulted in decreased IL-1α and IL-1β secretion, whereas inhibition of pannexin-1 with carbenoxolone suppressed IL-1β secretion only. Treatment with anti-diabetic drugs metformin and glibenclamide also reduced IL-1α and IL-1β secretion in infection and cytokine-primed adipose tissue. In conclusion, this study has demonstrated ER stress to activate the inflammasome in pregnant adipose tissue. Therefore, increased ER stress may contribute towards the pathophysiology of obesity in pregnancy and GDM.

  6. Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling.

    Science.gov (United States)

    Lavall, Daniel; Schuster, Pia; Jacobs, Nadine; Kazakov, Andrey; Böhm, Michael; Laufs, Ulrich

    2017-05-05

    The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling. © 2017 by The American Society for

  7. Cellular uptake of lead in the blood-cerebrospinal fluid barrier: Novel roles of Connexin 43 hemichannel and its down-regulations via Erk phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Song, Han; Zheng, Gang; Liu, Yang; Shen, Xue-Feng; Zhao, Zai-Hua [Department of Occupational and Environmental Health and the Ministry-of-Education' s Key Laboratory of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Aschner, Michael [Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Luo, Wen-Jing, E-mail: luowenj@fmmu.edu.cn [Department of Occupational and Environmental Health and the Ministry-of-Education' s Key Laboratory of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Chen, Jing-Yuan, E-mail: jy_chen@fmmu.edu.cn [Department of Occupational and Environmental Health and the Ministry-of-Education' s Key Laboratory of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China)

    2016-04-15

    As the structural basis of blood-cerebrospinal fluid barrier (BCB), epithelial cells in the choroid plexus (CP) are targets for lead (Pb). Pb is known to accumulate in the CP; however, the mechanism of Pb uptake in the choroidal epithelial cells remains unknown. Recently, hemichannels of Connexin 43 (Cx43), the most ubiquitously expressed gap junction proteins in the CP, were found to be important pathways for many substances. This study was designed to investigate the roles of Cx43 in Pb uptake in the epithelial cells. Autometallography was used to outline Pb's subcellular location, and the characteristics of Pb transport into CP cells, including concentration- and time-dependence were analyzed by atomic absorption spectroscopy. Knockdown/overexpression of Cx43 with transient siRNA/plasmids transfections before Pb exposure diminished/increased the Pb accumulation. In the Z310 cell-based doxycycline-inducible Cx43 expression cell line (iZCx43), doxycycline induced a significant increase (3-fold) in Pb uptake, corresponding to the increased Cx43 levels. Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations. Cx43-induced Pb uptake was attenuated after blockage of Cx43 hemichannels with its inhibitor, carbenoxolone. Additionally, down-regulation of Cx43 protein levels by Pb exposure paralleled cellular Pb concentrations in the time study. Concomitantly, expressions of phosphor-Src and phosphor-Erk were both significantly increased by Pb. However, inactivation of Erk, not Src pathway, reversed Pb-induced downregulation of Cx43. Taken together, these data establish that Pb can accumulate in the BCB and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Erk phosphorylation. - Highlights: • Pb is sequestrated in choroid plexus both in vivo and in vitro. • Cx43 knockdown/overexpression prevents/increases Pb accumulations. • Cx43 hemichannels are required for Pb uptake. • Pb-induced Erk

  8. Pre-weaning growth hormone treatment reverses hypertension and endothelial dysfunction in adult male offspring of mothers undernourished during pregnancy.

    Directory of Open Access Journals (Sweden)

    Clint Gray

    Full Text Available Maternal undernutrition results in elevated blood pressure (BP and endothelial dysfunction in adult offspring. However, few studies have investigated interventions during early life to ameliorate the programming of hypertension and vascular disorders. We have utilised a model of maternal undernutrition to examine the effects of pre-weaning growth hormone (GH treatment on BP and vascular function in adulthood. Female Sprague-Dawley rats were fed either a standard control diet (CON or 50% of CON intake throughout pregnancy (UN. From neonatal day 3 until weaning (day 21, CON and UN pups received either saline (CON-S, UN-S or GH (2.5 ug/g/day(CON-GH, UN-GH. All dams were fed ad libitum throughout lactation. Male offspring were fed a standard diet until the end of the study. Systolic blood pressure (SBP was measured at day 150 by tail cuff plethysmography. At day 160, intact mesenteric vessels mounted on a pressure myograph. Responses to pressure, agonist-induced constriction and endothelium-dependent vasodilators were investigated to determine vascular function. SBP was increased in UN-S groups and normalised in UN-GH groups (CON-S 121±2 mmHg, CON-GH 115±3, UN-S 146±3, UN-GH 127±2. Pressure mediated dilation was reduced in UN-S offspring and normalised in UN-GH groups. Vessels from UN-S offspring demonstrated a reduced constrictor response to phenylephrine and reduced vasodilator response to acetylcholine (ACh. Furthermore, UN-S offspring vessels displayed a reduced vasodilator response in the presence of L-NG-Nitroarginine Methyl Ester (L-NAME, carbenoxolone (CBX, L-NAME and CBX, Tram-34 and Apamin. UN-GH vessels showed little difference in responses when compared to CON and significantly increased vasodilator responses when compared to UN-S offspring. Pre-weaning GH treatment reverses the negative effects of maternal UN on SBP and vasomotor function in adult offspring. These data suggest that developmental cardiovascular programming is

  9. The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

    Science.gov (United States)

    Bele, Tanja; Fabbretti, Elsa

    2016-08-01

    P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

  10. Cellular uptake of lead in the blood-cerebrospinal fluid barrier: Novel roles of Connexin 43 hemichannel and its down-regulations via Erk phosphorylation

    International Nuclear Information System (INIS)

    Song, Han; Zheng, Gang; Liu, Yang; Shen, Xue-Feng; Zhao, Zai-Hua; Aschner, Michael; Luo, Wen-Jing; Chen, Jing-Yuan

    2016-01-01

    As the structural basis of blood-cerebrospinal fluid barrier (BCB), epithelial cells in the choroid plexus (CP) are targets for lead (Pb). Pb is known to accumulate in the CP; however, the mechanism of Pb uptake in the choroidal epithelial cells remains unknown. Recently, hemichannels of Connexin 43 (Cx43), the most ubiquitously expressed gap junction proteins in the CP, were found to be important pathways for many substances. This study was designed to investigate the roles of Cx43 in Pb uptake in the epithelial cells. Autometallography was used to outline Pb's subcellular location, and the characteristics of Pb transport into CP cells, including concentration- and time-dependence were analyzed by atomic absorption spectroscopy. Knockdown/overexpression of Cx43 with transient siRNA/plasmids transfections before Pb exposure diminished/increased the Pb accumulation. In the Z310 cell-based doxycycline-inducible Cx43 expression cell line (iZCx43), doxycycline induced a significant increase (3-fold) in Pb uptake, corresponding to the increased Cx43 levels. Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations. Cx43-induced Pb uptake was attenuated after blockage of Cx43 hemichannels with its inhibitor, carbenoxolone. Additionally, down-regulation of Cx43 protein levels by Pb exposure paralleled cellular Pb concentrations in the time study. Concomitantly, expressions of phosphor-Src and phosphor-Erk were both significantly increased by Pb. However, inactivation of Erk, not Src pathway, reversed Pb-induced downregulation of Cx43. Taken together, these data establish that Pb can accumulate in the BCB and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Erk phosphorylation. - Highlights: • Pb is sequestrated in choroid plexus both in vivo and in vitro. • Cx43 knockdown/overexpression prevents/increases Pb accumulations. • Cx43 hemichannels are required for Pb uptake. • Pb-induced Erk

  11. Co-culture of neural crest stem cells (NCSC and insulin producing beta-TC6 cells results in cadherin junctions and protection against cytokine-induced beta-cell death.

    Directory of Open Access Journals (Sweden)

    Anongnad Ngamjariyawat

    Full Text Available PURPOSE: Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death. PROCEDURES: Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1β and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry, nitrite production (Griess reagent, protein localization (immunofluorescence and protein phosphorylation (flow cytometry. RESULTS: We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii NCSC-derived laminin production; (iii decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv decreased beta-TC6 cell phosphorylation of ERK(T202/Y204, FAK(Y397 and FAK(Y576. Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs. CONCLUSION: In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta

  12. Evidence for aberrant astrocyte hemichannel activity in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL).

    Science.gov (United States)

    Burkovetskaya, Maria; Karpuk, Nikolay; Xiong, Juan; Bosch, Megan; Boska, Michael D; Takeuchi, Hideyuki; Suzumura, Akio; Kielian, Tammy

    2014-01-01

    Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3 that leads to vision loss, progressive cognitive and motor decline, and premature death. Morphological evidence of astrocyte activation occurs early in the disease process and coincides with regions where neuronal loss eventually ensues. However, the consequences of CLN3 mutation on astrocyte function remain relatively ill-defined. Astrocytes play a critical role in CNS homeostasis, in part, by their ability to regulate the extracellular milieu via the formation of extensive syncytial networks coupled by gap junction (GJ) channels. In contrast, unopposed hemichannels (HCs) have been implicated in CNS pathology by allowing the non-discriminant passage of molecules between the intracellular and extracellular milieus. Here we examined acute brain slices from CLN3 mutant mice (CLN3Δex7/8) to determine whether CLN3 loss alters the balance of GJ and HC activity. CLN3Δex7/8 mice displayed transient increases in astrocyte HC opening at postnatal day 30 in numerous brain regions, compared to wild type (WT) animals; however, HC activity steadily decreased at postnatal days 60 and 90 in CLN3Δex7/8 astrocytes to reach levels lower than WT cells. This suggested a progressive decline in astrocyte function, which was supported by significant reductions in glutamine synthetase, GLAST, and connexin expression in CLN3Δex7/8 mice compared to WT animals. Based on the early increase in astrocyte HC activity, CLN3Δex7/8 mice were treated with the novel carbenoxolone derivative INI-0602 to inhibit HCs. Administration of INI-0602 for a one month period significantly reduced lysosomal ceroid inclusions in the brains of CLN3Δex7/8 mice compared to WT animals, which coincided with significant increases in astrocyte GJ communication and normalization of astrocyte resting membrane potential to WT levels. Collectively, these findings suggest that alterations in

  13. Evidence for aberrant astrocyte hemichannel activity in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL.

    Directory of Open Access Journals (Sweden)

    Maria Burkovetskaya

    Full Text Available Juvenile Neuronal Ceroid Lipofuscinosis (JNCL is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3 that leads to vision loss, progressive cognitive and motor decline, and premature death. Morphological evidence of astrocyte activation occurs early in the disease process and coincides with regions where neuronal loss eventually ensues. However, the consequences of CLN3 mutation on astrocyte function remain relatively ill-defined. Astrocytes play a critical role in CNS homeostasis, in part, by their ability to regulate the extracellular milieu via the formation of extensive syncytial networks coupled by gap junction (GJ channels. In contrast, unopposed hemichannels (HCs have been implicated in CNS pathology by allowing the non-discriminant passage of molecules between the intracellular and extracellular milieus. Here we examined acute brain slices from CLN3 mutant mice (CLN3Δex7/8 to determine whether CLN3 loss alters the balance of GJ and HC activity. CLN3Δex7/8 mice displayed transient increases in astrocyte HC opening at postnatal day 30 in numerous brain regions, compared to wild type (WT animals; however, HC activity steadily decreased at postnatal days 60 and 90 in CLN3Δex7/8 astrocytes to reach levels lower than WT cells. This suggested a progressive decline in astrocyte function, which was supported by significant reductions in glutamine synthetase, GLAST, and connexin expression in CLN3Δex7/8 mice compared to WT animals. Based on the early increase in astrocyte HC activity, CLN3Δex7/8 mice were treated with the novel carbenoxolone derivative INI-0602 to inhibit HCs. Administration of INI-0602 for a one month period significantly reduced lysosomal ceroid inclusions in the brains of CLN3Δex7/8 mice compared to WT animals, which coincided with significant increases in astrocyte GJ communication and normalization of astrocyte resting membrane potential to WT levels. Collectively, these findings suggest that

  14. Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

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    Hayoz Sébastien

    2012-05-01

    Full Text Available Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP

  15. Interventions for men and women with their first episode of genital herpes.

    Science.gov (United States)

    Heslop, Rachel; Roberts, Helen; Flower, Deralie; Jordan, Vanessa

    2016-08-30

    ; largely due to lack of information supplied in the publications, and due to the age of the trials. This review found low quality evidence from two studies of oral acyclovir, when compared to placebo, reduced the duration of symptoms in individuals undergoing their first episode of genital herpes (mean difference (MD) -3.22, 95% confidence interval (CI) -5.91 to -0.54; I(2) = 52%). In two studies (112 participants), intravenous acyclovir decreased the median number of days that patients with first-episode herpes suffered symptoms. Oral valaciclovir (converted to acyclovir) also showed a similar length of symptom duration when compared to acyclovir in two studies.There is currently no evidence that topical acyclovir reduces symptoms (MD -0.61 days, 95% CI -2.16 to 0.95; 3 RCTs, 195 participants, I(2) statistic = 56%). There is also no current evidence that the topical treatments of cicloxolone cream, carbenoxolone sodium cream, adenosine arabinoside, idoxuridine in dimethyl sulfoxide, when compared to placebo reduced the duration of symptoms in people undergoing their first episode of herpes.Two studies reported no evidence of a reduction in the number of median days to recurrence following treatment with oral acyclovir versus placebo. Adverse events were generally poorly reported by all of the included studies and we were unable to quantitatively analyse this outcome. For those taking acyclovir, there were no serious adverse events; the most common adverse events reported for oral acyclovir were coryza, dizziness, tiredness, diarrhoea and renal colic. For intravenous acyclovir these were phlebitis, nausea and abnormal liver function tests and for topical acyclovir there was pain with the topical application.Those undergoing interferon treatment had significantly more adverse events compared to those taking placebo. There is low quality evidence from this review that oral acyclovir reduced the duration of symptoms for genital herpes. However, there is low quality evidence

  16. Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

    Science.gov (United States)

    Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B

    2016-04-01

    Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P gap junctions between