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Sample records for 86rb vblizi porogov

  1. Calcium-dependent 86 Rb efflux and ethanol intoxication: studies of human red blood cells and rodent brain synaptosomes.

    Science.gov (United States)

    Yamamoto, H A; Harris, R A

    1983-04-08

    Effects of ethanol on calcium-dependent potassium efflux were investigated in red blood cells (RBC) from humans and brain synaptosomes from rats and mice. 86 Rb was used as a tracer for potassium. Synaptosomes and RBC were lysed and resealed with 86 Rb and calcium-EGTA buffers to regulate intracellular levels of ionized calcium. In vitro addition of ethanol (100 mM) stimulated the calcium-dependent 86 Rb efflux of synaptosomes. This stimulation was blocked by apamin, an inhibitor of the calcium-dependent potassium current of nerve cells. In addition, intracerebroventricular injection of apamin inhibited ethanol-induced narcosis in mice, providing behavioral evidence for the importance of calcium-stimulated potassium efflux in alcohol intoxication. In vitro addition of ethanol, propanol or butanol increased calcium-dependent 86 Rb efflux of human RBC at low concentrations of free calcium, but did not change the calcium-independent efflux of 86 Rb. These results suggest that the calcium-dependent 86 Rb efflux of nerve endings may have an important role in the pharmacological and toxicological effects of ethanol.

  2. Alterations of 86Rb+ fluxes in poliovirus-infected HeLa cells and their dependence on virus replication

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    Schaefer, A.; Geck, P.; Zibirre, R.; Kuehne, J.; Koch, G.

    1984-07-30

    Components of the 86Rb+ influx were investigated subsequent to poliovirus infection in the presence and absence of guanidine-HCl, both under normal steady-state conditions and after Na+ preloading of the cells. Measurements of the ouabain-sensitive 86Rb+ uptake indicated a biphasic change in the activity of the Na+, K+ pump in the course of virus infection: a transient increase in the second hour postinfection, that was detectable only after Na+ preloading and inhibition after 3 hr. The enhanced activity of the Na+, K+ pump was not affected, while the decrease later was fully prevented by the antiviral agent guanidine-HCl. The piretanide-sensitive 86Rb+ uptake due to the Na+, K+, 2 Cl- cotransport system also became strongly inhibited beginning in the second hour postinfection. The inhibition of this transport system was partially antagonized by guanidine-HCl. The remaining 86Rb+ influx in the presence of ouabain and piretanide increased in the third hour postinfection. The latter change in 86Rb+ influx, indicating an increased permeability to monovalent cations was completely abolished by guanidine-HCl.

  3. Effects of aging on agonist-activated sup 86 Rb efflux in arteries of Fischer 344 rats

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    Cox, R.H.; Tulenko, T.N. (Univ. of Pennsylvania, Philadelphia (USA))

    1989-08-01

    Segments of thoracic aorta (DTA), tail artery (TA), and mesenteric artery branches (MAB) were obtained from male Fischer 344 rats at ages of 1, 2, 6, 12, 24, and 30 mo and were used to determine the effects of aging on agonist-activated {sup 86}Rb (and {sup 42}K) efflux. At all three arterial sites, basal efflux decreased during development (1-6 mo), but no further changes were observed with aging (6-30 mo). The initial efflux response to 10 microM norepinephrine (NE) in the presence of 1 microM propranolol exhibited either no change (DTA) or an increase (TA and MAB) during development (1-6 mo), but all three sites showed a large decrease during aging (6-30 mo). Changes in the steady-state response to NE paralleled changes in the basal efflux at all ages and arterial sites. The initial efflux response to 75 mM K+-physiological salt solution (PSS) for the DTA in the presence of 1 microM phentolamine and 1 microM propranolol decreased during development followed by an increase during aging, whereas for the TA and MAB, there were no significant changes with age. The steady-state efflux response to K+ decreased during development at all three sites but was increased only for the DTA during aging. The steady-state efflux response to K+ was not altered for the TA and MAB during aging. Efflux responses using {sup 42}K were qualitatively similar, but rate constants were quantitatively larger than those with {sup 86}Rb at all three arterial sites and at all ages.

  4. sup 86 Rb(K) influx and ( sup 3 H)ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

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    Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P. (Royal Free Hospital and School of Medicine, London (England))

    1989-08-01

    Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), {sup 86}Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific ({sup 3}H)ouabain binding by the human platelet. This {sup 86}Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active {sup 86}Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active {sup 86}Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

  5. Differences in modifications of cytoplasmic free Ca2+ concentration and 86Rb+ influx in human neoplastic B cells by antibodies to mu- relative to delta-Ig heavy chains.

    Science.gov (United States)

    Heikkilä, R; Ruud, E; Funderud, S; Godal, T

    1985-01-01

    Cytoplasmic free Ca2+ concentration and influx of 86Rb+ (K+ analogue) were determined during the first minutes after stimulation of neoplastic human B cells and B cell lines by antibodies to surface Ig. The Ca2+ concentration increased in the great majority of samples (41 of 48). All of four B cell lines also responded, providing formal evidence that accessory cells are not required for this early, surface Ig-mediated event. Antibodies to delta as well as mu, heavy chains (anti-delta and anti-mu) could induce both Ca2+ and 86Rb+ responses. 86Rb+ responders were found within the group of Ca2+ responders, but no quantitative relation was observed between the two responses. In cells expressing both sIgM and sIgD, antibodies to delta heavy chains were more potent than those to mu heavy chains in inducing Ca2+ responses, whereas the opposite pattern was seen with regard to 86Rb+ responses. These results demonstrate that sIgM and sIgD can deliver different biochemical signals to the cell. PMID:3921300

  6. Some effects of nicorandil on the smooth muscles of the rat and guinea pig

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    Weston, A.H.

    1987-01-01

    In rat isolated portal vein, nicorandil (0.1-500 microM) abolished spontaneous tension waves and inhibited mechanical responses to norepinephrine (0.1-100 microM) and KCl (5-80 mM). Intracellular electrical recording showed that nicorandil (0.1-1 microM) abolished spontaneous multispike complexes and at higher concentrations (up to 500 microM) raised the membrane potential to approximately -90 mV. Using /sup 86/Rb as a K+-marker, nicorandil (5-500 microM) increased the /sup 86/Rb efflux rate coefficient. In rat isolated aorta, nicorandil (8-32 microM) inhibited mechanical responses to norepinephrine (0.125-100 microM) and KCl (5-80 mM), but had no effect on /sup 86/Rb exchange. In guinea pig isolated taenia caeci, nicorandil (4-64 microM) relaxed spontaneous mechanical tone and increased /sup 86/Rb efflux in the absence and presence of apamin, 100 nM. It is concluded that the inhibitory effects of nicorandil in portal vein and taenia caeci are mediated at least in part by a mechanism which involves the opening of apamin-insensitive, /sup 86/Rb-permeable K+ channels. In aorta, however, the opening of such channels was not detected, and the inhibitory effects of nicorandil in this tissue are associated with an, as yet, undefined mechanism.

  7. GABA/sub B/ receptor activation inhibits Ca/sup 2 +/-activated potassium channels in synaptosomes: involvement of G-proteins

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    Ticku, M.K.; Delgado, A.

    1989-01-01

    /sup 86/Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca/sup 2 +/-activated K/sup +/-channels. Depolarization of /sup 86/Rb-loaded synaptosomes in physiological buffer increased Ca/sup 2 +/-activated /sup 86/Rb-efflux by 400%. The /sup 86/Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La/sup 3 +/ indicating the involvement of Ca/sup 2 +/-activated K/sup +/-channels. (-)Baclofen inhibited Ca/sup 2 +/-activated /sup 86/Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca/sup 2 +/-activated K/sup +/-channels. These results suggest that baclofen inhibits Ca/sup 2 +/-activated K/sup +/-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology.

  8. K sup + channel openers activate brain sulfonylurea-sensitive K sup + channels and block neurosecretion

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    Schmid-Antomarchi, H.; Amoroso, S.; Fosset, M.; Lazdunski, M. (Centre National de la Recherche Scientifique, Valbonne (France))

    1990-05-01

    Vascular K{sup +} channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate {sup 86}Rb{sup +} efflux from slices of substantia nigra. This {sup 86}Rb{sup +} efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K{sup +} channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K{sub 0.5}, the half-maximal effect of the enantiomer ({minus})-cromakalim, is as low as 10 nM, whereas K{sub 0.5} for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K{sup +} channels lead to inhibition of {gamma}-aminobutyric acid release. There is an excellent relationship between potency to activate {sup 86}Rb{sup +} efflux and potency to inhibit neurotransmitter release.

  9. Measurement of K/sup +/ conductance in gastric vesicles from secreting stomach

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    Rabon, E.; Gunther, R.D.

    1986-05-01

    Specific inhibitors were used to identify two components of /sup 86/Rb/sup +/ uptake in vesicles obtained from secreting rabbit stomachs. Rb/sup +/ transport was measured in vesicles as trace /sup 86/Rb/sup +/ uptake following removal of external K/sup +/ from vesicles equilibrated in potassium gluconate. /sup 86/Rb)2= uptake mediated by the gastric H/sup +/K/sup +/-ATPase was identified by sensitivity to vanadate, ATP and pyridyl (1,2a) imidazole (SCH28080). In contrast, /sup 86/Rb/sup +/ influx through a K/sup +/ conductance mechanism was inhibited by the protonophore (TCS) induced collapse of the K/sup +/ diffusion potential. K/sup +/ conductance sensitivity to quinine and the K/sup +/ channel blocker bis-Guanidinium (bis G-8) were demonstrated by inhibition of a K/sup +/ induced chase of intravesicular /sup 86/Rb/sup +/ previously loaded by /sup 86/Rb/sup +//K/sup +/ exchange in the presence of 2 ..mu..M SCH28080. The K/sup +/ conductance is Ba/sup 2 +/ and apamine insensitive and exhibits a monovalent cation specificity of Rb > Kapprox. = Cs >> Na, Li. KCl dependent H/sup +/ transport exhibited complete sensitivity to the H/sup +/, K/sup +/-ATPase inhibitors SCH28080 and vanadate. The measurements of Rb/sup +/ pathways distinctive for the H/sup +/, K/sup +/-ATPase and a K/sup +/ conductance support previous suggestions of a functional linkage between the H/sup +/, K/sup +/-ATPase and a K/sup +/ conductance in vesicles, obtained from stimulated stomach. The experimental discrimination between the two Rb/sup +/ pathways suggests that a separate mechanism is utilized for each transport pathway.

  10. Rubidium occlusion within tryptic peptides of the H,K-ATPase.

    Science.gov (United States)

    Rabon, E C; Smillie, K; Seru, V; Rabon, R

    1993-04-15

    86Rb+ binding to the H,K-ATPase was measured in the Mg(2+)-vanadate-inhibited enzyme at 4 degrees C. The concentration dependence of 86Rb+ binding in detergent-free preparations exhibited two components, one saturable with a K0.5 (Rb+) of 0.76 +/- 0.3 mM and a binding capacity of 2626 +/- 690 pmol of Rb+/mg of protein and the second nonsaturable, but linearly dependent, upon the 86Rb+ concentration. The concentration dependence of 86Rb+ binding was unaffected by digitonin treatment with a K0.5 (Rb+) of 0.63 +/- 0.09 mM and a binding capacity of 2824 +/- 152 pmol of Rb+/mg of protein, but the amplitude of the nonsaturable component was eliminated. The level of 86Rb+ binding was optimized by vanadate and decreased by ADP and ATP, suggesting that cation binding is stabilized in the E2-like conformation and antagonized in the E1 conformation. The Rb(+)-dependent stabilization of the E2 enzyme conformation was confirmed from the fluorescent quench response of the fluorescein isothiocyanate (FITC)-labeled enzyme, where 86Rb+ bound to the FITC-labeled enzyme with a K0.5 = 0.85 +/- 0.3 mM and a saturable binding capacity of 2121 pmol of 86Rb+/mg of protein and quenched the FITC fluorescence with a K0.5(Rb+) of 3.6 +/- 0.3 mM. The K(+)-competitive inhibitor, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ), also quenched FITC fluorescence with a K0.5(MDPQ) of 24.5 +/- 0.6 microM and competitively inhibited 86Rb+ binding with a K*0.5 = 35.8 microM (MDPQ). The MDPQ-induced quench of FITC fluorescence at Lys517 within the cytoplasmic M4/M5 nucleotide domain and displacement of 86Rb+ from a functionally defined extracytoplasmic binding domain indicate that structural determinants of the E2 conformational state exist within both cytoplasmic and extracytoplasmic domains of the H,K-ATPase and thus provide evidence of concerted conformational changes between the nucleotide and cation binding domains within the FITC-labeled H,K-ATPase. Membrane

  11. Ouabain inhibition of the sodium-potassium pump: estimation of ED50 in different types of human leucocytes in vitro

    DEFF Research Database (Denmark)

    Møller, B; Vaag, Allan; Johansen, Torben

    1990-01-01

    uptake of K+ (86Rb+) that is sensitive to ouabain, 10(-5) mol l-1. 2. Dose-response curves for inhibition of the Na(+)-K+ pump were obtained after exposure of the cells to various concentrations of ouabain for 2.5 h when the level of pump inhibition was considered to be at steady state. 3. The ED50...

  12. Cotransport of water by Na¿-K¿-2Cl¿ cotransporters expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Zeuthen, Thomas; Macaulay, Nanna

    2012-01-01

    The NKCC1 and NKCC2 isoforms of the mammalian Na¿–K¿–2Cl¿ cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic...

  13. ATP-sensitive K/sup +/ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone

    Energy Technology Data Exchange (ETDEWEB)

    de Weille, J.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1988-02-01

    The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide names from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and /sup 86/Rb/sup +/ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of APT-sensitive K/sup +/ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamine. Glibenclamide blocks the galanin-activated ATP-sensitive K/sup +/ channel. /sup 86/Rb/sup +/ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced /sup 86/Rb/sup +/ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K/sup +/ channel reported earlier. /sup 86/Rb/sup +/ efflux studies confirm the electrophysiological demonstration that galanin activates and ATP-dependent K/sup +/ channel.

  14. Cationic and secretory effects of glimepiride and glibenclamide in perifused rat islets

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    Lebrun, P.; Malaisse, W.J. (Laboratories of Pharmacology and Experimental Medicine, Brussels Free University, Brussels (Belgium))

    1992-01-01

    The effects of glimepiride and glibenclamide upon [sup 86]Rb outflow, [sup 45]Ca outflow and insulin release were examined in rat islets perfused at low (zero to 2.8 mM) or close-to-normal (8.3 mM) D- glucose concentrations. At the low hexose concentrations, a marked and not reversible decrease in [sup 86]Rb outflow contrasted with a rapid and reversible increase in [sup 45]Ca outflow. The latter increase was abolished in the absence of extracellular Ca[sup 2+], and was not associated with any pronounced stimulation of insulin release. Inversely, in the presence of 8.3 mM D-glucose, a comparable increase in [sup 45]Ca efflux now coincided with an increase in [sup 86]Rb efflux and a marked and not reversible stimulation of insulin release. Whether in terms of the time course or glucose dependency of the cationic and secretory responses, a coupled increase in both [sup 40]Ca inflow and [sup 45]Ca outflow thus coincided with either negative or positive changes in [sup 86]Rb outflow and either minimal or marked changes in insulin output. Such dissociated behaviors suggest that the insulinotropic action of hypoglycemic sulfonylureas is not necessarily attributable solely to a primary decrease in K[sup +] conductance. (au).

  15. Activation of the Na+/K(+)-pump in rat peritoneal mast cells following histamine release: a possible role in cell recovery

    DEFF Research Database (Denmark)

    Knudsen, T; Ferjan, I; Johansen, Torben

    1993-01-01

    1. The activity of the Na+/K(+)-pump in rat peritoneal mast cells was measured at various time intervals after induction of cellular histamine release by compound 48/80 or by the antigen-antibody reaction. The Na+/K(+)-pump activity was assessed as the ouabain-sensitive potassium uptake....... On the basis of the present results, we suggest a role for the Na+/K(+)-pump in the recovery process of the mast cell following histamine release....... of the cells using 86Rb+ as a tracer for potassium (K+(86Rb+)-uptake). 2. Stimulation of the cells with compound 48/80 induced a time and concentration dependent increase of the Na+/K(+)-pump activity. The pump activity was maximal 2 min after stimulation of the cells. Then, the activity gradually decreased...

  16. Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes

    DEFF Research Database (Denmark)

    Owens, T; Kaplan, J G

    1980-01-01

    Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation...... of thymidine into DNA; the expected dilution of these two responses by nude spleen cells did not occur. However, if the nude splenocytes were added immediately prior to assay to the enriched T cells that had been precultured in presence of Con A, the expected dilution of the activated T-cell responses occurred......; both 86Rb uptake and thymidine incorporation were reduced proportionally to the degree of dilution of the T cells by the nonresponding cells. These data indicate that during co-culture in presence of Con A there is interaction between the T cells, capable of responding to mitogens, and the nude spleen...

  17. Ouabain-binding and 86rubidium-uptake in lymphocytes of normal and borderline hypertensive subjects

    DEFF Research Database (Denmark)

    Nielsen, J R; Pedersen, K E; Johansen, Torben;

    1983-01-01

    activity were studied in lymphocytes of nine borderline hypertensives (27 (20-36) years) and nine controls (28 (20-36) years). Maximum 3H-ouabain binding and 86Rb-uptake were taken as measures of the number of pump sites and cation pump activity, respectively. The median number of sodium/potassium pump...... sites was 49.6 X 10(3) molecules/cell in the BH group compared to 32.4 X 10(3) in the control group (P less than 0.01). Median 90 min 86Rb-uptakes were 54.0 pmol/10(6) cells in BH subjects and 39.4 in controls (P less than 0.10). The increased number of sodium/potassium pump sites and the tendency...... to increased cation pump activity in lymphocytes of BH subjects in vitro may be interpreted as an adaptive change possibly induced by a circulating natriuretic substance....

  18. Comparative phloem Mobility of nickel in nonsenescent plants. [Pisum sativa L. ; Pelargonium zonale L

    Energy Technology Data Exchange (ETDEWEB)

    Neumann, P.M.; Chamel, A.

    1986-06-01

    /sup 63/Ni was applied to nonsenescent source leaves and found to be transported to sink tissues in pea (Pisum saativum L.) and geranium plants (Pelargonium zonale L.). The comparative mobilities (percent tracer transported out of source leaf division % /sup 86/Rb transported) for /sup 63/Ni in peas was 2.12 and in geranium 0.25. The value for the phloem mobile /sup 86/Rb was 1.00. By contrast, the comparative mobility of /sup 45/Ca, which is relatively immobile in the phloem, was low (0.05 in peas, 0.00 in geranium). Interruption of the phloem pathway between source and sink leaves by steam girdling almost completely inhibited /sup 63/Ni accumulation in the sink leaves of both species. The authors conclude that Ni is transported from nonsenescent source leaves to sink tissues via the phloem of leguminous and nonleguminous plants.

  19. Contribution to the study of {beta} disintegration and of nuclear structure using experiments on certain {beta}-{gamma} cascades: 198{sub Au}, 86{sub Rb}, 170{sub Tm}; Contribution a l'etude de la desintegration beta et a l'etude de la structure nucleaire a l'aide d'experiences sur certaines cascades beta-gamma: 198{sub Au}, 86{sub Rb}, 170{sub Tm}

    Energy Technology Data Exchange (ETDEWEB)

    Lachkar, J. [Commissariat a l' Energie Atomique, Bruyeres-le-Chatel (France). Centre d' Etudes; Paris-11 Univ., fabulte des Sciences 91 - Orsay (France)

    1969-07-01

    {beta}{gamma} directional angular correlations and shapes of inner beta spectra leading to the first excited level of the final nucleus enable one to determine the nuclear matrix elements typical of the {beta} transition. In the three observed first forbidden cases: {sup 170}Tm, {sup 86}Rb, {sup 198}Au, these matrix elements do not confirm the independent shell model theory. Other hypotheses are then suggested and discussed. (author) [French] Les experiences de correlation angulaire {beta}{gamma} et l'etude du spectre {beta} conduisant au premier niveau excite du noyau final permettent de determiner les elements de matrices nucleaires caracteristiques de cette transition. Dans les trois cas etudies (transitions une fois interdites): {sup 170}Tm, {sup 86}Rb, {sup 198}Au, ces elements de matrices ne peuvent etre retrouves a l'aide du modele en couches et a particules independantes. D'autres hypotheses sont alors emises et discutees. (auteur)

  20. Effect of low temperatures on glucose-induced insulin secretion and ionic fluxes in rat pancreatic islets.

    Science.gov (United States)

    Escolar, J C; Hoo-Paris, R; Castex, C; Sutter, B C

    1987-11-01

    The direct effect of cold on the inhibition of B cell secretion is well known in hibernating and experimentally hypothermic mammals. This temperature dependency may result from the inhibition of ion transport across the membranes. In order to verify this hypothesis, ionic effluxes and insulin secretion from rat islets loaded with 86Rb+ and 45Ca+ were measured during perifusion. At 37 degrees C, the rise in glucose concentration from zero to 16.7 mmol/l provoked a rapid decrease in 86Rb+ efflux, an early fall and subsequent rise in 45Ca2+ efflux and a typical biphasic pattern of insulin secretion. At 27 degrees C, glucose induced only a very slight increase in insulin secretion, while the fluxes of radioactive ions were not significantly modified in amplitude but were clearly delayed. At 17 degrees C, no insulin response to glucose was observed and the decrease in K+ conductance indicated by 86Rb+ flux decrease was less temperature-dependent than the movement of Ca2+. After supplementary stimulation with a high extracellular concentration of Ca2+, insulin secretion was enhanced at 27 degrees C and reached levels induced by glucose alone at 37 degrees C. An increase in hormone secretion occurred even at 17 degrees C, but only during a first phase of secretion. Regular increases in temperature potentiated insulin secretion and provoked changes in ionic fluxes which suggest that B cell depolarization (86Rb+ flux decrease) induced by glucose can occur at 15 degrees C but cannot induce the opening of voltage-dependent Ca2+ channels (increase in 45Ca2+ efflux) until temperatures higher than 27 degrees C are reached.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. In vitro -Untersuchungen zum Kaliumtransport durch das Epithel des Pansens beim Schaf

    OpenAIRE

    Oberhäuser, Simone

    2010-01-01

    The transport of potassium across the sheep rumen was studied with the USSING chamber technique by using 86Rb. a. In all experiments a net secretion of potassium was observed. There was a large variability of the unidirectional Rb fluxes. b. The manipulation of the Na transport and therefore the activity of the basolateral Na+-K+-ATPase by replacement of anions (Cl-, HCO3 -, SCFA) by gluconate or by a decrease of the incubation temperature did not influence transport of Rb significantl...

  2. Na,K-ATPase characterized in artificial membranes. 2. Successive measurement of ATP-driven Rb-accumulation, ouabain-blocked Rb-flux and palytoxin-induced Rb-efflux.

    Science.gov (United States)

    Anner, B M; Moosmayer, M

    1994-01-01

    The Na,K-ATPase is a multifunctional system anchored in the membrane of eukaryotic cells; it is responsible for the establishment and regulation of the Na/K balance of cell and organism by a stoichiometric mechanism linking Na extrusion to K uptake and ATP hydrolysis. The receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the system. Conversely, palytoxin, the most potent animal toxin, exerts its toxic effect by creating nonspecific leaks in the cell membrane leading to K-efflux and influx of Na and Ca ions. Ouabain prevents the pore-forming action of palytoxin in cells and therefore Na,K-ATPase is suspected to be the common receptor of ouabain and palytoxin. We have developed an artificial membrane system to determine structure-function relationships and ligand interactions of purified Na,K-ATPase: two-sided, bi-directional ATP-filled liposomes. In this system, ATP-driven 86Rb accumulation, arrest of 86Rb-uptake by ouabain, and palytoxin-induced 86Rb-leak were measured successively in the same preparation. Ouabain prevented the leak when the enzyme was ouabain-sensitive (rabbit kidney) but not when it was ouabain-resistant (rat kidney). On the basis of these data in conjunction with conformational analyses, allosteric conformational competition for the ouabain-palytoxin antagonism is proposed.

  3. A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: Heterologous regulation of the same K+ channel

    Energy Technology Data Exchange (ETDEWEB)

    Kamatchi, G.L.; Ticku, M.K. (Univ. of Texas Health Science Center, San Antonio (USA))

    1991-02-01

    The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between them when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.

  4. Nicotine-morphine interactions at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors.

    Science.gov (United States)

    Talka, Reeta; Salminen, Outi; Whiteaker, Paul; Lukas, Ronald J; Tuominen, Raimo K

    2013-02-15

    Nicotine and opioids share several behavioral and rewarding properties. Although both opioids and nicotine have their own specific mechanism of action, there is empirical and experimental evidence of interactions between these drugs. We studied receptor-level interactions of nicotine and morphine at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors. [(3)H]epibatidine displacement was used to determine if morphine binds competitively to nicotinic acetylcholine receptors. Functional interactions of morphine and nicotine were studied with calcium fluorometry and (86)Rb(+) efflux assays. Morphine displaced [(3)H]epibatidine from nicotinic agonist binding sites in all cell lines studied. The Ki values for morphine were 13.2μM in SH-EP1-hα4β2 cells, 0.16μM and 126μM in SH-SY5Y cells and 43.7μM in SH-EP1-hα7 cells. In SH-EP1-hα4β2 cells expressing α4β2 nicotinic acetylcholine receptors, morphine acted as a partial agonist of (86)Rb(+) efflux comparable to cytisine (with EC50 values of 53.3μM for morphine and 5.38μM for cytisine). The effect of morphine was attenuated concentration-dependently by the nicotinic antagonist mecamylamine. In the SH-SY5Y cell line expressing several subtypes of nicotinic acetylcholine receptors morphine had an inhibitory effect on nicotine induced (86)Rb(+) ion efflux mediated by α3(⁎) nicotinic acetylcholine receptors. These results suggest that morphine acts as a partial agonist at α4β2 nicotinic acetylcholine receptors and as a weak antagonist at α3(⁎) nicotinic acetylcholine receptors.

  5. Circulating inhibitor of ouabain-insensitive cation transport in malignantrenal hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Simon, G.

    1986-03-01

    The role of circulating humoral agents in the pathogenesis of vascular wall Na depletion in malignant hypertension (MHT) was investigated. Plasma was collected from 33 male F344 rats with malignant one-kidney, one clip HT and 22 normotensive control rats. MHT developed spontaneously and was characterized by inactivity, weight loss, edema, anemia or hemoconcentration, hyperkalemia, and renal insufficiency. For bioassay, monolayers of quiescent vascular smooth muscle cells from F344 rats were incubated in deproteinized or whole plasma for measurement of /sup 86/Rb uptake with or without 2 mM ouabain or 1 mM furosemide. Compared to controls, ouabain-insensitive /sup 86/Rb uptake was reduced from 8.2 +- 2.0 nmol/mg protein min/sup -1/ (mean +- SD) to 5.2 +- 1.4 in deproteinized plasma (p < 0.01, N = 12) and from 6.6 +- 1.9 to 4.0 +- 0.3 in whole plasma (p < 0.05, N=5) of rats with MHT, due in part to a reduction in furosemide-sensitive uptake (p < 0.01, N = 6). There were no differences in ouabain-sensitive /sup 86/Rb uptake of cells between groups. In rats with MHT the increased Na content of the aorta that characterizes benign one-kidney, one clip HT was reversed, and bladder wall Na content was reduced (p < 0.001, N = 9). In MHT, a furosemide-like, ouabain-insensitive cation transport inhibitor in blood and urine may be the cause of vascular wall Na loss and of natriuresis that triggers the syndrome.

  6. Phentolamine and yohimbine inhibit ATP-sensitive K+ channels in mouse pancreatic beta-cells.

    OpenAIRE

    Plant, T D; Henquin, J C

    1990-01-01

    1. The effects of phentolamine and yohimbine on adenosine 5'-triphosphate (ATP)-sensitive K+ channels were studied in normal mouse beta-cells. 2. In the presence of 3 mM glucose, many ATP-sensitive K+ channels are open in the beta-cell membrane. Under these conditions, phentolamine inhibited 86Rb efflux from the islets. This inhibition was faster with 100 than with 20 microM phentolamine but its steady-state magnitude was similar with both concentrations. Yohimbine (20-100 microM) also inhibi...

  7. Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes.

    Science.gov (United States)

    Agner, G; Kaulin, Y A; Schagina, L V; Takemoto, J Y; Blasko, K

    2000-06-01

    The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.

  8. Measurement of reaction cross-sections for 89Y at average neutron energies of 7.24-24.83 MeV

    Science.gov (United States)

    Zaman, Muhammad; Kim, Guinyun; Naik, Haladhara; Kim, Kwangsoo; Shahid, Muhammad

    2015-05-01

    We measured neutron-induced reaction cross-sections for 89Y(n,γ)90mY and 89Y(n,α)86Rb reactions with the average neutron energy region from 7.45 to 24.83 MeV by an activation and off-line γ-ray spectrometric technique using the MC-50 Cyclotron at Korea Institute of Radiological and Medical Sciences. The neutron-induced reaction cross-sections of 89Y as a function of neutron energy were taken from the TENDL-2013 library. The flux-weighted average cross-sections for 89Y(n,γ)90mY and 89Y(n,α)86Rb reactions were calculated from the TENDL-2013 values based on mono-energetic neutron and by using the neutron energy spectrum from MCNPX 2.6.0 code. The present results are compared with the flux-weighted values of TENDL-2013 and are found to be in good agreement

  9. Actions of ionomycin in rat parotid gland

    Energy Technology Data Exchange (ETDEWEB)

    Poggioli, J.; Leslie, B.A.; McKinney, J.S.; Weiss, S.J.; Putney, J.W. Jr.

    1982-04-01

    The effects of ionomycin (SQ 23,377), a carboxylic acid Ca-ionophore, on the rat parotid acinar cell were investigated. Ionomycin stimulated 86Rb efflux from parotid slices and was substantially more potent and efficacious than the Ca-ionophore, A-23187. The release of 86Rb was dependent on the concentration of ionomycin and of Ca. Ionomycin also stimulated 22Na uptake and 3H-protein secretion, but did not stimulate the incorporation of 32PO4 into phosphatidylinositol. These observations are consistent with an action of ionomycin in increasing cytosolic Ca by acting as an ionophore and not involving endogenous receptors. Pretreatment with ionomycin inhibited the transient, Ca-independent responses to carbachol or physalaemin. When ionomycin was added to parotid cells pre-equilibrated with 45Ca, a net loss of radiocalcium was observed. These observations suggest that ionomycin can release the receptor-regulated cellular Ca pool. Morphological studies did not reveal any nonspecific deleterious effects in the cells after incubation with 2.67 microM ionomycin.

  10. Inhibition of Na(+) -K+ pump activity by divalent cations in intact peritoneal mast cells of the rat

    DEFF Research Database (Denmark)

    Knudsen, T; Berthelsen, Carsten; Johansen, Torben

    1990-01-01

    1. The inhibition by the divalent cations magnesium, barium and strontium and the trivalent ion lanthanum of the Na(+) -K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure mast cell populations by measurement of the ouabain-sensitive uptake of the radioactive potassium......-resistant uptake was not changed. Half maximum decrease in the ouabain-sensitive K+(86Rb+)-uptake was observed with 1.8 mM magnesium, 1.2mM barium and 0.7 mM strontium. 4. The trivalent ion lanthanum blocked almost completely the ouabain-sensitive K+(86Rb+)-uptake at a concentration of 1 microM as does 1 m......M calcium. Combining either of these ions with magnesium had no further inhibitory effect on the ouabain-sensitive uptake. 5. In conclusion, in addition to the previously suggested modulation by calcium of the activity of the Na+ (-)K+ pump, evidence is provided in this investigation that the modulation may...

  11. The mode of inhibition of the Na+-K+ pump activity in mast cells by calcium

    DEFF Research Database (Denmark)

    Knudsen, T; Johansen, Torben

    1989-01-01

    1 The inhibition by calcium of the Na(+)-K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure populations of the cells by measuring the ouabain-sensitive uptake of the radioactive potassium analogue, 86rubidium (86Rb+). 2 Exposure of the cells to calcium induced a time...... not develop when the mast cells were incubated in a potassium-free medium, which is known to block Na(+)-K+ pump activity and allow accumulation of sodium inside the cells. Likewise, increasing the sodium permeability of the plasma membrane by monensin abolished the inhibition of the pump activity. In both...... peritoneal mast cells in a calcium-free medium increases the permeability of the plasma membrane to sodium, and the consequent increase in the intracellular concentration of sodium causes an increase in the activity of the pump. Addition of calcium to the cell suspension decreases the sodium permeability...

  12. Effects of β₂-agonists on force during and following anoxia in rat extensor digitorum longus muscle

    DEFF Research Database (Denmark)

    Fredsted, A; Gissel, H; Ortenblad, N

    2012-01-01

    of salbutamol on force recovery were prevented by blocking the Na(+),K(+)- pumps with ouabain or by blocking glycolysis with 2-deoxyglucose. Dibutyryl cAMP (1 mM) or theophylline (1 mM) also improved force recovery remarkably. In anoxic muscles, salbutamol decreased intracellular Na(+), increased (86)Rb uptake...... and K(+) content indicating stimulation of the Na(+),K(+) - pumps. In fatigued muscles salbutamol induced recovery of excitability. Thus, β(2)-agonists reduce the anoxia-induced loss of force leading to partial force recovery. These data strongly suggest that this effect is mediated by cAMP stimulation......Electrical stimulation of isolated muscles may lead to membrane depolarization, gain of Na(+), loss of K(+) and fatigue. These effects can be counteracted with β(2)-agonists possibly via activation of the Na(+),K(+)- pumps. Anoxia induces loss of force; however, it is not known whether β(2...

  13. The effect of ultraviolet radiation on early stages of activation of human lymphocytes: inhibition is independent of effects on DNA

    DEFF Research Database (Denmark)

    Castellanos, G; Owens, T; Rudd, C;

    1982-01-01

    before mitogen was added to the cultures, but were unaffected if irradiation occurred after 16 h of culture in presence of Con A. Cells irradiated with 84 ergs/mm2 at the onset of culture with mitogen did not show the early increase of cation pump function which is a characteristic of stimulated......Low doses (30-84 ergs/mm2, 1 erg = 10(7) J) of ultraviolet radiation (UV) caused severe inhibition of the proliferation of human lymphocytes in vitro. Greatest inhibition was produced when resting cells were irradiated immediately prior to stimulation with concanavalin A (Con A); this was true...... lymphocytes, when this was measured by means of 86Rb uptake after 2-4 h culture. The mitogen-stimulated activation of cation pump function has previously been shown to be unaffected by concentrations of cycloheximide and actinomycin D which produce virtually complete inhibition of protein and RNA synthesis...

  14. Some metabolic effects of overeating in man.

    Science.gov (United States)

    Welle, S L; Seaton, T B; Campbell, R G

    1986-12-01

    Metabolic responses to 20 days of overeating were examined in five healthy volunteers. Overfeeding caused a variable increase (1-18%) in basal metabolic rate but no change in metabolic rate during light exercise. Postprandial resting metabolic rate was 8-40% higher (mean 18%) during overeating. The increase in oxygen consumption during a norepinephrine infusion was the same before (20 +/- 2%) and after (17 +/- 3%) overfeeding. Overfeeding elevated basal insulin concentrations in all subjects and increased the insulin response to intravenous glucose in four of five subjects. Overfeeding did not significantly alter mean serum T3 concentrations or erythrocyte 86Rb uptake (an index of Na+,K+-ATPase activity). These data do not confirm reports that overfeeding increases metabolic rate more during exercise than during rest. They also suggest that the increase in resting metabolic rate during overfeeding is not caused by increased responsiveness to norepinephrine or increased serum T3 concentrations.

  15. Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

    Energy Technology Data Exchange (ETDEWEB)

    Lind, I.; Ahnert-Hilger, G.; Fuchs, G.; Gratzl, M.

    1987-07-01

    Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of /sup 86/Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.

  16. Water permeability of Na+-K+-2C1- cotransporters in mammalian epithelial cells

    DEFF Research Database (Denmark)

    Hammann, Steffen; Herrera-Perez, J.J.; Bundgaard, Magnus

    2005-01-01

    Water transport properties of the Na+-K+-2Cl- cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity...... changes of the cotransporter and interaction with Na+, K+ and Cl-. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive 86Rb+, the NKCCs of this tissue did not contribute...... any bumetanide-sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes....

  17. Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

    Directory of Open Access Journals (Sweden)

    C. Massart

    2014-01-01

    Full Text Available NADPH oxidases (NOXes and dual oxidases (DUOXes generate O2.− and H2O2. Diphenyleneiodonium (DPI inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog 99mTcO4− nor of the K+ cation mimic 86Rb+ in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

  18. Chiral recognition of pinacidil and its 3-pyridyl isomer by canine cardiac and smooth muscle: Antagonism by sulfonylureas

    Energy Technology Data Exchange (ETDEWEB)

    Steinberg, M.I.; Wiest, S.A.; Zimmerman, K.M.; Ertel, P.J.; Bemis, K.G.; Robertson, D.W. (Eli Lilly and Company, Indianapolis, IN (USA))

    1991-01-01

    Pinacidil, a potassium channel opener (PCO), relaxes vascular smooth muscle by increasing potassium ion membrane conductance, thereby causing membrane hyperpolarization. PCOs also act on cardiac muscle to decrease action potential duration (APD) selectively. To examine the enantiomeric selectivity of pinacidil, the stereoisomers of pinacidil (a 4-pyridylcyanoguanidine) and its 3-pyridyl isomer (LY222675) were synthesized and studied in canine Purkinje fibers and cephalic veins. The (-)-enantiomers of both pinacidil and LY222675 were more potent in relaxing phenylephrine-contracted cephalic veins and decreasing APD than were their corresponding (+)-enantiomers. The EC50 values for (-)-pinacidil and (-)-LY222675 in relaxing cephalic veins were 0.44 and 0.09 microM, respectively. In decreasing APD, the EC50 values were 3.2 microM for (-)-pinacidil and 0.43 microM for (-)-LY222675. The eudismic ratio was greater for the 3-pyridyl isomer than for pinacidil in both cardiac (71 vs. 22) and vascular (53 vs. 17) tissues. (-)-LY222675 and (-)-pinacidil (0.1-30 microM) also increased 86Rb efflux from cephalic veins to a greater extent than did their respective optical antipodes. The antidiabetic sulfonylurea, glyburide (1-30 microM), shifted the vascular concentration-response curve of (-)-pinacidil to the right by a similar extent at each inhibitor concentration. Glipizide also antagonized the response to (-)-pinacidil, but was about 1/10 as potent with a maximal shift occurring at 10 and 30 microM. Glyburide antagonized the vascular relaxant effects of 0.3 microM (-)-LY222675 (EC50, 2.3 microM) and reversed the decrease in APD caused by 3 microM (-)-LY222675 (EC50, 1.9 microM). Nitroprusside did not alter 86Rb efflux, and vascular relaxation induced by sodium nitroprusside was unaffected by sulfonylureas.

  19. HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels.

    Science.gov (United States)

    Zhang, Hai Xia; Akrouh, Alejandro; Kurata, Harley T; Remedi, Maria Sara; Lawton, Jennifer S; Nichols, Colin G

    2011-03-01

    Murine ventricular and atrial ATP-sensitive potassium (K(ATP)) channels contain different sulfonylurea receptors (ventricular K(ATP) channels are Kir6.2/SUR2A complexes, while atrial K(ATP) channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K(ATP) channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K(ATP) channels over atrial K(ATP) channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as (86)Rb(+) efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K(ATP) currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K(ATP) currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits (86)Rb(+) effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K(ATP) channels.

  20. Correlation between catecholamine release and sodium pump inhibition in the perfused adrenal gland of the cat

    Science.gov (United States)

    Garcia, A.G.; Garcia-Lopez, E.; Montiel, C.; Nicolas, G.P.; Sanchez-Garcia, P.

    1981-01-01

    1 Ca2+ reintroduction to retrogradely perfused and ouabain (10-4 M)-treated cat adrenal glands caused a catecholamine secretory response which was greater the longer the time of exposure to the cardiac glycoside. Such a response was proportional to the external Na+ concentration [Na+]o. 2 A qualitatively similar, yet smaller response was observed when glands were perfused with Krebs solution lacking K+ ions; thus, K+ deprivation mimicked the secretory effects of ouabain. Catecholamine secretion evoked by Ca2+ reintroduction in K+-free solution (0-K+) was also proportional to [Na+]o and greater the longer the time of exposure of the gland to 0-K+ solution. 3 The ionophore X537A also mimicked the ouabain effects, since Ca2+ reintroduction to glands treated with this agent (25 μM) caused a sharp secretory response. When added together with X537A, ouabain (10-4 M) did not modify the response to the ionophore. 4 N-ethylmaleimide (NEM), another Na+, K+-ATPase inhibitor, did not evoke the release of catecholamines; on the contrary, NEM (10-4 M) inhibited the catecholamine secretory response to high [K+]o, acetylcholine, Ca2+ reintroduction and ouabain. 5 Ouabain (10-4 M) inhibited the uptake of 86Rb into adreno-medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30-40 min) to reach its peak. NEM (10-4 M) blocked 86Rb uptake in a similar manner. 6 The results are further evidence in favour of the presence of a Na+-Ca2+ exchange system in the chromaffin cell membrane, probably involved in the control of [Ca2+]i and in the modulation of catecholamine secretion. This system is activated by increasing [Na+]i, either directly (ionophore X537A, increased [Na+]o) or indirectly (Na+ pump inhibition). However, the simple inhibition of Na+ pumping does not always lead to a catecholamine secretory response; such is

  1. Syringotoxin pore formation and inactivation in human red blood cell and model bilayer lipid membranes.

    Science.gov (United States)

    Szabó, Zsófia; Gróf, Pál; Schagina, Ludmila V; Gurnev, Philip A; Takemoto, Jon Y; Mátyus, Edit; Blaskó, Katalin

    2002-12-23

    The effect of syringotoxin (ST), a member of the cyclic lipodepsipeptides family (CLPs) produced by Pseudomonas syringae pv. syringae on the membrane permeability of human red blood cells (RBCs) and model bilayer lipid membranes (BLMs) was studied and compared to that of two recently investigated CLPs, syringomycin E (SRE) and syringopeptin 22A (SP22A) [Biochim. Biophys. Acta 1466 (2000) 79 and Bioelectrochemistry 52 (2000) 161]. The permeability-increasing effect of ST on RBCs was the least among the three CLPs. A time-dependent ST pore inactivation was observed on RBCs at 20 and 37 degrees C but not at 8 degrees C. From the kinetic model worked out parameters as permeability coefficient of RBC membrane for 86Rb(+) and pores mean lifetime were calculated. A shorter pores mean lifetime was calculated at 37 degrees C then at 20 degrees C, which gave us an explanation for the unusual slower rate of tracer efflux measured at 37 degrees C then that at 20 degrees C. The results obtained on BLM showed that the pore inactivation was due to a decrease in the number of pores but not to a change of their dwell time or conductance.

  2. Effect of ADH on rubidium transport in isolated perfused rat cortical collecting tubules

    Energy Technology Data Exchange (ETDEWEB)

    Schafer, J.A.; Troutman, S.L.

    1986-06-01

    Unidirectional fluxes of 86Rb+ were measured as an indicator of potassium transport in isolated rat cortical collecting tubules perfused and bathed at 38 degrees C with isotonic solutions in which Rb+ replaced K+. Under control conditions the lumen-to-bath flux (Jl----b) was significantly less than the bath-to-lumen flux (Jb----l), indicating net Rb+ secretion. Net secretion increased approximately 180% after addition of 100 microU/ml of arginine vasopressin (ADH) to the bathing solution, due to a rapid and reversible increase in Jb----l from 4.6 +/- 0.8 to 9.0 +/- 1.9 pmol X min-1 X mm-1 with no significant change in Jl----b. The ADH effect was completely inhibited by 2 mM luminal Ba2+. The average transepithelial voltage (Ve) was not significantly different from zero in the control period but became lumen negative (-5 to -10 mV) after ADH. With 10(-5) M amiloride in the lumen Ve was lumen positive (+2 to +4 mV) and was unaltered by ADH or Ba2+, yet ADH produced a significant but attentuated increase in Jb----l with no change in Jl----b. The results indicate that ADH augments net K+ secretion either by an increase in the Ba2+-sensitive conductance of the apical membrane or by an increase in the electrochemical potential driving force for net Rb+ secretion through this pathway.

  3. Increased activity of digoxin-like substance in low-renin hypertension in acromegaly

    Energy Technology Data Exchange (ETDEWEB)

    Soszynski, P.; Slowinska-Srzednicka, J.; Zgliczynski, S. (Medical Center for Postgraduate Education, Warsaw (Poland))

    1990-01-01

    Arterial hypertension is common in acromegaly, but the pathogenesis of this complication remains unknown. To determine the role of an endogenous Na,K pump inhibitor/digoxin-like substance (DLS) in the pathogenesis of hypertension in acromegaly 76 subjects: 28 with acromegaly, 20 with essential hypertension and 28 healthy controls were studied. Serum DLS was measured with the use of radioimmunoassay and bioassay by the inhibition of digoxin-sensitive erythrocyte 86-Rb uptake. In acromegaly, the activity of DLS was significantly increased and plasma renin activity decreased in the hypertensive group, as compared with that of the normotensive group and controls. Moreover, DLS was elevated in the low-renin group of essential hypertension, as compared with that of the normal/high-renin group or controls. The activity of DLS correlated positively with mean arterial pressure and negatively with plasma renin activity, but not with growth hormone levels. In conclusion, an endogenous sodium pump inhibitor/digoxin-like substance may play a role in the pathogenesis of low-renin hypertension in acromegaly.

  4. Measurement of the hyperfine splitting of {sup 133}Cs atoms in superfluid helium

    Energy Technology Data Exchange (ETDEWEB)

    Imamura, K., E-mail: kimamura@riken.jp [RIKEN Nishina Center (Japan); Furukawa, T. [Tokyo Metropolitan University, Department of Physics (Japan); Yang, X. F. [Peking University, School of Physics (China); Mitsuya, Y. [Meiji University, Department of Physics (Japan); Fujita, T. [Osaka University, Department of Physics (Japan); Hayasaka, M. [Tokyo Gakugei University, Department of Physics (Japan); Kobayashi, T. [RIKEN Center for Advanced Photonics (Japan); Hatakeyama, A. [Tokyo University of Agriculture and Technology, Department of Applied Physics (Japan); Ueno, H. [RIKEN Nishina Center (Japan); Odashima, H. [Meiji University, Department of Physics (Japan); Matsuo, Y. [Hosei University, Department of Advanced Sciences (Japan)

    2015-04-15

    We have been developing a new nuclear laser spectroscopy method named “OROCHI” (Optical RI-atom Observation in Condensed Helium as Ion-catcher). OROCHI utilizes superfluid helium (He II) not only as an efficient stopping medium of highly energetic ions but also as a host matrix of in-situ atomic laser spectroscopy. Using these characteristic of He II, we produce atomic spin polarization and measure Zeeman and hyperfine structure (HFS) splitting using laser-RF (radio frequency) / MW (microwave) double resonance method. From the measured energy splittings, we can deduce nuclear spins and moments. So far, we have conducted a series of experiments using both stable ({sup 85,87}Rb, {sup 133}Cs, {sup 197}Au, {sup 107,109}Ag) and unstable isotopes ({sup 84,86}Rb) to confirm the feasibility of OROCHI method, especially observing Zeeman resonance and determining nuclear spins. The measurement of HFS splitting of atoms introduced into He II is indispensable to clarify the nuclear properties by deducing nuclear moments as well as the study of nuclear spins. For this purpose, we perform a precision measurement of HFS of {sup 133}Cs atoms immersed in He II using laser ablation technique. In this paper, we describe the result of the experiment.

  5. Alpha adrenergic modulation of the Na/sup +/ pump of canine vascular smooth muscle

    Energy Technology Data Exchange (ETDEWEB)

    Navran, S.S.; Adair, S.E.; Allen, J.C.; Seidel, C.L.

    1986-03-01

    Some vasoactive agents, eg. beta adrenergic agonists and forskolin, stimulate the Na/sup 7/ pump by a cAMP- dependent mechanism. The authors have now demonstrated that phenylephrine (PE) stimulates the Na/sup 7/ pump in intact blood vessels as quantitated by an increased ouabain-sensitive /sup 86/Rb uptake. The stimulation is dose-dependent (ED/sub 50/, 3 x 10/sup -6/M) and blocked by phentolamine (I/sub 50/, 10/sup -7/M), prazosin (I/sub 50/, 10/sup -8/M) yohimbine (I/sub 50/, 10/sup -6/M) or elevated intracellular Na/sup +/. These data suggest that the Na/sup +/ pump stimulation is mediated through alpha/sub 1/ receptors which produce an influx of extracellular Na/sup +/. In vascular smooth muscle cell cultures PE stimulates the Na/sup +/ pump, but only when cells have been deprived of fetal calf serum (FCS). Since FCS is known to stimulate Na/sup +/influx, in the continuous presence of FCS, these cells may already be Na/sup +/-loaded and therefore refractory to further stimulation by alpha-adrenergic agents. Unlike those vasorelaxants whose mechanism involves stimulation of the Na/sup +/ pump, alpha adrenergic agents are vasoconstrictors and therefore the role of Na/sup +/ pump stimulation in this case may be as a mechanism of feedback inhibition of contractility.

  6. Intestinal circulation during inhalation anesthesia

    Energy Technology Data Exchange (ETDEWEB)

    Tverskoy, M.; Gelman, S.; Fowler, K.C.; Bradley, E.L.

    1985-04-01

    This study was designed to evaluate the influence of inhalational agents on the intestinal circulation in an isolated loop preparation. Sixty dogs were studied, using three intestinal segments from each dog. Selected intestinal segments were pumped with aortic blood at a constant pressure of 100 mmHg. A mixture of /sub 86/Rb and 9-microns spheres labeled with /sup 141/Ce was injected into the arterial cannula supplying the intestinal loop, while mesenteric venous blood was collected for activity counting. A very strong and significant correlation was found between rubidium clearance and microsphere entrapment (r = 0.97, P less than 0.0001). Nitrous oxide anesthesia was accompanied by a higher vascular resistance (VR), lower flow (F), rubidium clearance (Cl-Rb), and microspheres entrapment (Cl-Sph) than pentobarbital anesthesia, indicating that the vascular bed in the intestinal segment was constricted and flow (total and nutritive) decreased. Halothane, enflurane, and isoflurane anesthesia were accompanied by a much lower arteriovenous oxygen content difference (AVDO/sub 2/) and oxygen uptake than pentobarbital or nitrous oxide. Compared with pentobarbital, enflurane anesthesia was not accompanied by marked differences in VR, F, Cl-Rb, and Cl-Sph; halothane at 2 MAC decreased VR and increased F and Cl-Rb while isoflurane increased VR and decreased F. alpha-Adrenoceptor blockade with phentolamine (1 mg . kg-1) abolished isoflurane-induced vasoconstriction, suggesting that the increase in VR was mediated via circulating catecholamines.

  7. Effects of anaesthesia induction drugs on circulation in denervated intestinal loop preparation.

    Science.gov (United States)

    Tverskoy, M; Gelman, S; Fowler, K C; Bradley, E L

    1985-09-01

    The effect of anaesthesia induction drugs on the intestinal circulation was evaluated in an isolated loop preparation in 28 dogs. Selected intestinal loops were perfused with aortic blood by a pump at a constant pressure of 100 mmHg. A mixture of 86Rb and 9 microns spheres labeled with 141Ce was injected into the arterial cannula supplying the intestinal segment while mesenteric venous blood was collected for activity counting. Diazepam in a dose of 3 mg X kg-1 was accompanied by a significantly lower clearance (Cl-Rb), and permeability-surface area product (PS) than pentobarbitone; there were no differences between diazepam and pentobarbitone in total blood flow (BF), vascular resistance (VR) and oxygen consumption in the intestinal segments. Circulatory variable observed after midazolam, 8 mg X kg-1 and an additional 16 mg X kg-1, did not significantly differ from those seen during pentobarbitone. Ketamine in a dose of 8 mg X kg-1 was accompanied by a significantly lower BF, Cl-Rb, microsphere entrapment (Cl-Sph), PS, and higher VR and arterio-venous oxygen content difference. Sixteen mg X kg-1 of ketamine did not lead to any additional changes in determined variables of the intestinal circulation. Alpha-adrenoceptor blockade completely abolished vasoconstriction caused by ketamine, suggesting that the long-lasting vasoconstricting effect of ketamine on the intestinal circulation is mediated through catecholamines.

  8. O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Kim, M.S.; Akera, T.

    1987-02-01

    The role of O2 free radicals in the reduction of sarcolemmal Na+-K+-ATPase, which occurs during reperfusion of ischemic heart, was examined in isolated guinea pig heart using exogenous scavengers of O2 radicals and an inhibitor of xanthine oxidase. Ischemia and reperfusion reduced Na+-K+-ATPase activity and specific (3H)ouabain binding to the enzyme in ventricular muscle homogenates and also markedly lowered sodium pump activity estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices. These effects of ischemia and reperfusion were prevented to various degrees by O2-radical scavengers, such as superoxide dismutase, catalase, dimethyl-sulfoxide, histidine, or vitamin E or by the xanthine oxidase inhibitor, allopurinol. The degree of protection afforded by these agents paralleled that of reduction in enhanced lipid peroxidation of myocardial tissue as estimated from malondialdehyde production. These results strongly suggest that O2 radicals play a crucial role in the injury to sarcolemmal Na+-K+-ATPase during reperfusion of ischemic heart.

  9. Development of new techniques for using an atomic reactor in the study of agricultural environment and expansion of its utilization

    Energy Technology Data Exchange (ETDEWEB)

    Yuita, Koichi; Yamazaki, Shinichi; Akiyama, Tsuyoshi; Ota, Takeshi; Taniyama, Ichiro; Sakurai, Yasuhiro; Makino, Tomoyuki; Takahashi, Yoshiaki [National Inst. of Agro-Environmental Sciences, Tsukuba, Ibaraki (Japan)

    1997-02-01

    This study was made aiming to establish a method for simultaneous determinations of elements in soil. In the last year, the determination for elements having half lives of 2 weeks or less was performed and so, this study attempted to establish a determination method for 11 elements with longer half lives. When the determination by radiation analysis was carried out after cooling period of 1 week for {sup 76}As, {sup 122}Sb, {sup 131}Ba, {sup 140}La, {sup 147}Nd, {sup 153}Sm and {sup 175}Yb, and of 2 weeks for {sup 46}Sc, {sup 51}Cr, {sup 60}Co, {sup 86}Rb, {sup 141}Ce, {sup 152}Eu, {sup 177}Lu, {sup 181}Hf and {sup 182}Ta, quantification of these rare elements were able with a good accuracy and in a relatively short time by using the correction with titanium as the internal standard element. Then, to elucidate the relationship between the elution profiles of heavy metals in soil and the characteristics of organic compounds in soil, the conditions for neutron irradiation for direct labelling of heavy metals and those for water elution of those metals were investigated. The present results indicate that the profile of water elution might be influenced by the characteristics of organic compounds in soil. (M.N.)

  10. Choroid plexus potassium cotransport: modulation by osmotic stress and external potassium.

    Science.gov (United States)

    Keep, R F; Xiang, J

    1995-06-01

    The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide-sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 +/- 0.02, 0.44 +/- 0.04, and 0.84 +/- 0.06 min-1 in 240, 300, and 424 mOsm/kg solutions, respectively (p brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide-sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 +/- 1, 19 +/- 3, 13 +/- 2, and 6 +/- 1 nmol/mg/min after 0, 10, 30, and 60-min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide-sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.

  11. Altered erythrocyte Na-K pump in anorectic patients

    Energy Technology Data Exchange (ETDEWEB)

    Pasquali, R.; Strocchi, E.; Malini, P.; Casimirri, F.; Ambrosioni, E.; Melchionda, N.; Labo, G.

    1985-07-01

    The status of the erythrocyte sodium pump was evaluated in a group of patients suffering from anorexia nervosa and a group of healthy female control subjects. Anorectic patients showed significantly higher mean values of digoxin-binding sites/cell (ie, the number of Na-K-ATPase units) with respect to control subjects while no differences were found in the specific /sup 86/Rb uptake (which reflects the Na-K-ATPase activity) between the two groups. A significant correlation was found between relative weight and the number of Na-K-ATPase pump units (r = -0.66; P less than 0.0001). Anorectic patients showed lower serum T3 concentrations (71.3 +/- 53 ng/dL) with respect to control subjects (100.8 +/- 4.7 ng/dL; P less than 0.0005) and a significant negative correlation between T3 levels and the number of pump units (r = -0.52; P less than 0.003) was found. This study therefore shows that the erythrocyte Na-K pump may be altered in several anorectic patients. The authors suggest that this feature could be interrelated with the degree of underweight and/or malnutrition.

  12. Theoretical Study of the Molecular and Electronic Structures of CPDT-TCNQ and Its Difluoro and Dimethyl Derivatives

    Institute of Scientific and Technical Information of China (English)

    GONG Zhi-Jun; LI Qian-Shu

    2006-01-01

    CPDT-TCNQ and its derivatives are good candidates for charge-transfer acceptors.In this work, the electronic ground and excited states of CPDT-TCNQ as well as its difluoro and dimethyl derivatives are studied. The ground state optimized structures and energies were obtained using a restricted (closed-shell) density functional theory (DFT) as approximated by the various hybrid functionals (RB3LYP, RB3P86, RB3PW91). The 6-31G** and 6-31+G** basis sets were employed in calculations. All derivatives are planar and exhibit a quinoid structure in their electronic ground states. The energy and oscillator strength of the first 15 singlet-singlet electronic transitions have been investigated by applying the time-dependent density functional theory (TD-DFT) approximations to the correspondingly optimized ground state geometries. The results show the strongest absorption in electronic spectra of molecules due to the HOMO-LUMO electronic transition of the thiophene backbone.

  13. Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B.

    Science.gov (United States)

    Genisyuerek, Selda; Papatheodorou, Panagiotis; Guttenberg, Gregor; Schubert, Rolf; Benz, Roland; Aktories, Klaus

    2011-03-01

    Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.

  14. Distribution of 13N following intravenous injection of [13N]ammonia in the rat.

    Science.gov (United States)

    Freed, B R; Gelbard, A S

    1982-01-01

    Ammonia labeled with cyclotron-produced 13N was injected intravenously in rats and the content of 13N in 14 major organs and tissues was determined at eight intervals ranging from 0.2 to 50 min after injection. The distribution of 13N at 12 s was employed to estimate the unidirectional tissue extraction for ammonia. The estimated fractional extraction for most of the tissues studied ranged from 70 to 100%. The 12-s 13N concentrations in a number of tissues (with lungs and brain the principal exceptions) were found to be quite similar to those reported for 42K+ and 86Rb+ in the rat, suggesting a similar mechanism to transcapillary extraction. Most of the injected dose was initially extracted by the musculature, lungs, and kidneys. The lungs and kidneys released the bulk of their extracted ammonia-derived nitrogen within 10 min of injection. The gut, heart, and spleen also recirculated extracted nitrogen, but on a much smaller scale than the lungs and kidneys. The recirculated label was accumulated mainly by muscle, liver, and skin. The results suggest that the lungs and kidneys are important sources of systemically recirculated ammonia metabolites in the rat.

  15. Limits on the release of Rb isotopes from a zeolite based 83mKr calibration source for the XENON project

    CERN Document Server

    Hannen, V; Arneodo, F; Baudis, L; Beck, M; Bokeloh, K; Ferella, A D; Giboni, K; Lang, R F; Lebeda, O; Ortjohann, H -W; Schumann, M; Spalek, A; Venos, D; Weinheimer, C

    2011-01-01

    The isomer 83mKr with its half-life of 1.83 h is an ideal calibration source for a liquid noble gas dark matter experiment like the XENON project. However, the risk of contamination of the detector with traces of the much longer lived mother isotop 83Rb (86.2 d half-life) has to be ruled out. In this work the release of 83Rb atoms from a 1.8 MBq 83Rb source embedded in zeolite beads has been investigated. To do so, a cryogenic trap has been connected to the source for about 10 days, after which it was removed and probed for the strongest 83Rb gamma-rays with an ultra-sensitive Germanium detector. No signal has been found. The corresponding upper limit on the released 83Rb activity means that the investigated type of source can be used in the XENON project and similar low-background experiments as 83mKr generator without a significant risk of contaminating the detector. The measurements also allow to set upper limits on the possible release of the isotopes 84Rb and 86Rb, traces of which were created alongside ...

  16. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  17. Influence of fentanyl and morphine on intestinal circulation

    Energy Technology Data Exchange (ETDEWEB)

    Tverskoy, M.; Gelman, S.; Fowler, K.C.; Bradley, E.L.

    1985-06-01

    The influence of fentanyl and morphine on the intestinal circulation was evaluated in an isolated loop preparation in 37 dogs anesthetized with pentobarbital intravenously. Selected intestinal segments were pumped with aortic blood at a constant pressure of 100 mm Hg. A mixture of /sup 86/Rb and 9-micron spheres labeled with /sup 141/Ce was injected into the arterial cannula supplying the intestinal loop, while mesenteric venous blood was collected for activity counting. A strong correlation was found between the clearances of rubidium and microspheres (r = 0.97, P less than 0.0001), suggesting that the shunting of 9-micron spheres through the intestines reflects the shunting of blood through nonnutritive vessels. Intravenous fentanyl decreased oxygen uptake (O/sub 2/up), and vascular resistance (VR), and increased blood flow (BF), rubidium and microsphere clearances (Cl-Rb, Cl-Sph, respectively), and permeability--surface area product (PS) in a dose-related fashion. Intravenous morphine in a dose of 1 mg X kg-1 increased Cl-Rb (nutritive BF) without changes in total (nutritive and nonnutritive) BF. This increase in nutritive BF is probably related to morphine-induced histamine release. Morphine in a dose of 5 mg X kg-1 was accompanied by vasoconstriction that was completely abolished by alpha-adrenoceptor blockade. The data suggest that morphine-induced intestinal vasoconstriction is mediated via a release of epinephrine, apparently from the adrenal medulla. It is concluded that changes in the intestinal circulation during anesthesia with narcotics might play a certain role in the cardiovascular homeostasis during anesthesia and surgery. An increase in oxygen content in portal venous blood, resulting from a decrease in intestinal oxygen uptake, should facilitate hepatic oxygenation.

  18. Effects of Ouabain on Proliferation of Human Endothelial Cells Correlate with Na+,K+-ATPase Activity and Intracellular Ratio of Na+ and K.

    Science.gov (United States)

    Tverskoi, A M; Sidorenko, S V; Klimanova, E A; Akimova, O A; Smolyaninova, L V; Lopina, O D; Orlov, S N

    2016-08-01

    Side-by-side with inhibition of the Na+,K+-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na+ and K+ ratio ([Na+]i/[K+]i). Thus, we compared the dose- and time-dependences of the effect of ouabain on intracellular [Na+]i/[K+]i ratio, Na+,K+-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na+]i/[K+]i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na+,K+-ATPase activity by 25-30%, as measured by the rate of (86)Rb(+) influx. Higher ouabain concentrations inhibited Na+,K+-ATPase, increased [Na+]i/[K+]i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na+]i/[K+]i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na+]i/[K+]i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na+ and K+ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na+,K+-ATPase and decrease in the [Na+]i/[K+]i ratio.

  19. Relationship between alpha-1 receptors and cations in rat liver plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Smart, J.L.

    1986-01-01

    The influence of cations on binding of (/sup 3/H)-prazosin (PRZ), an alpha-1 specific antagonist, to alpha receptor sites in rat liver plasma membranes was examined. All cations tested were able to produce dose-dependent shifts to lower affinity binding sites for PRZ. The maximum number of binding sites was also observed to be altered. Inclusion of cations resulted in a slower observed rate constant for association as well as a delay in the dissociation of specifically bound PRZ following the addition of phentolamine. In contrast, the ability of (-)-norepinephrine to displace PRZ was enhanced by the addition of cations. The influence of alpha-1 receptor stimulation on Na/sup +//K/sup +/-ATPase activity in rat liver was examined by two methods - rat liver plasma membrane Na/sup +//K/sup +/-ATPase activity following liver perfusion in situ and /sup 86/Tb uptake in rat liver slices. The activity of the Na/sup +/ pump was found to be biphasic following exposure to phenylephrine (PE), an alpha-1 agonist. Stimulation (35%) was present over the first two minutes, while activity was inhibited over the interval of 5 to 10 minutes of continued PE exposure. Both phases were blocked by prazosin. The influence of DAG and protein kinase C (PKC) in alpha-1 receptor modulation of the Na/sup +/ pump was studied by employing 4-beta-phorbol (PMA), a phorbol ester which activates PKC. Perfusion of livers with PMA in situ or incubation with slices yielded inhibition of ATPase activity in membranes and /sup 86/Rb uptake in that was qualitatively and quantitatively similar to PE. These results suggest cations may influence receptor function in vivo and in vitro and the inhibitory effects of PE on the sodium pump may be mediated through PKC.

  20. Adrenergic and histaminergic neural interactions in dog paws

    Energy Technology Data Exchange (ETDEWEB)

    Baker, C.H.; Davis, D.L.; Sutton, E.T.; Lindsey, B.G. (Univ. of South Florida, Tampa (USA))

    1988-09-01

    The mechanisms underlying the vascular responses of superficial fibular nerve stimulation (SFNS) have not been defined. Right hindpaws of anesthetized heparinized dogs were vascularly and neurally isolated, enclosed in a volume recorder, and perfused with controlled pressure. Vascular volume (VV) ({sup 131}I-labeled albumin) and rate of tissue volume changes (V{sub T}) (plethysmography) were determined. SFNS increased blood flow resistance, reduced capillary filtration coefficient (CFC) and permeability-surface area product (PS) of {sup 86}Rb, increased VV, and reduced {sup 131}I-albumin recovery. SFNS during terbutaline increased resistance, CFC, PS, and VV were unchanged, {sup 131}I-albumin recovery was complete, and V{sub T} increased at one-fourth the control rate. Phentolamine and yohimbine blocked all responses to SFNS. Prazosin with SFNS attenuated hemodynamic changes and V{sub T} increased to two-thirds of control, decreased VV, albumin, and Rb recovery but not PS and CFC. SFNS during pyrilamine maleate reduced V{sub T} increase to two-thirds of control rate and blocked decreases in PS and CFC. Metiamide did not change the SFNS responses, except to reduce vascular volume and V{sub T}. The combined histamine H{sub 1} and H{sub 2} blockers reduced V{sub T} increase to one-third of control and attenuated albumin loss, prevented histamine dilation, attenuated vasopressin and norepinephrine but not angiotensin constriction. SFNS stimulation increased precapillary resistance by {alpha}{sub 1}- and {alpha}{sub 2}-receptors and venous resistance by {alpha}{sub 2}-receptors and increased permeability by histamine release from endothelium.

  1. Influence of fentanyl and morphine on intestinal circulation.

    Science.gov (United States)

    Tverskoy, M; Gelman, S; Fowler, K C; Bradley, E L

    1985-06-01

    The influence of fentanyl and morphine on the intestinal circulation was evaluated in an isolated loop preparation in 37 dogs anesthetized with pentobarbital intravenously. Selected intestinal segments were pumped with aortic blood at a constant pressure of 100 mm Hg. A mixture of 86Rb and 9-micron spheres labeled with 141Ce was injected into the arterial cannula supplying the intestinal loop, while mesenteric venous blood was collected for activity counting. A strong correlation was found between the clearances of rubidium and microspheres (r = 0.97, P less than 0.0001), suggesting that the shunting of 9-micron spheres through the intestines reflects the shunting of blood through nonnutritive vessels. Intravenous fentanyl decreased oxygen uptake (O2up), and vascular resistance (VR), and increased blood flow (BF), rubidium and microsphere clearances (Cl-Rb, Cl-Sph, respectively), and permeability--surface area product (PS) in a dose-related fashion. Intravenous morphine in a dose of 1 mg X kg-1 increased Cl-Rb (nutritive BF) without changes in total (nutritive and nonnutritive) BF. This increase in nutritive BF is probably related to morphine-induced histamine release. Morphine in a dose of 5 mg X kg-1 was accompanied by vasoconstriction that was completely abolished by alpha-adrenoceptor blockade. The data suggest that morphine-induced intestinal vasoconstriction is mediated via a release of epinephrine, apparently from the adrenal medulla. It is concluded that changes in the intestinal circulation during anesthesia with narcotics might play a certain role in the cardiovascular homeostasis during anesthesia and surgery. An increase in oxygen content in portal venous blood, resulting from a decrease in intestinal oxygen uptake, should facilitate hepatic oxygenation.

  2. The nicotinic receptor in the rat pineal gland is an alpha3beta4 subtype.

    Science.gov (United States)

    Hernandez, Susan C; Vicini, Stefano; Xiao, Yingxian; Dávila-García, Martha I; Yasuda, Robert P; Wolfe, Barry B; Kellar, Kenneth J

    2004-10-01

    The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [3H]epibatidine ([3H]EB) binds with an affinity of approximately 100 pM to nAChRs in the pineal gland, and the density of these sites is approximately 5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at alpha3beta4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block 86Rb+ efflux in the cells expressing heterologous alpha3beta4 nAChRs. Measurements of mRNA indicated the presence of transcripts for alpha3, beta2, and beta4 nAChR subunits but not those for alpha2, alpha4, alpha5, alpha6, alpha7, or beta3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [3H]EB-labeled nAChRs contained alpha3 and beta4 subunits associated in one complex. The beta2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the alpha3beta4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.

  3. The Na{sup +}/K{sup +} -pump in rat peritoneal mast cells: Some aspects of regulatio of activity and cellular fusion

    Energy Technology Data Exchange (ETDEWEB)

    Knudsen, T. [Odense Univ., Dept. of Pharmacology, Inst. of Medical Biology, The Faculty of Health Scineces (Denmark)

    1995-12-31

    The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also also have important clinical implications. The aim of the present work has been to document the existence of the Na{sup +}/K{sup +}-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na{sup +}/K{sup +}-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with {sup 86}Rb{sup +} as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE-directed stimulation of the cell was used as a parameter of cellular degranulation. Histamine was measured by spectrofluorometry. Besides describing aspects of the function and regulation of the Na{sup +}/K{sup +}-pump in the rat peritoneal mast cell, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases. (au) 253 refs.

  4. Mechanism of the pulmonary vasoconstrictor action of digoxin in the dog.

    Science.gov (United States)

    Mecca, T E; Elam, J T; Caldwell, R W

    1985-01-01

    The pulmonary vascular effects of a subarrhythmic dose of digoxin (60 micrograms/kg i.v.) were examined in the canine in situ perfused lung. Digoxin produced an increase in pulmonary vascular resistance (66.1%) and pulmonary arterial pressure (8.2 mm Hg) at 70 min after injection in the constant-flow, blood-perfused lung preparation. The digoxin-treated group exhibited higher plasma levels of norepinephrine compared with control dogs. The pulmonary vasoconstrictor response to digoxin was abolished by prior treatment with the alpha-adrenergic antagonists phenoxybenzamine and phentolamine. This vasoconstriction does not involve inhibition of synthesis or action of vasodilator prostaglandins by digoxin, as pretreatment with indomethacin did not attenuate, and even tended to increase, the pressor response to digoxin. The response was prevented by prior treatment with blockers of nonneuronal uptake of catecholamines normetanephrine and hydrocortisone, but not with cocaine, a blocker of neuronal uptake. In the lung preparation perfused with Krebs buffer solution, digoxin failed to produce vasoconstriction when administered intravenously (60 micrograms/kg) or in the perfusate at a concentration of 8 ng/ml, the blood level at the peak of the pressor response. Sodium-pump activity (ouabain-sensitive 86Rb+ uptake) of intralobular pulmonary arteries excised after 90 min of exposure to digoxin was the same as activity in arteries from control dogs. In conclusion, digoxin produces a pulmonary vasoconstriction through an alpha-adrenergic mechanism. Since the pressor response was observed only in the blood-perfused lung, blood-borne catecholamines are apparently involved.

  5. Potassium transport in opossum kidney cells: effects of Na-selective and K-selective ionizable cryptands, and of valinomycin, FCCP and nystatin.

    Science.gov (United States)

    Loiseau, A; Leroy, C; Castaing, M

    1997-11-13

    The effects of two ionizable cryptands, the Na-selective (221)C10 and the K-selective (222)C10, and of valinomycin, FCCP and nystatin on K+ fluxes in opossum kidney (OK) cells have been quantified. The Na,K-ATPase (ouabain-sensitive 86Rb influx) was stimulated by nystatin (> or = 20%), and inhibited by the other ionophores (50-80%), by barium (K-channel blocker) (61%) and by amiloride (Na entry blocker) (34%). The Vmax of the Na,K-ATPase phosphatase activity was unmodified by the ionophores, indicating the absence of direct interaction with the enzyme. The ATPi content was unmodified by the inhibitors and nystatin, but was lowered by (221)C10 (47%), (222)C10 (75%), valinomycin (72%) and FCCP (88%). Amiloride was found to partially remove the inhibition caused by (222)C10 (51%) and valinomycin (49%). Rb efflux was stimulated by nystatin (32%), unmodified by valinomycin, and was inhibited by (221)C10 (19%), (222)C10 (19%) and FCCP (10%). Barium (39%) and amiloride (32%) inhibited this efflux and, in their presence, the nystatin effect persisted, whereas that of the other ionophores vanished. At pH 6.4, the Rb efflux decreased by 14% of its value at pH 7.4, with no additional inhibition by cryptands. Cryptands are shown to inhibit the pH-sensitive K+-conductance, probably by inducing a K+-H+ exchange at the plasma membrane, and by uncoupling oxidative phosphorylation by inducing the entry of K+ and H+ (and possibly Ca2+) ions into the mitochondria.

  6. Membrane-Mediated Decrease in Root Exudation Responsible for Phorphorus Inhibition of Vesicular-Arbuscular Mycorrhiza Formation

    Science.gov (United States)

    Graham, James H.; Leonard, Robert T.; Menge, John A.

    1981-01-01

    The mechanism responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation in sudangrass (Sorghum vulgare Pers.) was investigated in a phosphorus-deficient sandy soil (0.5 micrograms phosphorus per gram soil) amended with increasing levels of phosphorus as superphosphate (0, 28, 56, 228 micrograms per gram soil). The root phosphorus content of 4-week-old plants was correlated with the amount of phosphorus added to the soil. Root exudation of amino acids and reducing sugars was greater for plants grown in phosphorus-deficient soil than for those grown in the phosphorus-treated soils. The increase in exudation corresponded with changes in membrane permeability of phosphorus-deficient roots, as measured by K+ (86Rb) efflux, rather than with changes in root content of reducing sugars and amino acids. The roots of phosphorus-deficient plants inoculated at 4 weeks with Glomus fasciculatus were 88% infected after 9 weeks as compared to less than 25% infection in phosphorus-sufficient roots; these differences were correlated with root exudation at the time of inoculation. For plants grown in phosphorus-deficient soil, infection by vesicular-arbuscular mycorrhizae increased root phosphorus which resulted in a decrease in root membrane permeability and exudation compared to nonmycorrhizal plants. It is proposed that, under low phosphorus nutrition, increased root membrane permeability leads to net loss of metabolites at sufficient levels to sustain the germination and growth of the mycorrhizal fungus during pre- and postinfection. Subsequently, mycorrhizal infection leads to improvement of root phosphorus nutrition and a reduction in membrane-mediated loss of root metabolites. PMID:16661955

  7. Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Ton, Hoai T; Smart, Amanda E; Aguilar, Brittany L; Olson, Thao T; Kellar, Kenneth J; Ahern, Gerard P

    2015-08-01

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

  8. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  9. Stichodactyla helianthus peptide, a pharmacological tool for studying Kv3.2 channels.

    Science.gov (United States)

    Yan, Lizhen; Herrington, James; Goldberg, Ethan; Dulski, Paula M; Bugianesi, Randal M; Slaughter, Robert S; Banerjee, Priya; Brochu, Richard M; Priest, Birgit T; Kaczorowski, Gregory J; Rudy, Bernardo; Garcia, Maria L

    2005-05-01

    Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well-characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. Chinese hamster ovary (CHO)-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. Stichodactyla helianthus peptide (ShK), isolated from S. helianthus venom and a known high-affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit 86Rb+ efflux from CHO-K1.hKv3.2b (IC50 approximately 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus laevis oocytes or in planar patch-clamp studies, ShK inhibited hKv3.2b channels with IC50 values of approximately 0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic beta cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with the blockade of Kv3 channels (i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential after hyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections). Taken together, these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations.

  10. Confirmation of mutant alpha 1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats.

    Science.gov (United States)

    Ruiz-Opazo, N; Barany, F; Hirayama, K; Herrera, V L

    1994-09-01

    As the sole renal Na,K-ATPase isozyme, the alpha 1 Na,K-ATPase accounts for all active transport of Na+ throughout the nephron. This role in renal Na+ reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)1079-->thymine (T) transversion, resulting in the substitution of glutamine276 with leucine and associated with decreased net 86Rb+ (K+) influx, was identified in Dahl salt-sensitive/JR rat kidney alpha 1 Na,K-ATPase cDNA. However, because a Taq polymerase chain reaction amplification-based reanalysis did not detect the mutant T1079 but rather only the wild-type A1079 alpha 1 Na,K-ATPase allele in Dahl salt-sensitive rat genomic DNA, we reexamined alpha 1 Na,K-ATPase sequences using Taq polymerase error-independent amplification-based analyses of genomic DNA (by polymerase allele-specific amplification and ligase chain reaction analysis) and kidney RNA (by mRNA-specific thermostable reverse transcriptase-polymerase chain reaction analysis). We also performed modified 3' mismatched correction analysis of genomic DNA using an exonuclease-positive thermostable DNA polymerase. All the confirmatory test results were concordant, confirming the A1079-->T transversion in the Dahl salt-sensitive alpha 1 Na,K-ATPase allele and its transcript, as well as the wild-type A1079 sequence in the Dahl salt-resistant alpha 1 Na,K-ATPase allele and its transcript. Documentation of a consistent Taq polymerase error that selectively substituted A at T1079 (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T1079 alpha 1 cDNA M13 subclones. This Taq polymerase error results in the reversion of mutant sequence back to the wild-type alpha 1 Na,K-ATPase sequence. This identifies a site- and nucleotide-specific Taq polymerase misincorporation, suggesting that a structural

  11. Transmembrane carboxyl residues are essential for cation-dependent function in the gastric H,K-ATPase.

    Science.gov (United States)

    Rabon, E C; Hoggatt, M; Smillie, K

    1996-12-13

    The K+-dependent ATPase activity of the H,K-ATPase was irreversibly inhibited by the carboxyl activating reagent, dicyclohexylcarbodiimide (DCCD). The inhibition was first order and displayed a concentration dependence with the K0.5 (DCCD) = 0.65 +/- 0.04 mM. KCl protected 70% of the ATPase activity from DCCD-dependent inhibition in a concentration-dependent manner with a K0.5 (K+) = 0.58 +/- 0.1 mM KCl. DCCD modification selectively inhibited the K+-dependent rather than ATP-dependent partial reactions including eosin fluorescence responses and ligand-stabilized initial tryptic cleavage patterns of the membrane-associated enzyme. DCCD modification also inhibited the binding of 86Rb+ and the fluorescent responses of the K+-competitive, fluorescent inhibitor 1-(2-methylphenyl)-4-methylamino-6-methyl-2, 3-dihydropyrrolo[3,2-c]quinoline. [14C]DCCD was incorporated into the H,K-ATPase in a time course identical to that describing the inactivation of the K+-dependent ATPase activity of the H,K-ATPase. A component of the [14C]DCCD incorporated into the H,K-ATPase was K+-sensitive where K+ reduced the [14C]DCCD incorporated into the enzyme by 1.6 nmol of [14C]DCCD/mg of protein. Membrane-associated tryptic peptides resolved from the [14C]DCCD-modified H,K-ATPase exhibited various K+ sensitivities with peptides at 23, 9.6, 8.2, 7.1, and 6.1 kDa containing 10-78%, 23-52%, 24-36%, 2%, and 3-4% K+-sensitivity, respectively. The N-terminal sequence of the K+-sensitive, approximately 23- and 9.6-kDa peptides was LVNE857, a C-terminal fragment of the ATPase alpha-subunit. The mass of the smaller peptide limited the residue assignment to the transmembrane M7/M8 domains and an intervening extracytoplasmic loop. An N-terminal sequence, SD840IM, was obtained from a 3.3-kDa, [14C]DCCD-labeled peptide resolved from a V8 digest of the partially purified alpha-subunit. This mass was sufficient to include LVNE but would exclude M8 and the intervening loop between M7 and M8. Glu857 is a

  12. Aespoe Hard Rock Laboratory. Final report of the first stage of the tracer retention understanding experiments

    Energy Technology Data Exchange (ETDEWEB)

    Winberg, A. [Conterra AB, Uppsala (Sweden); Andersson, Peter [Geosigma AB, Uppsala (Sweden); Hermanson, Jan [Golder Grundteknik, Solna (Sweden); Byegaard, Johan [Chalmers Univ. of Technology, Goeteborg (Sweden). Dept. of Nuclear Chemistry; Cvetkovic, V. [Royal Inst. of Tech., Stockholm (Sweden). Dept. of Water Resources Engineering; Birgersson, Lars [Kemakta Konsult AB, Stockholm (Sweden)

    2000-03-15

    from its surrounding. The near proximity of the experimental array to the tunnel (10-15 m) implies a strong gradient (approximately 10%) in the structure, which has to be overcome and controlled during the experiments. A methodology for characterising fracture pore space using resin injection, excavation using large diameter coring and subsequent analysis with photo-microscopic and image analysis techniques was developed and tested at a separate site. The results show that epoxy resin can be injected over several hours, and that the estimated areal spread is in the order of square metres. The mean apertures of the two investigated samples were 239 and 266 microns, respectively. Assessment of spatial correlation show practical ranges in the order of a few millimetres. Performed tracer tests with conservative tracers in Feature A show that the feature is connected between its interpreted intercepts in the array. The parameters evaluated from the conservative tests; flow porosity, dispersivity and fracture conductivity are similar, indicating a relative homogeneity. Previous work has identified cationic tracers, featured by sorption through ion exchange, as the most suitable tracers for sorbing tracer experiments at ambient Aespoe conditions. Laboratory experiments on generic Aespoe material and site-specific material included batch sorption experiments on various size fractions of the geological material, and through diffusion experiments on core samples of variable length on a centimetre length scale. The sorbtivity was found to be strongly affected by the biotite content and the sorption was also found to increase with contact time. The sorbtivity was found to follow the relative order; {sup 22}Na{sup +} < {sup 47}Ca{sup 2+} {approx_equal} {sup 85}Sr{sup 2+} << {sup 86}Rb{sup +} {approx_equal} {sup 133}Ba{sup 2+} The field tracer tests, using essentially the same cocktail of sorbing tracers as in the laboratory, were found to show the same relative sorbtivity as seen