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Sample records for 86rb vblizi porogov

  1. Special K: testing the potassium link between radioactive rubidium (86Rb) turnover and metabolic rate.

    PubMed

    Tomlinson, Sean; Mathialagan, Priya D; Maloney, Shane K

    2014-04-01

    The measurement of (86)Rb turnover recently has been suggested as a useful method for measuring field metabolic rate in small animals. We investigated a proposed mechanism of (86)Rb turnover, its analogy to K(+), by comparing the turnover of (86)Rb in a model insect, the rhinoceros beetle Xylotrupes gideon, fed a diet of plum jam or plum jam enriched with K(+) or Rb(+). The turnover of (86)Rb in the beetles on the K(+) and the Rb(+) diets was higher than that for beetles on the jam diet (F2,311=32.4; P=1.58×10(-13)). We also exposed the beetles to different ambient temperatures to induce differences in metabolic rate ( ) while feeding them the jam and K(+) diets. was higher at higher ambient temperature (Ta) for both jam (F1,11=14.56; P=0.003) and K(+) (F1,8=15.39; P=0.004) dietary groups, and the turnover of (86)Rb was higher at higher Ta for both jam (F1,11=10.80; P=0.007) and K(+) (F1,8=12.34; P=0.008) dietary groups. There was a significant relationship between (86)Rb turnover and for both the jam (F1,11=35.00; P=1.0×10(-3)) and the K(+) (F1,8=64.33; P=4.3×10(-5)) diets, but the relationship differed between the diets (F1,19=14.07; P=0.001), with a higher (86)Rb turnover in beetles on the K(+)-enriched than on the jam diet at all Ta. We conclude that (86)Rb turnover is related to K(+) metabolism, and that this is the mechanism of the relationship between (86)Rb turnover and . Studies relating (86)Rb turnover to should maintain dietary [K] as close as possible to that of natural diets for the most accurate calibrations for free-ranging animals.

  2. Effects of amino acids on membrane potential and 86Rb+ fluxes in pancreatic beta-cells

    SciTech Connect

    Henquin, J.C.; Meissner, H.P.

    1981-03-01

    The membrane potential of beta-cells was studied with microelectrodes in mouse islets and their potassium permeability was evaluated by measuring 86Rb+ fluxes in rat islets. In the absence of glucose, L-leucine, its metabolite ketoisocaproate, and its nonmetabolized analogue 2-aminonorbornane-2-carboxylic acid (BCH) depolarized beta-cells and triggered bursts of electrical activity like glucose. The effect of leucine was weak, but was potentiated by a low concentration of glucose or by theophylline; the effect of ketoisocaproate was stronger and faster than that of an equimolar concentration of glucose. Arginine alone produced only a fast depolarization of beta-cells, insufficient to trigger electrical activity. Leucine and arginine potentiated the activity induced by glucose. In a glucose-free medium, alanine only slightly depolarized beta cells, whereas isoleucine and phenylalanie had no effect. Leucine, ketoisocaproate, and BCH reversibly decreased 86Rb+ efflux from islets perifused in the absence of glucose and increased 86Rb+ uptake. By contrast, both in the absence or presence of glucose, arginine increased 86Rb+ efflux and decreased 86Rb+ uptake. It is proposed that leucine, ketoisocaproate, and BCH, as glucose, deplolarize beta-cells by decreasing their potassium permeability, whereas arginine acts differently. The appearance of bursts of electrical activity with secretagogues unrelated to glucose suggests that they reflect an intrinsic property of the beta-cell membrane.

  3. Lymphocyte (Na,K) ATPase-dependent 86Rb+ uptake in human obesity.

    PubMed

    Bozzo, C; Goria, M; Marena, S; Avagnina, S; Pagano, G

    1988-01-01

    Sodium and potassium ion-activated adenosinetriphosphatase (EC number 3.6.1.3) activity, measured as the uptake of 86 rubidium (an analogue of potassium) was determined on peripheral lymphocytes isolated from 20 normotensive obese subjects and 20 normal weight subjects. No difference in the total uptake of 86Rb or in the Na, K-ATPase-dependent uptake was observed in either group. Furthermore, no correlation between the body mass index (BMI) and the Na,K-ATPase-dependent 86Rb uptake was observed. However the Na,K-ATPase mediated 86Rb uptake was always positively correlated with basal blood insulin levels and the insulin sensitivity index. It may be concluded that no lymphocyte dysfunction of Na,K-ATPase was present in our obese patients and that its activity is controlled by insulin in both normal-weight and obese subjects.

  4. Efflux of 86Rb from rat and mouse pancreatic islets: the role of membrane depolarization.

    PubMed Central

    Matthews, E. K.; Shotton, P. A.

    1984-01-01

    The efflux of 86Rb from rat or mouse perifused islets preloaded with the isotope has been used as an index of the potassium permeability of the islet beta-cell membrane. Cellular transmembrane potentials were altered by changing [K]O or by direct electrical stimulation and the effects on potassium permeability examined. Omission of KCl from the medium perifusing rat islets induced a biphasic change in 86Rb efflux, a brief decline being superseded by a pronounced increase in efflux. Re-introduction of KCl, 4.7 mM, caused a further increase in 86Rb efflux preceding a return to control values. Increasing [K]O from 4.7 mM to 10 mM, 20 mM or 47 mM caused a phasic increase in 86Rb efflux with the magnitude of both the peak and average rate of efflux being dependent upon the extent of the change in [K]O. The increase in 86Rb efflux produced by [K]O, 47 mM, was attenuated by Co2+, 2.56 mM (51% inhibition) or quinine, 10 microM (47% inhibition), but efflux remained significantly (P less than 0.001) above control values. Electrical stimulation of single microdissected mouse pancreatic islets by currents of 0.1 to 0.5 mA evoked a rapid, phasic increase in 86Rb efflux. The magnitude of the response was unaffected by EGTA, 2 mM, or nupercaine, 100 microM. These observations are discussed in relation to the mechanisms controlling the potassium permeability, membrane potential and insulin secretion of the pancreatic islet beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6391599

  5. Role of diacylglycerol in adrenergic-stimulated sup 86 Rb uptake by proximal tubules

    SciTech Connect

    Baines, A.D.; Drangova, R.; Ho, P. )

    1990-05-01

    We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.

  6. Study of the sensitivity of neonates to digoxin: contribution of erythrocyte /sup 86/Rb uptake test

    SciTech Connect

    Zannad, F.; Marchal, F.; Royer, R.J.; Vert, P.; Robert, J.

    1981-01-01

    In general, there is little agreement how digoxin should be used in newborn, and the results of studies in this field seem contradictory. This study attempts a quantitative assessment of the number and the sensitivity of cellular receptors for digoxin in the organism, by the in vitro measurement of erythrocyte /sup 86/Rb neonates compared with adults and old people. Red blood cells are first incubated with differing concentrations of digoxin, and then incubated with /sup 86/Rb. The initial level of /sup 86/Rb uptake (Rbi) is that observed in the absence of digoxin. The 50% index of captation (IC50) is the digoxin concentration in nanograms per ml at which /sup 86/Rb uptake is half Rbi. Three grups of patients were studied: Group I: 12 neonates, less that 5 days old; Group II: 11 adults (26 to 57 years old); Group III: 9 elderly people (71 to 82 years old). Rbi was significantly lower in neonates (Mean +/- SD: 25.8% +/- 3.5, P less than 0.001) and in the elderly (29.9% +/- 3.1) than in adults (36.8% +/- 4.6). IC50 was significantly lower in the elderly (12.1 mg/ml +/- 2.4) than in the adult patients (20.5 ng/ml +/- 5.5, P less than 0.001). In the newborns, values of IC50 were widely scattered (16.2 ng/ml +/- 7.2). The authors suggest that since Rbi reflects Na+, K+-ATPase activity, this activity is diminished in newborn and old people, and indicates that they have fewer cellular recaptors for digoxin than adults. In the elderly, the low IC50 would imply increased sensitivity to digoxin. In neonates, the wide range of values for IC50 suggests considerable individual variation in sensitivity to digoxin. The results aer consistent with the recently recomnended lower dosages of digoxin i neonates.

  7. Exodus of 42K+ and 86Rb+ from rat thymic and human blood lymphocytes exposed to phytohemagglutinin.

    PubMed

    Segel, G B; Gordon, B R; Lichtman, M A; Hollander, M M; Klemperer, M R

    1976-03-01

    We have found that PHA produces an alteration in the lymphocyte membrane which allows 86Rb+ or 42K+ in prelabeled lymphocytes to exchange for cations present in washing solutions. These observations suggested that PHA might induce an increase in the exodus of intracellular potassium during incubation in physiologic media. We, therefore, examined 86Rb+ and 42K+ efflux from rat and human lymphocytes during incubation in tissue culture medium. The rate constant for efflux, Ke, was significantly increased by PHA. 86Rb+ efflux was increased by 27% in rat thymic lymphocytes and by 78% in human blood lymphocytes following PHA treatment.

  8. Paradoxical inhibitory effect of cromakalim on sup 86 Rb outflow from pancreatic islet cells

    SciTech Connect

    Lebrun, P.; Antoine, M.H.; Devreux, V.; Hermann, M.; Herchuelz, A. )

    1990-12-01

    Cromakalim appears to be the most potent pharmacologic agent belonging to the new class of smooth muscle relaxants: the K+ channel openers. The present study aimed at characterizing the effects of cromakalim on 86Rb outflow, 45Ca outflow and insulin release from prelabeled and perifused rat pancreatic islets. Cromakalim provoked a concentration-dependent reduction in 86Rb outflow. This inhibitory effect was attenuated in islets exposed throughout to glibenclamide or to a Ca+(+)-free medium. In islets exposed to glucose and extracellular Ca++, cromakalim induced a dose-dependent reduction in 45Ca outflow. The drug also inhibited the increase in 45Ca outflow mediated by K+ depolarization. Lastly, cromakalim elicited a concentration-dependent inhibition of insulin release from islets perifused in the presence of glucose and extracellular Ca++. The present data suggest that the paradoxical inhibitory effect of cromakalim on 86Rb outflow probably reflects the capacity of the drug to reduce the activity of the ATP-sensitive K+ channels and to indirectly inhibit the Ca+(+)-activated K+ channels. Furthermore, the cromakalim-induced changes in 45Ca outflow are compatible with an inhibitory effect of the drug on the voltage-dependent Ca++ channels.

  9. Leiurus quinquestriatus venom inhibits BRL 34915-induced /sup 86/Rb/sup +/ efflux from the rat portal vein

    SciTech Connect

    Quast, U.; Cook, N.S.

    1988-01-01

    The effect of the crude venom of the Israeli scorpion Leiurus quinquestriatus hebraeus on the /sup 86/Rb/sup +/ efflux stimulated by the K/sup +/ channel opener BRL 34915 in the rat portal vein was examined. Applied alone, the venom greatly increased the spontaneous mechanical activity of and the concomitant /sup 86/Rb/sup +/ efflux from the vessel. When the excitability of the vein was suppressed by the dihydropyridine calcium antagonist, PN 200-110, the /sup 86/Rb/sup +/ efflux stimulated by BRL 34915 could be shown to be inhibited by the venom. From the concentration dependence of this inhibition an IC/sub 50/ value of 0.17 +/- 0.01 mg/ml was estimated. This venom is thus the most potent blocker of BRL 34915-evoked /sup 86/Rb/sup +/ efflux reported so far. 17 references, 2 figures.

  10. Comparison of effects of cromakalim and pinacidil on mechanical activity and 86Rb efflux in dog coronary arteries

    SciTech Connect

    Masuzawa, K.; Asano, M.; Matsuda, T.; Imaizumi, Y.; Watanabe, M. )

    1990-05-01

    Effects of two K+ channel openers, cromakalim and pinacidil, on mechanical activity and on 86Rb efflux were compared in strips of dog coronary arteries. Cromakalim and pinacidil produced the relaxation in 20.9 mM K(+)-contracted strips with a pD2 of 6.53 and 5.95, respectively. In 65.9 mM K(+)-contracted strips, high concentrations of pinacidil, but not cromakalim, produced relaxation. Ca+(+)-induced contractions in 80 mM K(+)-depolarized strips were also inhibited by pinacidil but not by cromakalim. Glibenclamide, a blocker of ATP-regulated K+ (KATP) channels, competitively antagonized the relaxant responses to cromakalim with a pA2 value of 7.62. However, the antagonism by glibenclamide of the relaxant responses to pinacidil was not a typical competitive type, suggesting the contribution of other effects than the KATP channel opening activity to the relaxant effects of pinacidil. In resting strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux in a dose-dependent manner. The increase in the 86Rb efflux induced by cromakalim was greater than that by pinacidil. When the effects of cromakalim and pinacidil on the 86Rb efflux were determined in the 20.9 or 65.9 mM K(+)-contracted strips, both drugs increased the 86Rb efflux. Under the same conditions nifedipine, a Ca(+)+ channel blocker, produced the relaxation that is accompanied by the decrease in 86Rb efflux. The increase in the 86Rb efflux induced by cromakalim was much greater than that by pinacidil.

  11. Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of /sup 86/Rb+ efflux

    SciTech Connect

    Hunter, D.D.; Nathanson, N.M.

    1985-09-01

    An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute /sup 86/Rb+ as the tracer ion. Cardiac cells accumulate /sup 86/Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of /sup 86/Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for /sup 42/K+. The mAChR agonist carbamylcholine (carbachol) stimulated /sup 86/Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of /sup 86/Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of /sup 42/K+.

  12. Ba2+-inhibitable /sup 86/Rb+ fluxes across membranes of vesicles from toad urinary bladder

    SciTech Connect

    Garty, H.; Civan, M.M.

    1987-01-01

    /sup 86/Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6 mM. The effects of externally added cations on /sup 86/Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. The Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.

  13. Cerebrovascular permeability to 86Rb in the rat after exposure to pulsed microwaves

    SciTech Connect

    Goldman, H.; Lin, J.C.; Murphy, S.; Lin, M.F.

    1984-01-01

    Microwaves (pulsed, 2,450 MHz) at an average power density of 3 W/cm2 were applied directly to the head for 5, 10, or 20 min, producing a peak specific absorption rate of 240 W/kg in the brain, which, after a 10-min exposure, resulted in brain temperatures in excess of 43 degrees C. A bolus of 86Rb in isotonic saline was injected intravenously and an arterial sample was collected for 20 s to determine cardiac output. Compared with unexposed controls, uptake of 86Rb increased most in those regions directly in the path of the irradiation, namely, the occipital and parietal cortex, as well as the dorsal hippocampus, midbrain, and basal ganglia. In a separate group of animals, regional brain-vascular spaces were found to increase with brain temperature. These results support previous observations indicating that reliably demonstrable increases of blood-brain barrier permeability are associated with intense, microwave-induced hyperthermia, and that the observed changes are not due to field-specific interaction.

  14. Experimental regional myocardial ischaemia and myocardial uptake of potassium analogues; intercomparison of 42K, 86Rb and 201Tl.

    PubMed

    Kasalický, J; Mrhová, O; Stýs, V

    1983-01-01

    The uptake of 42K, 86Rb and 201Tl by non-ischaemic and ischaemic myocardium was determined in rats with coronary artery ligature lasting 10, 30, 60 and 120 min, and in control rats without ischaemia. Whereas the myocardial concentration of 201Tl and 42K in control rats was similar and higher than that of 86Rb, 201Tl was superior to the other two radionuclides due to its significantly higher accumulation in non-ischaemic myocardium and the higher ratio of non-ischaemic to ischaemic radioactivity. The 86Rb accumulation in non-ischaemic myocardium and non-ischaemic/ischaemic ratio began to decrease from its maximum at 10 min. 201Tl, 42K and 86Rb blood levels in intact animals decreased rapidly after intravenous injection to low and nearly stabilized values at 5 min. Na+K+-ATPase activity in the ischaemic myocardium was high in the acutely ischaemic myocardium and decreased to below control levels after 4 h of ischaemia; changes in activity could not influence the low uptake of potassium analogues in fresh ischaemic myocardium.

  15. /sup 22/Na+ and /sup 86/Rb+ transport in vascular smooth muscle of SHR, Wistar Kyoto, and Wistar rats

    SciTech Connect

    Kuriyama, S.; Denny, T.N.; Aviv, A.

    1988-06-01

    To gain further insight into differences in cellular Na+ and K+ regulation between the spontaneously hypertensive rat (SHR), Wistar Kyoto (WKY), and American Wistar (W) rats, 22Na+ and 86Rb+ washouts were performed under steady-state conditions in cultured vascular smooth muscle cells from the three rat strains. SHR vascular smooth muscle cells showed significantly higher bumetanide sensitive 86Rb+ washout rate constant (x 10(-4)/min; mean +/- SEM) than WKY cells (-38.6 +/- 2.84 and -23.8 +/- 3.58, respectively; p less than 0.005). SHR vascular smooth muscle cells also exhibited significantly higher values than WKY cells in the total 22Na+ washout rate constant (x 10(-2)/min) (-61.0 +/- 1.57 vs. -53.8 +/- 1.24; p less than 0.005). The amiloride sensitive component of the 22Na+ washout rate constant accounted for these differences (-18.6 +/- 1.04 for SHR and -12.1 +/- 2.00 for WKY; p less than 0.05). There were no apparent differences in cellular Na+ concentrations between WKY and SHR cells. In general, the 86Rb+ and 22Na+ washout parameters of W rat cells were quite similar to those of cells from SHR. We conclude that the bumetanide-sensitive 86Rb+ washout (the Na+ K+-cotransport), the overall, and the amiloride-sensitive 22Na+ washout (the latter primarily represents the Na+/H+ antiport) are higher in SHR than WKY rat vascular smooth muscle cells. These findings indicate innate differences in cellular Na+ and K+ transport in vascular smooth muscle cells of the SHR and WKY rat. The mechanisms responsible for these differences are yet to be determined.

  16. Erythrocyte sodium concentration and sup 86 Rb uptake in weanling Dahl rats

    SciTech Connect

    McCormick, C.P.; Hennessy, J.F.; Rauch, A.L.; Buckalew, V.M. Jr. )

    1989-08-01

    Alterations in Na, K ATPase pump activity as well as erythrocyte (RBC) intracellular sodium concentration (Nai) have been demonstrated in humans and rats with established hypertension. The contribution of hypertension itself to these changes is unclear. Accordingly, we investigated RBC ion transport and plasma ouabain-like factor (OLF) in four- to five-week old normotensive Dahl salt-sensitive (DS) and salt-resistant (DR) rats on low salt diet. Although both strains were normotensive, systolic blood pressure (SBP) of DS (123 {plus minus} 2 mm Hg) was higher than that of DR (116 {plus minus} 1 mm Hg). No interstrain difference was evident in RBC pump activity measured as ouabain-sensitive 86rubidium ({sup 86}Rb) uptake (DS = 0.277 {plus minus} .030 and DR = 0.271 {plus minus} .029 mumol/10(9)RBC/h) even though RBC Nai was greater in DS than DR (14.9 {plus minus} 2.0 v 10.7 {plus minus} 1.0 mEq/L; P less than 0.05). Plasma OLF was higher in DS than DR (28.9 {plus minus} 4.7 v 16.5 {plus minus} 2.3 pmol/mL; P less than 0.05), but did not correlate with RBC pump activity in either strain. RBC Nai was directly correlated with pump activity in DS (r = 0.84, P less than 0.01) and demonstrated a trend to correlate in DR (r = 0.71, P = 0.07). RBC Nai was also directly correlated with SBP in DR (r = 0.73, P less than 0.05) and DS (r = 0.70, P = 0.05). We conclude that RBC Nai is genetically determined in Dahl rats and is elevated in normotensive DS who are at risk for hypertension development.

  17. Effect of aldosterone on /sup 86/Rb fluxes in cultured kidney cells (A6)

    SciTech Connect

    Fidelman, M.L.; Duncan, R.L.; Watlington, C.O.

    1988-01-01

    This study was designed to evaluate the relative contributions of hormone induced changes in active and passive K+ transport in an epithelial cell line in continuous culture derived from toad kidney (A6) using /sup 86/Rb as a tracer for measuring unidirectional K+ fluxes. The effects of 24 h exposure to aldosterone (A) and aldosterone plus insulin (A+I) on unidirectional K+ fluxes were evaluated under short-circuited conditions and under open circuit conditions. In epithelia exposed to A, a small but significant amount of active K+ secretion was found, although it was not significantly greater than in control epithelia. The bidirectional fluxes in both A and A+I treated epithelia, under short-circuited conditions, increased by a similar amount over control values indicating an increase in apparent permeability of passive transepithelial K+ transport. Under open circuit conditions, A stimulated net K+ transport by about 5-fold over controls. The increase in K+ secretion produced by A under open circuit conditions could be explained by the combined effects of an increase in transepithelial K+ permeability and an increase in the transepithelial electrical potential difference (PD). The presence of I produced no additional effects to that of A on K+ transport under the conditions used in this study. It is concluded that the substantial increase in K+ secretion induced in A6 cells by 24 h exposure to A is primarily passive in nature. It is possible that the changes in both PD and transepithelial K+ permeability, which can account for the observed increase in K+ secretion, are secondary to the stimulation of active Na+ transport.

  18. Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components

    SciTech Connect

    Ortiz, O.E.; Lew, V.L.; Bookchin, R.M.

    1986-03-01

    We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.

  19. 86Rb+ Efflux Mediated by α4β2*-Nicotinic Acetylcholine Receptors with High and Low Sensitivity to Stimulation by Acetylcholine Display Similar Agonist-Induced Desensitization

    PubMed Central

    Marks, Michael J.; Meinerz, Natalie M.; Brown, Robert W. B.; Collins, Allan C.

    2010-01-01

    The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration-effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine stimulated 86Rb+ efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC50 values for each agonist for desensitization of 86Rb+ efflux both with high (EC50≈3 μM) and low (EC50≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective KD values for receptor binding. Even though the two components of α4β2*-nAChR mediated 86Rb+ efflux from mouse brain differ markedly in EC50 values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/hr and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high sensitivity and low sensitivity acetylcholine-stimulated 86Rb+ efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal 86Rb+ efflux from thalamus, IC50 values in both brain regions were unaffected by chronic nicotine treatment. PMID:20599770

  20. Evaluation of in vivo detection properties of 22Na, 65Zn, 86Rb, 109Cd and 137Cs in plant tissues using real-time radioisotope imaging system

    NASA Astrophysics Data System (ADS)

    Sugita, Ryohei; Kobayashi, Natsuko I.; Hirose, Atsushi; Tanoi, Keitaro; Nakanishi, Tomoko M.

    2014-02-01

    In plant research, radioisotope imaging provides useful information about physiological activities in various tissues and elemental transport between plant organs. To expand the usage of imaging techniques, a new system was developed to visualize beta particles, x-rays and gamma-rays emitted from plant bodies. This real-time radioisotope imaging system (RRIS) visualizes radioactivity after conversion into light with a CsI(Tl) scintillator plate. Herein, the RRIS detection properties of the gamma-ray emitters 22Na, 65Zn, 86Rb, 109Cd and 137Cs were evaluated in comparison with those of radioluminography (RLG) using an imaging plate. The lower quantitative detection limit (Bq mm-2) during a 15 min period ranged from 0.1 to 4, depending on the nuclide, similar to that of RLG. When the quantitative ability to detect radiation from various Arabidopsis tissues was analyzed, the quantitative capability in silique and the thick internode tended to be low. In an EGS5 simulation, beta particles were the greatest contributors to RRIS imaging of 22Na, 86Rb and 137Cs, and low-energy x-rays contributed significantly to 65Zn and 109Cd detection. Thus, both self-absorption and air space between the sample and scintillator surface could impair quantitative RRIS imaging. Despite these issues, RRIS is suggested for quantitative time-course measurements of radionuclide motion within plants.

  1. Characterization of bicarbonate-dependent potassium uptake in cultured corneal endothelial cells

    SciTech Connect

    Savion, N.; Farzame, N.; Berlin, H.B.

    1989-04-01

    Bovine corneal endothelial (BCE) cells in culture demonstrated 86Rb+ uptake which was mostly ouabain-sensitive with some (15 to 50%) ouabain-insensitive uptake that was dependent on the presence of bicarbonate in the incubation medium. Bovine smooth muscle (SM) cells demonstrated ouabain-sensitive 86Rb+ uptake but the ouabain-insensitive 86Rb+ uptake was not bicarbonate-dependent. Although omission of bicarbonate from the incubation buffer resulted in some reduction in the pH, this change was not responsible for the reduction in the ouabain-insensitive 86Rb+ uptake. Furthermore, the removal of bicarbonate decreased the 86Rb+ influx but not its efflux. This ouabain-insensitive and bicarbonate-dependent 86Rb+ influx in BCE cells proceeded at a linear rate for at least 60 min and increased as a function of bicarbonate concentration such that almost maximal uptake was observed at a concentration of about 10 to 15 mM. Saturation of the bicarbonate-dependent 86Rb+ pump in BCE cells occurred at a concentration of 2 mM Rb+ in the incubation buffer, similar to the previously observed value for the Na+, K+-ATPase. Competition experiments with both unlabeled Rb+ and K+ demonstrated that likewise in the Na+, K+-ATPase the 86Rb+ influx represented physiological influx of K+. Furthermore, the energy requirements of the bicarbonate-dependent 86Rb+ uptake were similar to those of the 86Rb+ uptake via the Na+, K+-ATPase. The results described in this work demonstrated a novel bicarbonate-dependent K+ pump in addition to the Na+, K+-ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Possible involvement of ATP-sensitive K+ channels in the relaxant response of dog middle cerebral artery to cromakalim

    SciTech Connect

    Masuzawa, K.; Asano, M.; Matsuda, T.; Imaizumi, Y.; Watanabe, M. )

    1990-11-01

    To determine the functions of ATP-sensitive K+ (KATP) channels in cerebral arterial smooth muscle, the effects of cromakalim, an opener of these channels, on tension and 86Rb efflux were investigated in endothelium-removed strips of dog middle cerebral arteries (MCAs). Cromakalim relaxed the strips that were precontracted with 20.9 mM K+ with a small maximum response. The relaxant responses to cromakalim were competitively antagonized by glibenclamide, a blocker of KATP channels. In strips precontracted with 65.9 mM K+, cromakalim failed to relax the strips. The addition of cromakalim to a resting strip caused a dose-dependent relaxation. In the resting strips of MCAs preloaded with 86Rb, cromakalim did not increase the 86Rb efflux. With 42K as the tracer ion, cromakalim still had no effect on the efflux from the resting strips. On the other hand, cromakalim increased the 86Rb and 42K efflux from the strips of dog coronary arteries (CAs). In 20.9 mM K(+)-contracted strips of MCAs, cromakalim significantly decreased the 86Rb efflux. However, after the inactivation of Ca(++)-activated K+ channels by the addition of 1 x 10(-7) M nifedipine to the 20.9 mM K(+)-contracted strips of MCAs, cromakalim produced a small but significant increase in the 86Rb efflux. Similarly, when the resting strips of MCAs were placed in the Ca(++)-free 12 mM-Mg(+)+ solution, cromakalim increased the 86Rb efflux. In 65.9 mM K(+)-contracted strips, cromakalim increased the 86Rb efflux from both arteries. However, the extent of the increase in 86Rb efflux was significantly smaller in the MCA than in the CA.

  3. Bradykinin and vasopressin stimulate Na/sup +/-K/sup +/-Cl/sup -/ cotransport in cultured endothelial cells

    SciTech Connect

    Brock, T.A.; Brugnara, C.; Canessa, M.; Gimbrone, M.A. Jr.

    1986-06-01

    The authors have characterized a Na/sup +/-K/sup +/-Cl/sup -/ cotransporter in vascular endothelial cells (EC) cultured from different blood vessels and species that is inhibited by the diuretics furosemide and bumentanide. Inward /sup 86/Rb influx transported by the Na/sup +/-K/sup +/ pump in cultured EC from bovine and pig aorta, bovine vena cava, and baboon cephalic vein but not in human umbilical or saphenous vein EC. External Na/sup +/ or Cl/sup -/-stimulated, ouabain-insensitive /sup 86/Rb influx is equal to furosemide or bumetanide-sensitive /sup 86/Rb influx. Ouabain-insensitive /sup 22/Na influx is also partially inhibited by these drugs and stimulated by increasing external K/sup +/ or Cl/sup -/. Net Na/sup +/ extrusion occurs via the Na/sup +/-K/sup +/-Cl/sup -/ cotransporter in the absence of external K/sup +/, whereas net Na/sup +/ influx occurs at higher external K/sup +/. Maximal concentrations (100 nM) of bradykinin and vasopressin increase the initial rate of bumetanide-sensitive /sup 86/Rb influx by approx.60 and 70%. Addition of either ethyleneglycol-bis(..beta..-aminotethylether)-N,N'-tetraacetic acid or LaCl/sub 3/ (to block calcium influx) prevents bradykinin-stimulated /sup 86/Rb influx. When intracellular calcium is elevated using ionomycin (100 nM), a Ca/sup 2 +/ionophore, bumetanide-sensitive /sup 86/Rb influx increases approx.twofold. In contrast, isoproterenol (100 ..mu..M) and forskolin (50 /sup +/M), adenylate cyclase stimulators, decrease furosemide-sensitive /sup 86/Rb influx. Thus in certain types of cultured EC, a Na/sup +/-K/sup +/-Cl/sup -/ cotransporter mediates a fraction of K/sup +/ influx quantitatively as important as the Na/sup +/-K/sup +/ pump (ouabain-sensitive /sup 86/Rb influx) and appears to be modulated by Ca/sup 2 +/ and cyclic nucleotides.

  4. Erythrocyte cation transport and age: effects of digoxin and furosemide

    SciTech Connect

    Kelly, J.G.; Copeland, S.; McDevitt, D.G.

    1983-08-01

    The uptake of rubidium 86 (/sup 86/Rb) by human erythrocytes was measured at various ages. Effects of digoxin and furosemide on this process were examined and, in the case of digoxin, related to its numbers of specific cellular binding sites. There were no significant effects of age on absolute cellular Rb uptake, digoxin-sensitive Rb uptake, or numbers of cellular binding sites for digoxin, but the ability of digoxin to inhibit digoxin-sensitive /sup 86/Rb uptake increased with age. The ability of furosemide to inhibit digoxin-insensitive /sup 86/Rb uptake did not change with age. Results suggest a dynamic contribution to altered sensitivity to digoxin in elderly persons.

  5. Effect of imposed serum deprivation on growth of the mouse 3T3 cell. Dissociation from changes in potassium ion transport as measured from [36Rb]rubidium ion uptake

    PubMed Central

    Tupper, Joseph T.; Zografos, Linda

    1978-01-01

    Decreased serum concentrations that substantially alter the growth of normal 3T3 cells alter neither the active and non-active components of unidirectional 86Rb+ influx nor the intracellular K+ content when compared with cells in exponential growth. Thus the changes in K+ transport (measured with 86Rb+ as an analogue for K+ movements) that occur on density-dependent growth inhibition of the mouse 3T3 cell are not mimicked by serum deprivation of the cells before density inhibition. PMID:728075

  6. Direct Regulation of Prokaryotic Kir Channel by Cholesterol*

    PubMed Central

    Singh, Dev K.; Rosenhouse-Dantsker, Avia; Nichols, Colin G.; Enkvetchakul, Decha; Levitan, Irena

    2009-01-01

    Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by 86Rb+ uptake. Our results show that 86Rb+ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses 86Rb+ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated 86Rb+ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific. PMID:19740741

  7. Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin.

    PubMed

    Haber, R S; Pressley, T A; Loeb, J N; Ismail-Beigi, F

    1987-07-01

    The Na+ ionophore monensin was used to study the Na+- and K+-dependence of ouabain-inhibitable 86Rb+ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na+ that were stable for up to 2 h. In experiments in which cellular Na+ was thus "clamped" at various levels, the activation curve for ouabain-inhibitable 86Rb+ uptake as a function of intracellular Na+ was found to be steepest near basal Na+ levels (Hill coefficient approximately equal to 2.4), indicating that these cells can respond to relatively large changes in passive Na+ entry by increasing the race of Na-K pump function with only minimal increases in cellular Na+. Exposure of cells to monensin also permitted examination of the extracellular-K+ dependence of ouabain-inhibitable 86Rb+ uptake in the presence of saturating intracellular Na+ and yielded a Hill coefficient of approximately 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable 86Rb+ uptake in intact cells was similar to the enzymatic Vmax of the Na+-K+-ATPase in cell lysates, suggesting that the Na+-K+-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.

  8. Effect of angiotensin II, ATP, and ionophore A23187 on potassium efflux in adrenal glomerulosa cells

    SciTech Connect

    Lobo, M.V.; Marusic, E.T.

    1986-02-01

    Angiotensin II stimulus on perifused bovine adrenal glomerulosa cells elicited an increase in 86Rb efflux from cells previously equilibrated with the radioisotope. When 45Ca fluxes were measured under similar conditions, it was observed that Ca and Rb effluxes occurred within the first 30 s of the addition of the hormone and were independent of the presence of external Ca. The 86Rb efflux due to angiotensin II was inhibited by quinine and apamin. The hypothesis that the angiotensin II response is a consequence of an increase in the K permeability of the glomerulosa cell membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated 86Rb or K loss (as measured by an external K electrode). This increased K conductance was also seen with 10(-4) M ATP. Quinine and apamin greatly reduced the effect of ATP or A23187 on 86Rb or K release in adrenal glomerulosa cells. The results suggest that Ca-dependent K channels or carriers are present in the membranes of bovine adrenal glomerulosa cells and are sensitive to hormonal stimulus.

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

    SciTech Connect

    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.

  10. Volume regulation by human lymphocytes. Role of calcium

    SciTech Connect

    Grinstein, S.; Dupre, A.; Rothstein, A.

    1982-05-01

    Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. /sup 86/Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++-containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased /sup 86/Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in /sup 86/Rb+ efflux. Quinine also inhibited the volume changes and the increased /sup 86/Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in /sup 86/Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic (Ca++), triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).

  11. Sodium pump activity and calcium relaxation in vascular smooth muscle of deoxycorticosterone acetate-salt rats

    SciTech Connect

    Soltis, E.E.; Field, F.P.

    1986-11-01

    The Na/sup +/-K/sup +/ pump activity was determined in femoral arterial smooth muscle from deoxycorticosterone acetate (DOCA)-salt hypertensive rats using potassium relaxation and ouabain-sensitive /sup 86/Rb uptake as indices. The membrane-stabilizing effect of calcium and its relation to Na/sup +/-K/sup +/ pump activity also were examined. Femoral arteries from DOCA-salt rats exhibited a greater relaxation in response to potassium addition after contraction with norepinephrine in a low potassium (0.6 mM) Krebs solution. The concentration of potassium required to produce a 50% relaxation was significantly less in DOCA-salt rats. Ouabain-sensitive /sup 86/Rb uptake was significantly greater at 3, 10, and 20 minutes of /sup 86/Rb incubation in femoral arteries from DOCA-salt rats. Linear regression analysis revealed a significant correlation between the uptake of /sup 86/Rb and time of incubation in both control and DOCA-salt rats. A significant difference in the slopes of the regression lines showed that the rate of uptake was greater in DOCA-salt rats. No difference was observed in ouabain-insensitive /sup 86/Rb uptake. A dose-dependent relaxation in response to increasing concentrations of calcium following contraction to norepinephrine was observed in femoral arteries from control and DOCA-salt rats. The relaxation was directly dependent on the level of extracellular potassium and was blocked by ouabain. Femoral arteries from DOCA-salt rats relaxed to a significantly greater extent in response to calcium at each level of potassium when compared with controls. These results provide further evidence for an increase in Na/sup +/-K/sup +/ pump activity in vascular smooth muscle from DOCA-salt hypertensive rats.

  12. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast.

    PubMed

    Díaz, Paula; Sibley, Colin P; Greenwood, Susan L

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3-5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10-1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  13. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

    PubMed Central

    Díaz, Paula; Sibley, Colin P.; Greenwood, Susan L.

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  14. The effect of Portuguese Man-of-war (Physalia physalis) venom on calcium, sodium and potassium fluxes of cultured embryonic chick heart cells.

    PubMed

    Edwards, L; Luo, E; Hall, R; Gonzalez, R R; Hessinger, D A

    2000-03-01

    Portuguese Man-of-war venom markedly increases calcium (45Ca2+) influx into primary, cultured, embryonic chick heart cells. This action is dose-dependent, but is unaffected by organic calcium blockers (diltiazem, verapamil, nifedipine, nimodipine and mibefradil). On the other hand, certain trivalent (La3+, Gd3+) and divalent (Zn2+, Ni2+, Cu2+, Mn2+) metals inhibit venom-induced calcium influx. Sodium (22Na+) influx into chick heart cells is also significantly increased by Man-of-war venom. Flecainide does not block venom-induced sodium influx. The efflux of the potassium analogue, 86Rb+, from heart cells is also significantly increased by the venom. The venom, however, has little or no effect on rubidium (86Rb+) or 2-deoxy-D-[2-3H] glucose influx. PMID:10669022

  15. Regulation of ATP-sensitive K sup + channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    SciTech Connect

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M. )

    1989-04-01

    The actions of somatostatin and of the phorbol ester 4{beta}-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and {sup 86}Rb{sup +} flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K{sup +} channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive {sup 86}Rb{sup +} efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K{sup +} channels are discussed.

  16. AtCCX3 Is an Arabidopsis Endomembrane H+-Dependent K+ Transporter1[W][OA

    PubMed Central

    Morris, Jay; Tian, Hui; Park, Sunghun; Sreevidya, Coimbatore S.; Ward, John M.; Hirschi, Kendal D.

    2008-01-01

    The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na+, K+, and Mn2+ transport. We also report high-capacity uptake of 86Rb+ in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of 86Rb+ uptake in AtCCX3 cells by excess Na+, K+, and Mn2+. Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl2. Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na+ accumulation and increased 86Rb+ transport. Uptake of 86Rb+ increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H+-dependent K+ transporter with apparent Na+ and Mn2+ transport properties distinct from those of previously characterized plant transporters. PMID:18775974

  17. Mechanism of action of chlorhexidine diacetate and phenoxyethanol singly and in combination against gram-negative bacteria.

    PubMed

    Fitzgerald, K A; Davies, A; Russell, A D

    1992-01-01

    Chlorhexidine diacetate and the aromatic alcohol, phenoxyethanol in combination had an enhanced bacteriostatic action against Escherichia coli and Pseudomonas aeruginosa strains. Investigations of potassium (K+) ion leakage by means of a potassium electrode and a radioactive method, employing 86Rb, indicated that the combination accelerated the rate of leakage from the cell. Leakage of pentose was also found to be enhanced in the presence of the combination compared with either drug alone.

  18. Early effects of aldosterone on Na-K pump in rat cortical collecting tubules

    SciTech Connect

    Fujii, Y.; Takemoto, F.; Katz, A.I. )

    1990-07-01

    Sustained exposure to aldosterone (Aldo) increases the abundance and activity of the Na-K pump in cortical collecting tubules (CCT). However, the onset and mechanism of the early interaction of Aldo with the CCT pump, especially in adrenal-intact animals, are unclear. We evaluated the short-term effects of the hormone on Na-K-adenosinetriphosphatase (ATPase) activity and on ouabain-sensitive 86Rb uptake, a measure of the transporting rate of the pump, in microdissected CCT from adrenal-intact rats. Incubation with Aldo (10(-8) M, 2 h) had no effect on Na-K-ATPase activity (Vmax), whereas it produced at least a twofold increase in 86Rb uptake. This effect was generated by physiological concentrations of the hormone (threshold 10(-10) M; apparent K1/2 approximately 10(-9) M), after a short lag of less than or equal to 30 min. Incubation with Aldo in the presence of amiloride or nystatin or in a Na-free medium (choline chloride) did not prevent the enhanced 86Rb uptake seen after Aldo alone; possible interpretations of these observations are discussed. We conclude that Aldo produces a rapid stimulation of pump function in CCT that precedes its induction of new pump synthesis; the physiological significance of this effect is suggested by its occurrence in tubules from adrenal-intact animals within the time frame and concentration range of the hormone's effects on electrolyte transport.

  19. Na sup + pump in renal tubular cells is regulated by endogenous Na sup + -K sup + -ATPase inhibitor from hypothalamus

    SciTech Connect

    Cantiello, H.F.; Chen, E.; Ray, S.; Haupert, G.T. Jr. )

    1988-10-01

    Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na{sup +}-K{sup +}-ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK{sup 1}) were studied using {sup 86}Rb{sup +} uptake and ({sup 3}H)ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na{sup +}-K{sup +}-ATPase in LLC-PK{sub 1} cells. In contrast to membrane studies, no prolonged incubation with LLC-PK{sub 1} was needed to observe inhibition of Na{sup +}-K{sup +}-ATPase. HF caused a 33% inhibition of ouabain-sensitive {sup 86}Rb{sup +} influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK{sub 1} {sup 86}Rb{sup +} uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na{sup +}-K{sup +}-ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes.

  20. Effects of prostaglandin inhibition on intrarenal hemodynamics in acutely saline-loaded rats.

    PubMed

    Düsing, R; Melder, B; Kramer, H J

    1977-09-01

    We studied the effect of inhibition of the prostaglandin (PG)-synthesizing enzyme system in female Sprague-Dawley rats following acute expansion of the extracellular fluid volume (ECV). In 57 conscious rats expansion of the ECV with isotonic saline corresponding to an increase in body weight of 10% was induced. Prior to ECV expansion 31 rats received indomethacin (10 mg/kg of body wt) by stomach tube. In six non-ECV-expanded rats indomethacin had no effect on glomerular filtration rate (GFR) and renal plasma flow (RPF). In ECV-expanded rats pretreated with indomethacin, GFR was unaltered but 125I-hippuran clearance decreased, and filtration fraction significantly increased. Intrarenal 86Rb distribution was similar in control and ECV-expanded rats. Indomethacin caused a slight increase in relative cortical 86 RB activity in non-ECV-expanded rats, but had no effect on intrarenal 86Rb distribution in ECV-expanded rats. No difference in intracortical glomerular perfusion was noted between control and ECV-expanded rats. In indomethacin-treated ECV-expanded rats an increase in relative inner cortical perfusion was observed. Absolute perfusion remained unaltered. Thus the decrease in total RPF was entirely due to decreased perfusion of outer cortical nephrons. Renal prostaglandins therefore may play a permissive role for physical factors to promote renal sodium excretion in acute ECV expansion via changes in intrarenal hemodynamics. PMID:890884

  1. Potassium-dependent changes in the expression of membrane-associated proteins in barley roots

    SciTech Connect

    Fernando, M.; Kulpa, J.; Siddiqi, M.Y.; Glass, A.D.M. )

    1990-04-01

    Barley (Hordeum vulgare L. cv Halcyon) seedlings which has been grown in full strength complete inorganic nutrient media (containing 6 millimolar K{sup +}) had high internal K{sup +} concentrations and low values of K{sup +} ({sup 86}Rb{sup +}) influx when influx was measured from solutions containing 100 micromolar K{sup +}. Transfer of these plants to solutions lacking K{sup +} resulted in significant reductions of root and shoot K{sup +} concentrations and values of K{sup +} ({sup 86}Rb{sup +}) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K{sup +}, the changes induced by K{sup +} deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K{sup +} ({sup 86}Rb{sup +}) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K{sup +} status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washing in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar.

  2. Efflux of potassium from isolated rod outer segments: a photic effect

    PubMed Central

    Cavaggioni, A.; Sorbi, R. T.; Turini, S.

    1973-01-01

    1. Illumination of the isolated outer segments of rod photoreceptors loaded with 42K or 86Rb reduces the efflux of these ions. 2. During the perfusion of the isolated rod outer segments with a solution containing only 2·36 mM-Na the effect of light is absent, and the amplitude of the photic effect is linearly related to the logarithm of the extracellular Na concentration. 3. In darkness, raising the concentration of K in the fluid of perfusion gives an increase of the efflux of 86Rb and increasing the extracellular concentration of Ca yields a retention. The efflux of 86Rb and 42K is greater in darkness when sucrose or choline substitute for Na. 4. It is suggested that in darkness the isolated outer segments are permeable both to Na and to K. Light appears to decrease the permeability for Na ions. There is no evidence that the permeability for K ions is modified by light. PMID:4543342

  3. Oligosaccharide composition of the neurotoxin responsive Na/sup +/ channel and the requirement of sialic acid for activity

    SciTech Connect

    Negishi, M.; Shaw, G.W.; Glick, M.C.

    1986-05-01

    The neurotoxin responsive Na/sup +/ channel was purified to homogeneity in an 18% yield from a clonal cell line of mouse neuroblastoma, N-18, metabolically labeled with L-(/sup 3/H)fucose. The Na/sup +/ channel, a glycoprotein, M/sub r/=200,000 (gradient 7-14% PAGE) was digested with Pronase and the glycopeptides were characterized by serial lectin affinity chromatography. greater than 90% of the oligosaccharides contained sialic acid and 18% were biantennary, 39% were triantennary and 30% tetraantennary. The glycoprotein was reconstituted into artificial phospholipid vesicles and /sup 86/Rb flux was stimulated (65%) by 200 ..mu..M veratridine and 1.2 ..mu..g of scorpion venom and was inhibited (95%) by 5 ..mu..M tetrodotoxin. The requirement of sialic acid for Na/sup +/ channel activity was demonstrated since neuraminidase (0.01 U) treatment of the reconstituted glycoprotein eliminated the response of /sup 86/Rb flux to the stimulating neurotoxins. In other experiments, treatment of N-18 cells with 10 ..mu..M swainsonine, an inhibitor of glycoprotein processing, altered the oligosaccharide composition of the Na/sup +/ channel. When the abnormally glycosylated Na/sup +/ channel was reconstituted into artificial phospholipid vesicles, /sup 86/Rb flux in response to neurotoxins was impaired. Thus, glycosylation of the polypeptide with oligosaccharides of specific composition and structure is essential for expression of the biological activity of the neurotoxin responsive Na/sup +/ channel.

  4. Volume regulatory potassium transport in rabbit and human sickle erythrocytes in vitro

    SciTech Connect

    Al-Rohil, N.S.

    1988-01-01

    One approach to the therapy of sickle cell anemia is to decrease the hemoglobin concentration by inducing a slight swelling of the cell to retard the rate of hemoglobin polymerization. We found that a prolonged incubation of rabbit or human SS red cell in hypotonic medium caused an inactivation of the inactivation of swelling-stimulated potassium transport. The inactivation may have important practical consequences for the therapy of sickle cell anemia. Large cytoskeleton-free vesicles were prepared in order to study the possible role of the spectrin-actin membrane skeleton in the swelling-stimulated and N-ethylmaleimide (NEM)-stimulated transport. NEM pretreatment stimulated {sup 86}Rb efflux in vesicles by a factor of 2.4 + 0.55 (mean {plus minus} S.D.). The NEM effect on {sup 86}Rb efflux was specific in that the {sup 22}Na efflux into a Na medium was not stimulated but actually inhibited. The {sup 86}Rb efflux from the vesicles was not stimulated by hypotonic media. This finding is consistent with a role of the membrane skeleton in the detection and/or transduction of the signal by which cell swelling activates the transport.

  5. Some effects of way-120,491 on electrical and mechanical activity and on sup 42 K/ sup 85 Rb efflux in rat blood vessels

    SciTech Connect

    Edwards, G.; Weston, A.H. ); Oshiro, G. )

    1990-02-26

    The effects of WAY-120,491 (-)-(3A,4R-trans)-2-(2,3-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromethoxy)-2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one have been examined in vitro using portal veins and aortic segments from male Sprague-Dawley rats. In aorta, WAY-120,491 (3.3-33nM) produced a dose-dependent relaxation of 20mM KC1 contractions, an effect antagonized by glibenclamide. WAY-120,491 had no effect on tension induced by 80mM CK1. When segments of aorta were loaded with {sup 86}Rb and {sup 42}K, WAY-120,491 (330nM-33{mu}M) increased the efflux of both isotopes, an effect antagonized by glibenclamide. In portal vein, WAY 120-491 (3.3-330nM) inhibited spontaneous tension development by abolishing electrical multispike complexes. In tissues loaded with {sup 86}Rb, WAY-120,491 (330nM-33{mu}M) increased the rate of {sup 86}Rb exchange. It is concluded that these effects of WAY-120,491 in rat blood vessels are consistent with the opening of plasmalemmal K-channels and that WAY-120,491 belongs to the pharmacological grouping known as the K-channel openers.

  6. Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K(+) transport in a higher plant cell.

    PubMed

    Clint, G M; Blatt, M R

    1989-12-01

    Fusicoccin (FC) has long been known to promote K(+) uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H(+) pumping stimulated in FC, could help drive K(+) uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K(+) ((86)Rb(+)) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180-192). Thus, the activities of specific pathways for K(+) movement across the membrane could be modified in FC. We have explored a role for K(+) channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical ((86)Rb(+)) flux and in electrical current under voltage clamp was followed, using the K(+) channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K(+)-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K(+) channel current contributed appreciably to charge balance maintainingV m, and adding 10 mM TEA to block the current depolarized (positive-going)V m; TEA also reduced(86)Rb(+) efflux by 68-80%. Following treatments with 10 μM FC, both K(+) channel current and(86)Rb(+) efflux decayed, irreversbly and without apparent lag, to 10%-15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced(86)Rb(+) influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K(+) (Rb(+)), despite membrane potentials which were 30-60 mVpositive of the K(+) equilibrium

  7. Prenatal exposure of rats to nicotine causes persistent alterations of nicotinic cholinergic receptors

    PubMed Central

    Gold, Allison B.; Keller, Ashleigh B.; Perry, David C.

    2010-01-01

    We examined for immediate and persistent changes in nAChRs in cerebral cortex, thalamus and striatum of male rats caused by prenatal exposure to nicotine from gestational day 3 to postnatal day 10 (PN10), and how such exposure affected the responses of adolescents to subsequent nicotine challenge. Receptor numbers were assessed by [3H]epibatidine binding and receptor function was measured by acetylcholine-stimulated 86Rb efflux (cerebral cortex and thalamus) and nicotine-stimulated dopamine release (striatum). Immediate effects of prenatal nicotine, assessed in PN10 animals, were not detected for any parameter. A subsequent 14 day nicotine exposure in adolescence revealed persistent changes caused by prenatal nicotine exposure. Nicotine exposure in adolescents caused up-regulation of binding in all three regions; however, this up-regulation was lost in thalamus from animals prenatally exposed to nicotine. Nicotine exposure in adolescents caused decreased nicotine-stimulated dopamine release in striatum; this effect was also lost in animals prenatally exposed to nicotine. Comparison of parameters in PN10 and PN42 rats revealed developmental changes in the CNS cholinergic system. In thalamus, binding increased with age, as did the proportion of 86Rb efflux with high sensitivity to acetylcholine. In cortex, binding also increased with age, but there was no change in total 86Rb efflux, and the proportion of high to low sensitivity efflux declined with age. Nicotine-stimulated striatal dopamine release (both total and α-conotoxin MII-resistant release) increased with age in naïve animals, but not in those prenatally exposed to nicotine. These findings demonstrate that prenatal exposure to nicotine causes alterations in the regulation of nAChRs by nicotine that persist into adolescence. These changes may play a role in the increased risk for nicotine addiction observed in adolescent offspring of smoking mothers. PMID:19028470

  8. Effect of insulin on Na+,K(+)-ATPase in rat collecting duct.

    PubMed Central

    Féraille, E; Rousselot, M; Rajerison, R; Favre, H

    1995-01-01

    1. The collecting duct is involved in the whole antinatriuretic effect of insulin, as indicated in vitro by the stimulatory effect of the hormone on ouabain-sensitive 86Rb+ uptake. Since Na+,K(+)-ATPase drives Na+ reabsorption, the contribution of the Na+ pump to the effect of insulin was investigated in rat isolated cortical and outer medullary collecting duct. 2. Insulin enhanced ouabain-sensitive 86Rb+ uptake in the absence, as well as in the presence, of either 5 x 10(-4) M amiloride or 10(-3) M hydrochlorothiazide (HCT). Maximal ouabain-sensitive 86Rb+ uptake, measured in Na(+)-loaded tubules, was also enhanced by insulin. The insulin effect persisted both in the absence of external Na+, when the Na+,K(+)-ATPase operates in a Rb(+)-Rb+ exchange mode, and in tubules depolarized by a high external concentration (20 mM) of Rb+ or by addition of 3 mM Ba2+. 3. Insulin treatment did not alter the intracellular Na and K concentrations, the specific binding of [3H]ouabain measured in intact tubules, or the hydrolytic activity of Na+,K(+)-ATPase measured after permeabilization of the tubule cells. 4. In conclusion, in the rat collecting duct, insulin increased Na+,K(+)-ATPase-mediated cation transport independently of Na+ availability, membrane potential and recruitment of pump units. The effect of insulin was lost after cell permeabilization, suggesting the presence of a cytosolic factor which controls the turnover of Na+,K(+)-ATPase. Images Figure 1 PMID:8568653

  9. Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses

    SciTech Connect

    Van Winkle, L.J.; Campione, A.L. )

    1991-07-01

    The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptuses at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity.

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

    PubMed

    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.

  11. The mechanism of patulin's cytotoxicity and the antioxidant activity of indole tetramic acids

    SciTech Connect

    Riley, R.T.; Showker, J.L. )

    1991-06-01

    In LLC-PK1 cells exposed to patulin (50 microM), lipid peroxidation, abrupt calcium influx, extensive blebbing, and total LDH release appeared to be serially connected events with each representing a step in the loss of structural integrity of the plasma membrane. The aforementioned patulin-induced events were prevented by concurrent incubation with butylated hydroxytoluene, deferoxamine, and cyclopiazonic acid, a fungal metabolite. Patulin also caused depletion of nonprotein sulfhydryls, increased 86Rb+ efflux, dome collapse, and eventually the loss of cell viability. These events were not prevented by antioxidants, results consistent with the hypothesis that they were also serially connected but occurring parallel to those previously mentioned. The earliest events observed in patulin-treated cells were the decrease in nonprotein sulfhydryls and increase in 86Rb+ efflux (5 min) which occurred before statistically significant alterations in protein-bound sulfhydryls. The increased potassium efflux (86Rb+ efflux) occurred via a pathway distinct from BaCl2, quinine, or tetraethylammonium sensitive potassium channels. This is the first published report of the antioxidant activity of indole tetramic acids (cyclopiazonic acid and cyclopiazonic acid imine). The protective effect of tetramic acids in LLC-PK1 cells was restricted to indole tetramic acids, and their prevention of lipid peroxidation did not involve iron chelation. The results of this study demonstrate that cyclopiazonic acid is a potent inhibitor of azide-insensitive, ATP-dependent, a23187-sensitive calcium uptake by the lysate of LLC-PK1 cells. This result is consistent with the hypothesis that the endoplasmic reticulum calcium transport ATPase is a sensitive target for cyclopiazonic acid in LLC-PK1 cells.

  12. Ionic, electrical, and secretory effects of protein kinase C activation in mouse pancreatic B-cells: studies with a phorbol ester

    SciTech Connect

    Bozem, M.; Nenquin, M.; Henquin, J.C.

    1987-09-01

    The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to study the effects of protein kinase C activation on stimulus-secretion coupling in mouse pancreatic B-cells. At a nonstimulatory concentration of glucose (3 mM), 100 nM TPA, but not 10 nM TPA, slightly and slowly increased insulin release and /sup 45/Ca/sup 2 +/ efflux and decreased /sup 86/Rb/sup +/ efflux, but did not affect the membrane potential of B-cells. At a threshold concentration of glucose (7 mM), 100 nM TPA markedly increased insulin release without triggering electrical activity in B-cells. At a stimulatory concentration of glucose (10 mM), TPA caused a dose-dependent irreversible increase in insulin release, /sup 45/Ca/sup 2 +/ efflux, and /sup 86/Rb/sup +/ efflux and slightly augmented islet cAMP levels. Omission of extracellular Ca/sup 2 +/ abolished the effects of 10 nM TPA and partially inhibited those of 100 nM TPA on insulin release and /sup 45/Ca/sup 2 +/ efflux. In contrast, their effect on /sup 86/Rb/sup +/ efflux was paradoxically augmented. Glucose-induced electrical activity in B-cells was only marginally affected by TPA; the duration of the slow waves with spikes was not modified, but a small shortening of the polarized intervals raised their frequency and slightly increased the overall activity. This increase was significant only with 10 nM TPA, whereas only 100 nM TPA brought about a minute increase in /sup 45/Ca/sup 2 +/ influx. These results thus show that TPA induces insulin release or potentiates glucose-induced insulin release without mimicking or amplifying the initial ionic and electrical signals triggered by glucose. They suggest that protein kinase C activation affects stimulus-secretion coupling by modulating intracellular and/or nonelectrogenic membrane events.

  13. Radioassay of dual-labeled samples with a Cherenkov counting technique

    NASA Astrophysics Data System (ADS)

    Fujii, Haruo; Takiue, Makoto

    1998-03-01

    A new Cherenkov counting technique which allows radioactivities of a dual-labeled sample to be determined simultaneously by using a wavelength shifter has been proposed, and tested for the pairs 32P-36Cl and 86Rb-36Cl. The minimum requirements for this method are a single channel liquid scintillation counter, a wavelength shifter and a reference sample for determining the Cherenkov counting efficiency. The simple procedure for sample preparation and measurement makes the technique very useful for routine radioassay with the help of a desk-top computer.

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

    PubMed

    Massart, C; Giusti, N; Beauwens, R; Dumont, J E; Miot, F; Sande, J Van

    2013-01-01

    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 (99m)TcO4 (-) nor of the K(+) cation mimic (86)Rb(+) 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. PMID:24371722

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

    NASA Astrophysics Data System (ADS)

    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

  16. Evidence for coordinate genetic control of Na,K pump density in erythrocytes and lymphocytes

    SciTech Connect

    DeLuise, M.; Flier, J.S.

    1985-08-01

    The erythrocyte is widely used as a model cell for studies of the Na,K pump in health and disease. However, little is known about the factors that control the number of Na,K pumps expressed on the erythrocytes of a given individual, nor about the extent to which erythrocytes can be used to validly assess the pump density on other cell types. In this report, the authors have compared the interindividual variance of Na,K pump density in erythrocytes of unrelated individuals to that seen with identical twins. Unlike unrelated individuals, in whom pump parameters, i.e., ouabain binding sites, /sup 86/Rb uptake, and cell Na concentration vary widely, identical twin pairs showed no significant intrapair variation for these values. Thus, a role for genetic factors is suggested. In addition, the authors established and validated a method for determining Na,K pump density and pump-mediated /sup 86/Rb uptake in human peripheral lymphocytes. Using this method, they show that whereas Na,K pump density differs markedly between erythrocytes (mean of 285 sites per cell) and lymphocytes (mean 40,600 sites per cell), there is a strong and highly significant correlation (r = 0.79, P less than 0.001) between the pump density in these cell types in any given individual. Taken together, these studies suggest that genetic factors are important determinants of Na,K pump expression, and that pump density appears to be coordinately regulated in two cell types in healthy individuals.

  17. Charge distribution in the reactor-neutron-induced fission of 232Th

    NASA Astrophysics Data System (ADS)

    Erten, H. N.; Grütter, A.; Rössler, E.; von Gunten, H. R.

    1982-05-01

    The independent yields of 82Br, 86Rb, 96Nb, 98Nbm, 128Sbg, and 136Cs were determined in the reactor-neutron-induced fission of 232Th using radiochemical techniques. Results: (2.3+/-2.3)×10-4% for 82Br, <3.8×10-4% for 86Rb, <4.2×10-5% for 96Nb, (2.48+/-0.53)×10-3% for 98Nbm, (2.34+/-0.37)×10-3% for 128Sbg, and (1.70+/-0.13) ×10-4% for 136Cs. Using the extended Zp model of Wahl with the yield data from this work and the literature the following parameters were obtained for the charge distribution in 232Th fission: width of Gaussian dispersion σ¯Z=0.52+/-0.01, ΔZP (=ZP-ZUCD)=0.45+/-0.02. The even-odd proton and neutron enhancement factors were found to be small. These parameters and systematics of even-odd proton and neutron effects in low energy fission are discussed. NUCLEAR REACTIONS, FISSION Radiochemical fission yields 232Th(n,f), calculated charge dispersion parameters, and odd-even effects.

  18. Vasodilatation with pinacidil. Mode of action in rat resistance vessels

    SciTech Connect

    Videbaek, L.M.; Aalkjaer, C.; Mulvany, M.J. )

    1988-01-01

    Pinacidil is a newly developed antihypertensive vasodilator, proposed to belong to the new group of smooth muscle relaxants, the K+ channel openers. The in vitro effects of pinacidil on induced tone, smooth muscle membrane potential and {sup 86}Rb and {sup 42}K efflux from rat resistance vessels (internal diameter about 200 microns) were studied. Tone induced with noradrenaline was concentration-dependently inhibited by pinacidil. Responses to electrical field stimulation were also inhibited. However, tone induced with high K+ depolarization, noradrenaline in the presence of high K+, caffeine-induced contractions and noradrenaline contractions in the presence of felodipine were little affected by pinacidil. Pinacidil caused concentration-dependent hyperpolarisation of the resting smooth muscle. Pinacidil caused only a small and transient increase of the {sup 86}Rb efflux rate constant, while the same concentrations of pinacidil produced a significant increase in the {sup 42}K efflux rate constant. Our results seem to indicate that the relaxant effect of pinacidil is the result of an increase in K+ permeability, thus causing hyperpolarisation and relaxation. The opened K+ channels appear to be selective for K+ over Rb+.

  19. Bisphenols that stimulate cells to release alkali metal cations: a structure-activity study.

    PubMed

    Hopp, L; Megee, S O; Lloyd, J B

    1998-10-22

    The laxative action of phenolphthalein (5) is believed to result from induction of potassium and water efflux from the colon epithelium. In cultured cells, K+ efflux is promoted by 5 and by a contaminant (1) present in commercial phenol red. Six compounds with chemical structures related to those of 5 and 1 were tested for ability to induce the release of 86Rb from COS-7 cells preloaded with this isotope: 4,4'-(9-fluorenylidene)diphenol (2), 4, 4'-(9-fluorenylidene)dianiline, 4, 4'-(9-fluorenylidene)bisphenoxyethanol, 1,1'-bi-2-naphthol, 4, 4'-biphenol, and bis(4-hydroxyphenyl)methane. With one exception these compounds were all inactive at a concentration of 10 microM. However, 2 caused profound 86Rb efflux at concentrations as low as 100 nM. Concentrations of 5 1-2 orders of magnitude higher were needed to achieve similar levels of activity. The three compounds known to be active in this experimental system share a common feature that is absent in all the inactive compounds: a five-membered ring structure, one of whose carbon atoms is disubstituted with p-hydroxyphenyl residues. Because 2 and 5 are readily available, comparative studies on the mechanism of action of these biphenols at the cellular level can now be undertaken. PMID:9784117

  20. Potassium and Sodium Absorption Kinetics in Roots of Two Tomato Species 1

    PubMed Central

    Wrona, Anne F.; Epstein, Emanuel

    1985-01-01

    Excised roots of the tomato species, Lycopersicon esculentum Mill. cv Walter (the commercial species) and of Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull. (a wild species from the Galapagos Islands), were used in comparative studies of their absorption of K+ and Na+. Uptake of 86Rb-labeled K+ and 22Na-labeled Na+ by excised roots of `Walter' and L. cheesmanii varied as a function of genotype and tissue pretreatment with or without K+. Excised roots of `Walter' consistently absorbed more 86Rb-labeled K+ than those of L. cheesmanii. Absorption of K+ from solutions ranging from 0.01 to 0.2 millimolar KCl showed saturation kinetics in both K+-pretreated and K+-depleted roots of `Walter,' and for K+-depleted roots of L. cheesmanii. K+-pretreated roots of L. cheesmanii had exceedingly low rates of K+ uptake with strikingly different, linear kinetics. Pretreatment with K+ caused a decrease in rates of K+ uptake in both genotypes. Potassium depleted roots of L. cheesmanii absorbed Na+ at a greater rate than those of `Walter,' whereas K+-pretreated roots of `Walter' absorbed Na+ at a greater rate than those of L. cheesmanii. The results confirm and extend previous conclusions to the effect that closely related genotypes may exhibit widely different responses to the two alkali cations, K+ and Na+. PMID:16664530

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

    PubMed

    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.

  2. Inhibition of cation channel function at the nicotinic acethylcholine receptor from Torpedo: Agonist self-inhibition and anesthetic drugs

    SciTech Connect

    Forman, S.A.

    1989-01-01

    Modulation of the nicotinic acethylcholine receptor from Torpedo by cholinergic agonists, local anesthetics, and n-alkanols was studied using {sup 86}Rb{sup +} flux studies in sealed native Torpedo electroplaque membrane vesicles. Reliable concentration-response and kinetic data were obtained using manual ten sec filtration assays in vesicles partially blocked with alpha-bungarotoxin to remove spare receptors and quenched-flow assays to assess initial {sup 86}Rb{sup +} flux rates or the rate of drug-induced receptor inactivation. Concentration response relationships for the agonists acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, and (-)-nicotine are all bell-shape due to stimulation of cation channel opening at low concentrations and inhibition of channels at higher concentrations. The rate of agonist-induced fast desensitization (k{sub d}) increases with (acetylcholine) in parallel with channel activation, suggesting that desensitization proceeds from the open state and/or states in rapid equilibrium with it. At self-inhibitory acetylcholine concentrations, a new rapid inactivation (rate = k{sub f}) is observed before fast desensitization. The rate and extent of rapid inactivation is compatible with bimolecular association between acethylcholine and inhibitory site with K{sub B} = 40 mM.

  3. Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

    SciTech Connect

    Forman, S.A.; Miller, K.W. )

    1989-02-21

    The relationship between the high-affinity procaine channel inhibition site and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid {sup 86}Rb{sup +} quenched-flux assays at 4 {degree}C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with {alpha}-bungarotoxin ({alpha}-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations alone, AChR undergoes one phase of inactivation in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting ACh concentrations rapid inactivation complete in 30-75 ms is followed by fast desensitization at the same k{sub d} observed without procaine. The dependence of k{sub r} on (procaine) is consistent with a bimolecular association between procaine and its AChR site. Inhibition of AChR function by mixtures of procaine plus self-inhibiting concentrations of ACh or suberyldicholine was studied by reducing the level of {alpha}-BTX block in vesicles. The data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and k{sub r}'s dependence on (ACh) in channel-activating range closely parallels that of {sup 86}Rb{sup +} flux response to ACh.

  4. Extrarenal potassium adaptation: role of skeletal muscle

    SciTech Connect

    Blachley, J.D.; Crider, B.P.; Johnson, J.H.

    1986-08-01

    Following the ingestion of a high-potassium-content diet for only a few days, the plasma potassium of rats rises only modestly in response to a previously lethal dose of potassium salts. This acquired tolerance, termed potassium adaptation, is principally the result of increased capacity to excrete potassium into the urine. However, a substantial portion of the acute potassium dose is not immediately excreted and is apparently translocated into cells. Previous studies have failed to show an increase in the content of potassium of a variety of tissues from such animals. Using /sup 86/Rb as a potassium analogue, we have shown that the skeletal muscle of potassium-adapted rats takes up significantly greater amounts of potassium in vivo in response to an acute challenge than does that of control animals. Furthermore, the same animals exhibit greater efflux of /sup 86/Rb following the termination of the acute infusion. We have also shown that the Na+-K+-ATPase activity and ouabain-binding capacity of skeletal muscle microsomes are increased by the process of potassium adaptation. We conclude that skeletal muscle is an important participant in potassium adaptation and acts to temporarily buffer acute increases in the extracellular concentration of potassium.

  5. Regulation of the sodium-potassium pump in cultured rat skeletal myotubes by intracellular sodium ions

    SciTech Connect

    Brodie, C.; Sampson, S.R.

    1989-07-01

    The properties of the Na-K pump and some of the factors controlling its amount and function were studied in rat myotubes in culture. The number of Na-K pump sites was quantified by measuring the amount of (/sup 3/H)ouabain bound to whole-cell preparations. Activity of the pump was determined by measurement of ouabain-sensitive /sup 86/Rb-uptake and component of membrane potential. Chronic treatment of myotubes with tetrodotoxin (TTX), which lowers (Na)i, decreased the number of Na-K pumps, the ouabain-sensitive 86Rb uptake, and the size of the electrogenic pump component of Em. In contrast, chronic treatment with either ouabain or veratridine, which increases (Na+)i, resulted in an elevated level of Na-K pump sites. This effect was blocked by inhibitors of protein synthesis. Neither rates of degradation nor affinity of pump sites in cells treated with TTX, veratridine, or ouabain differred from those in control cells. The number and activity of Na-K pump sites were unaffected by chronic elevation in (Ca)i or chronic depolarization. We conclude that alterations in the level in intracellular Na ions play the major role in regulation of Na-K pump synthesis in cultured mammalian skeletal muscle.

  6. Effect of Rb+ on cromakalim-induced relaxation and ion fluxes in guinea pig trachea.

    PubMed

    Foster, K A; Arch, J R; Newson, P N; Shaw, D; Taylor, S G

    1992-11-01

    The effects of cromakalim, verapamil and salbutamol have been examined in guinea pig trachealis smooth muscle in both Krebs physiological salt solution and Krebs solution where K+ has been replaced by Rb+. Cromakalim-induced relaxation in the presence of Rb+ was reduced in extent and became transient, whilst the relaxation response to verapamil was enhanced and that to salbutamol unaffected. The transient relaxation occurring in Rb+ was blocked by quinidine and glibenclamide. The presence of extracellular Rb+ also prevented cromakalim-stimulated efflux of both 86Rb+ and 42/43K+. There was, however, no effect on cromakalim-stimulated 86Rb+ uptake. It is proposed that cromakalim is opening two populations of potassium channel in guinea pig tracheal smooth muscle, one of which is susceptible to blockade by Rb+ and one of which is not. The latter channel appears to play the dominant role in cromakalim-stimulated uptake, and is responsible for the transient relaxation response in the presence of rubidium, whilst the former is responsible for the maintained relaxation. PMID:1468491

  7. A rabbit jejunal isolated enterocyte preparation suitable for transport studies.

    PubMed Central

    Brown, P D; Sepúlveda, F V

    1985-01-01

    A method is described for isolating viable enterocytes from rabbit jejunum. Estimates of sucrase and gamma-glutamyl transferase activities in cells isolated by this method suggest that they originate from the upper villus only. Isolated cells accumulate both alpha-methyl-D-glucoside and alanine, maintaining high intracellular concentrations for at least 60 and 40 min respectively. Accumulation of alpha-methyl-D-glucoside is inhibited by the presence of phloridzin. The cells accumulate 42K and 86Rb in an identical manner. This uptake, which is maintained for at least 60 min, is inhibited in the presence of ouabain. Passive efflux of 42K and 86Rb occurs with rate constants which are virtually identical. The efflux follows a single exponential suggesting that it originates from only one intracellular compartment. It is suggested that the preparation can be used to study the effect of sugars and amino acids on K efflux. The advantages of using such a preparation are discussed. PMID:2862277

  8. Alkali-Ion-Crown Ether in Art and Conservation: The Applied Bioinorganic Chemistry Approach

    PubMed Central

    Hilfrich, Uwe; Taylor, Harold; Weser, Ulrich

    2004-01-01

    Dried varnish is rich in many ester moieties, which may be broken down into small, soluble compounds by esterase activity or alkaline hydrolysis. Two methods for varnish removal have been developed, including the treatment of either lipase or RbOH / PEG-400 crown ether which allow aged oil varnishes or paint coverings to be removed or thinned. These techniques are designed to proceed in a controlled manner without damaging lower paint or base layers. Unfortunately, lipase did not react with the aged ester groups of dried linseed oil varnish. Surprisingly, the varnish came off in the presence of Tris buffer alone which, in addition, formed reactive metal complexes. A better choice was the use of high Mr alkali ion polyethylene glycol–400 (PEG-400) crown ether type chelates. PEG-400 complexes alkali ions including rubidium and other alkaliions impeding the diffusion of their basic counter ions into lower varnish or paint layers. Possible migration of alkali metal ions into the paint layer during alkaline varnish removal was determined by labelling the cleansing solutions with 86Rb. Fortunately, varnish is degraded on the surface only. Lower paint or varnish layers are not attacked even if chemically similar to the varnish or over painting to be removed as virtually no 86Rb was detected on the paint surface. PMID:18365066

  9. Volume-induced increase of anion permeability in human lymphocytes.

    PubMed

    Grinstein, S; Clarke, C A; Dupre, A; Rothstein, A

    1982-12-01

    Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.

  10. Methadone is a non-competitive antagonist at the α4β2 and α3* nicotinic acetylcholine receptors and an agonist at the α7 nicotinic acetylcholine receptor.

    PubMed

    Talka, Reeta; Salminen, Outi; Tuominen, Raimo K

    2015-04-01

    Nicotine-methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non-competitive antagonist at rat α3β4 nicotinic acetylcholine receptors (nAChR) and an agonist at human α7 nAChRs. In this study, we used cell lines expressing human α4β2, α7 and α3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [(3) H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and (86) Rb(+) efflux and changes in intracellular calcium [Ca(2+) ]i were used to assess changes in the functional activity of the receptors. Methadone displaced [(3) H]epibatidine from nicotinic agonist-binding sites in SH-EP1-hα7 and SH-SY5Y cells, but not in SH-EP1-hα4β2 cells. The Ki values for methadone were 6.3 μM in SH-EP1-hα7 cells and 19.4 μM and 1008 μM in SH-SY5Y cells. Methadone increased [Ca(2+) ]i in all cell lines in a concentration-dependent manner, and in SH-EP1-hα7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH-EP1-hα4β2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre-treatment abolished the nicotine-induced response in [Ca(2+) ]i in all cell lines expressing nAChRs. In SH-EP1-hα4β2 and SH-SY5Y cells, methadone had no effect on the (86) Rb(+) efflux, but it antagonized the nicotine-induced (86) Rb(+) ion efflux in a non-competitive manner. These results suggest that methadone is an agonist at human α7 nAChRs and a non-competitive antagonist at human α4β2 and α3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co

  11. [The influx of K(+) ions in leaves of Elodea densa, dependence on light, potassium concentration, and temperature].

    PubMed

    Jeschke, W D

    1970-06-01

    1. The influx of potassium ions in leaves of Elodea densa during short periods of time was measured using (42)K and (86)Rb as tracers. The K(+) influx was linear with time (Fig. 1) without a contribution by Donnan adsorption even in 1 min experiments. 2. Light increased the K(+) influx in air by a factor of up to 30-50 compared to dark/air. Light-induction of the K(+) influx is similar to the light-induction of photosynthesis except for the initial O2 outburst. The half-time of induction, however, is somewhat larger for K(+) influx than for photosynthesis (Fig.2). 3. The isotherms of K(+) influx exhibit the dual mechanism documented for many other species (Figs. 3 and 4). 4. Similar dual isotherms of K(+) influx are obtained in dark/air, light/air, and light/N2, suggesting similar transport mechanisms in light and dark (Figs. 3 and 4). 5. Using (86)Rb as a tracer for K(+), lower values of influx are obtained than with (42)K, the preference for (42)K being higher at low concentrations (Figs. 5,6). However, the light-stimulation (Fig. 5) and the effect of inhibitors on K(+) influx (Table 4) are also found with (86)Rb, indicating that it may be used for such measurements. 6. A change of temperature results in a dual Arrhenius plot (Fig. 7) of K(+) influx with two different apparent activation energies in the light as well as in the dark. The values of E app in the range of strong dependence on temperature are almost equal in light and dark. 7. The causes of the increased K(+) influx in the light are discussed. The influx is inhibited by uncoupling agents and inhibitors of the energy transfer (Table 3) suggesting a dependence on ATP production. On the basis of the carrier concept and using the equations of coenzyme kinetics, a change of the apparent K m (') and V max (') values caused by light can be predicted in the direction found experimentally (Fig. 8). However, the necessary rise of ATP concentration in the light is higher than can be anticipated in vivo. The

  12. Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

    PubMed Central

    Stewart, Andrew K.; Shmukler, Boris E.; Vandorpe, David H.; Rivera, Alicia; Heneghan, John F.; Li, Xiaojin; Hsu, Ann; Karpatkin, Margaret; O'Neill, Allison F.; Bauer, Daniel E.; Heeney, Matthew M.; John, Kathryn; Kuypers, Frans A.; Gallagher, Patrick G.; Lux, Samuel E.; Brugnara, Carlo; Westhoff, Connie M.

    2011-01-01

    Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+ and 86Rb+, with secondarily increased 86Rb+ influx sensitive to ouabain and to bumetanide. Increased RhAG-associated 14C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+, 86Rb+, and 14C-MA were pharmacologically distinct, and Li+ uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH4+ and Gd3+. RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA+). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH4Cl, but MA/MA+ elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+ substitution or bath addition of 5 mM NH4Cl or MA/MA+. These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+ and MA/MA+; 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA+ transport, and decreased NH3/NH4+-associated depolarization; and 3) RhAG transports NH3/NH4+ and MA/MA+ by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms. PMID:21849667

  13. Intermediate Conductance Ca2+-Activated K+ Channels Modulate Human Placental Trophoblast Syncytialization

    PubMed Central

    Díaz, Paula; Wood, Amber M.; Sibley, Colin P.; Greenwood, Susan L.

    2014-01-01

    Regulation of human placental syncytiotrophoblast renewal by cytotrophoblast migration, aggregation/fusion and differentiation is essential for successful pregnancy. In several tissues, these events are regulated by intermediate conductance Ca2+-activated K+ channels (IKCa), in part through their ability to regulate cell volume. We used cytotrophoblasts in primary culture to test the hypotheses that IKCa participate in the formation of multinucleated syncytiotrophoblast and in syncytiotrophoblast volume homeostasis. Cytotrophoblasts were isolated from normal term placentas and cultured for 66 h. This preparation recreates syncytiotrophoblast formation in vivo, as mononucleate cells (15 h) fuse into multinucleate syncytia (66 h) concomitant with elevated secretion of human chorionic gonadotropin (hCG). Cells were treated with the IKCa inhibitor TRAM-34 (10 µM) or activator DCEBIO (100 µM). Culture medium was collected to measure hCG secretion and cells fixed for immunofluorescence with anti-IKCa and anti-desmoplakin antibodies to assess IKCa expression and multinucleation respectively. K+ channel activity was assessed by measuring 86Rb efflux at 66 h. IKCa immunostaining was evident in nucleus, cytoplasm and surface of mono- and multinucleate cells. DCEBIO increased 86Rb efflux 8.3-fold above control and this was inhibited by TRAM-34 (85%; p<0.0001). Cytotrophoblast multinucleation increased 12-fold (p<0.05) and hCG secretion 20-fold (p<0.05), between 15 and 66 h. Compared to controls, DCEBIO reduced multinucleation by 42% (p<0.05) and hCG secretion by 80% (p<0.05). TRAM-34 alone did not affect cytotrophoblast multinucleation or hCG secretion. Hyposmotic solution increased 86Rb efflux 3.8-fold (p<0.0001). This effect was dependent on extracellular Ca2+, inhibited by TRAM-34 and 100 nM charybdotoxin (85% (p<0.0001) and 43% respectively) but unaffected by 100 nM apamin. In conclusion, IKCa are expressed in cytotrophoblasts and their activation inhibits the formation

  14. Vacuolar ATPase driven potassium transport in highly metastatic breast cancer cells.

    PubMed

    Salyer, Sarah A; Olberding, Jordan R; Distler, Anthony A; Lederer, Eleanor D; Clark, Barbara J; Delamere, Nicholas A; Khundmiri, Syed J

    2013-10-01

    Breast cancer is the second leading cause of death in women and thus has received a great deal of attention by researchers. Recent studies suggested decreased occurrence of cancer in patients treated with cardiac glycosides (CGs) for heart conditions. Because CGs induce their cellular effects via the Na(+), K(+) ATPase (Na-K), we treated four breast cancer cell lines (MCF-7, T47D, MDA-MB453, and MDA-MB231) and a non-cancerous breast ductal epithelial cell line (MCF-10A) with ouabain, a well-characterized CG, and measured cell proliferation by measuring bromodeoxyuridine incorporation. Ouabain (1μM) decreased cell proliferation in all cell lines studied except MDA-MB453 cells. Western blot of Na-K α and β subunits showed α1, α3, and β1 expression in all cell lines except MDA-MB453 cells where Na-K protein and mRNA were absent. Potassium uptake, measured as rubidium ((86)Rb) flux, and intracellular potassium were both significantly higher in MDA-MB453 cells compared to MCF-10A cells. RT-qPCR suggested a 7 fold increase in voltage-gated potassium channel (KCNQ2) expression in MDA-MB453 cells compared to MCF-10A cells. Inhibition of KCNQ2 prevented cell growth and (86)Rb uptake in MDA-MB453 cells but not in MCF-10A cells. All cancer cells had significantly higher vacuolar H-ATPase (V-ATPase) activity than MCF-10A cells. Inhibition of V-ATPase decreased (86)Rb uptake and intracellular potassium in MDA-MB453 cells but not in MCF-10A cells. The findings point to the absence of Na-K, high hERG and KCNQ2 expression, elevated V-ATPase activity and sensitivity to V-ATPase inhibitors in MDA-MB453. We conclude that cancer cells exhibit fundamentally different metabolic pathways for maintenance of intracellular ion homeostasis.

  15. Coronary blood flow in rats native to simulated high altitude and in rats exposed to it later in life.

    PubMed

    Turek, Z; Turek-Maischeider, M; Claessens, R A; Ringnalda, B E; Kreuzer, F

    1975-03-22

    In rats exposed to a simulated high altitude of 3500 m for their whole prenatal and postnatal life a severe cardiac hypertrophy develops. In rats born and first staying 5 weeks at sea level and then being exposed to simulated high altitude, only a unilateral right cardiac hypertrophy occurs. In both groups nutritional coronary blood flow was estimated in left ventricle, right ventricle, and septum and was compared with control animals of similar age. Coronary blood flow was measured at hypoxia in all groups. At first cardiac output was determined by the Fick principle, then 86Rb was applied and the animals were killed after 55 sec. Activity of 86Rb was measured in both cardiac ventricles and septum and the fractional uptake was calculated. According to Sapirstein (1956, 1958) the distribution of 86-RB follows the distribution of cardiac output and from both these data the nutritional blood flow to the parts of the heart may be estimated. Cardiac output was similar in rats exposed to simulated high altitude later in life ('newcomers') and in control animals, but it was significantly lower in rats born in the low pressure chamber ('natives'). Fractions of cardiac output supplying cardiac ventricles and septum in rats from both hypoxic groups were significantly higher than in control animals. In the 'natives' they were significantly higher than in the 'newcomers'. The fractions of cardiac output in both 'newcomers' and 'natives' remained significantly higher than those of the control animals, also when calculated per gram of heart tissue. Nutritional coronary blood flow (in ml/min) was higher in both ventricles and septum of the 'newcomers' and in the right ventricle of the 'natives', and lower in the septum of the 'natives', when compared with control animals. Coronary blood flow per gram of heart tissue (in ml/min.g) was significantly higher in all cardiac parts of the 'newcomers', but it was about the same in all cardiac parts of the 'natives' when compared with

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

    SciTech Connect

    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.

  17. Purothionin from wheat endosperm reversibly blocks myogenic differentiation of chick embryonic muscle cells in culture

    SciTech Connect

    Kyu Bong Kwak; Young Sup Lee; Se Won Suh; Doo Bong Ha; Chin Ha Chung ); Chin Sang Chung )

    1989-08-01

    Purothionin from wheat endosperm is a cysteine-rich, basic polypeptide of about 5,000 Da, which modifies membrane permeability of cultured mammalian cells. This peptide was found to block fusion of chick embryonic muscle cells in culture but allows proliferation and alignment. A purothionin concentration of 6 {mu}m/ml was necessary for the complete prevention of myotube formation. Under similar conditions, incorporation of ({sup 35}S) methionine occurred normally but the synthesis of muscle-specific proteins including creatin kinase and acetylcholine receptor was strongly inhibited. In addition, purothionin blocked the uptake of {sup 86}Rb{sup +}, immediately after its addition to the cultured myoblasts. These results suggest that purothionin exerts its regulatory effect on the transition from proliferative to differentiative myoblasts by interfering with membrane permeability or intercellular contact and recognition, which are necessary for the initiation of muscle differentiation.

  18. Phospholipase A and the interaction of Rickettsia prowazekii and mouse fibroblasts (L-929 cells)

    SciTech Connect

    Winkler, H.H.; Miller, E.T.

    1982-10-01

    L-929 cells were killed when approximately 50 viable Rickettsia prowazekii organisms per L-cell were centrifuged onto a monolayer. The glycerophospholipids of the L-cell were hydrolyzed to lysophosphatides and free fatty acids. Concomitantly, there was a loss of membrane integrity as shown by release of lactate dehydrogenase and 86Rb and permeability to trypan blue dye. No glycerophospholipid hydrolysis or cytotoxicity occurred when the rickettsiae were inactivated by heat, UV irradiation, N-ethylmaleimide, or metabolic inhibitors before their addition to the L-929 cells. On the other hand, treatment of the L929 cells with the cytoskeleton agents colchicine or cytochalasin B or with N-ethylmaleimide inhibited neither the phospholipase A activity nor the loss of membrane integrity. Cytochalasin B-treated cells could be damaged by even small numbers of rickettsiae. We suggest that this phospholipase A activity is used by the rickettsiae to escape from the phagosomes into the cytoplasm of host cells.

  19. Distributions of Li+, Na+ K+, Rb+, and Cs+ tracer ions in erythrocytes at 38 degrees C in relation to entry rates of these ions into cells at 0 degree C.

    PubMed

    Salminen, S; Ekman, A; Rastas, J

    2000-01-01

    Forces that are able to transport Na+ and K+ into two compartments were investigated. A modified Nernst-Planck equation for coupled flows of electric current, water, and ions was integrated. The result shows that if alkali ions in the ion channel of the cell membrane are separated by their electric-current-induced inward flows against an electro-osmotic outward flow of water, the logarithms of the stationary cell/medium distributions of these ions should be proportional to the inverse of their diffusion mobilities. The relationship was tested in human erythrocytes. From inward and outward movements of tracer alkali ions, calculations were made to obtain their stationary distributions at infinite time. The cell/medium distributions determined in this way at 38 degrees C are Li+ = 0.59, 22Na+ = 0.044, 42K+ = 10.0, 86Rb+ = 11.9, and 137Cs+ = 3.07. The entry rates of ions into the cell at 0 degrees C are understood to represent their diffusion mobilities in the pump channel. The entry rates are Li+ = 1.44, 2Na+ = 1, 42K+ = 2.22, 86Rb+ = 2.39, and 137Cs+ = 1.72 relative to that of 22Na+. There is an expected negative correlation between the logarithms of the stationary cell/ medium distributions at 38 degrees C and the inverse of the entry rates into the cell at 0 degrees C for the five ions. It is suggested that the proposed physical forces cause the separation of alkali ions in the channel of Na,K-ATPase. PMID:11156287

  20. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

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

    SciTech Connect

    Steinberg, M.I.; Wiest, S.A.; Zimmerman, K.M.; Ertel, P.J.; Bemis, K.G.; Robertson, D.W. )

    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.

  2. Energy-dependent cell volume maintenance in UC-11MG human astrocytomas.

    PubMed

    Lomneth, R; Gruenstein, E I

    1989-10-01

    Swelling of astrocytes in the brain is a major cause of the morbidity and mortality associated with stroke and head trauma. Using a human astrocytoma cell line (UC-11MG) as a model system, we studied cell volume changes caused by ATP depletion under conditions mimicking hypoxia. ATP levels were reduced to less than 10% of control using the metabolic inhibitors KCN or antimycin in combination with glucose deprivation. This was sufficient to eliminate ouabain-sensitive 86Rb+ uptake, indicating the Na+-K+-adenosinetriphosphatase was not operating. Furosemide-sensitive 86Rb+ uptake was reduced by approximately 60%, indicating Na+-K+-2Cl- cotransport was also sensitive to ATP loss. ATP depletion resulted in a 30-40% reduction of cell volume within 60 min. ATP depletion also resulted in a net loss of intracellular K+. This loss of K+ could be blocked by Ba2+, indicating the K+ loss was through a conductive channel. When the net K+ loss was blocked by Ba2+, the volume decrease was also prevented. The cells remained viable throughout the time period as judged by exclusion of ethidium bromide by 99% of the cells and recovery of ATP levels to 75% of control within 60 min. We conclude that ATP depletion, following inhibition of glycolysis and oxidative phosphorylation, causes astrocytes to shrink because of a more rapid loss of K+ than uptake of Na+. Thus it appears that ATP depletion alone is not sufficient to account for the rapid phase of astrocytic swelling observed during cerebral ischemia. PMID:2801931

  3. Potassium Fluxes in Chlamydomonas reinhardtii (I.Kinetics and Electrical Potentials).

    PubMed Central

    Malhotra, B.; Glass, ADM.

    1995-01-01

    Potassium influx and cellular [K+] were measured in the unicellular green alga Chlamydomonas reinhardtii after pretreatment in either 10 or 0 mM external K+ ([K]0). K+ (42K+ or 86Rb+) influx was mediated by a saturable, high-affinity transport system (HATS) at low [K+]0 and a linear, low-affinity transport system at high [K+]o. The HATS was typically more sensitive to metabolic inhibition (and darkness) than the low-affinity transport system. Membrane electrical potentials were determined by measuring the equilibrium distribution of tetraphenylphosphonium. These values, together with estimates of cytoplasmic [K+] (B. Malhotra and A.D.M. Glass [1995] Plant Physiol 108: 1537-1545), demonstrated that at 0.1 mM [K+]0 K+ uptake must be active. At higher [K+]0 (>0.3 mM) K+ influx appeared to be passive and possibly channel mediated. When cells were deprived of K+ for 24 h, the Vmax for the HATS increased from 50 x 10-6 to 85 x 10-6 nmol h-1 cell-1 and the Km value decreased from 0.25 to 0.162 mM. Meanwhile, cellular [K+] declined from 24 x 10-6 to 9 x 10-6 nmol cell-1. During this period influx increased exponentially, reaching its peak value after 18 h of K+ deprivation. This increase of K+ influx was not expressed when cells were exposed to inhibitors of protein synthesis. The use of 42K+ and 86Rb+ in parallel experiments demonstrated that Chlamydomonas discriminated in favor of K+ over Rb+, and this effect increased with the duration of K+ deprivation. PMID:12228559

  4. Bumetanide-sensitive NaCl uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and hypertonicity.

    PubMed

    Liedtke, C M

    1992-05-01

    Loop diuretic-sensitive NaCl(K) cotransport plays a fundamental role in absorption and secretion of electrolytes in epithelial tissues. Cotransport activity was measured as uptake of 22Na, 36Cl, or 86Rb at 27 degrees C in isolated rabbit tracheal epithelial cells. Uptake of radiotracer was linear from 1 to 2 min after initiation of radiotracer transport. Bumetanide at 10 microM final concentration did not affect tracer uptake. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agent clonidine increased sodium and chloride uptake at least 5.5-fold. Bumetanide blocked sodium uptake by 85% and chloride uptake by 72%. 86Rb uptake was not affected by l-epinephrine, clonidine, or bumetanide. The alpha 2-adrenergic antagonist yohimbine blocked the effects of l-epinephrine and clonidine on 22Na and 36Cl uptake. In Ca(2+)-depleted transport medium, baseline levels of sodium and chloride uptake increased 3.8- and 2.4-fold, respectively, in a bumetanide-independent manner. Nevertheless, l-epinephrine and clonidine induced a net stimulation of sodium and chloride uptake similar to that found in Ca(2+)-replete medium. This response was reduced by bumetanide and yohimbine. The Ca(2+)-elevating agent ionomycin increased bumetanide-sensitive sodium and chloride uptake 7.2- and 6.2-fold, respectively. Replacement of chloride with gluconate or sodium with N-methyl-D-glucamine in the extracellular medium inhibited l-epinephrine and clonidine-stimulated bumetanide-sensitive sodium and chloride uptake, respectively. Osmotic shrinkage in hyperosmotic (500 mM NaCl with all other electrolytes at normal concentration) transport medium markedly increased bumetanide-inhibitable sodium and chloride uptake.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1590411

  5. Effects of acute and chronic uremia on active cation transport in rat myocardium

    SciTech Connect

    Druml, W.; Kelly, R.A.; England, B.K.; O'Hara, D.S.; Mitch, W.E. )

    1990-12-01

    As abnormalities of active cation transport could contribute to the genesis of uremic cardiomyopathy, we investigated myocardial sodium pump function in rats with acute renal failure (ARF) and with a model of experimental chronic renal failure (CRF) that has metabolic similarities to advanced chronic uremia in humans. CRF rats were hypertensive and had left ventricular hypertrophy (33% higher heart:body weight ratio; P less than 0.01) at four weeks compared to pair-fed sham-operated rats. Importantly, both ouabain- and furosemide-sensitive 86Rb uptake rates were unchanged in left ventricular myocardial slices from CRF, and the intracellular sodium concentration was not different from that of control rats even though skeletal muscle sodium was increased, as we found previously. Insulin-stimulated, ouabain-sensitive 86Rb influx was also preserved. There also were no abnormalities in myocardium cation transport in rats with ARF. However, (3H)ouabain binding was decreased 45% in CRF rats (P less than 0.01); it was unchanged in acute uremia. Decreased ouabain binding in chronic uremia was due entirely to fewer low affinity (3H)ouabain binding sites (the binding affinity for ouabain was unaffected). We conclude that in chronic, (but not acute) renal failure, sodium pump number is reduced in myocardium but intracellular sodium is unchanged and active cation flux rates are maintained. These results emphasize that in rats with chronic uremia, intracellular sodium homeostasis is preserved in myocardium, despite the presence of marked abnormalities of active cation transport in skeletal muscle that are characteristic of chronic uremia.

  6. Maturation of Rb+ and PAH accumulation by rabbit anterior uvea and choroid plexus

    SciTech Connect

    Krupin, T.; Fritz, C.; Becker, B.

    1985-02-01

    In vitro accumulation of radioactive para-aminohippuric acid (/sup 3/H-PAH) and rubidium (/sup 86/Rb+) by the anterior uvea, ciliary processes, and the choroid plexus was evaluated in tissues from newborn and various aged rabbits. Accumulation of PAH was present in the anterior uvea at 1 day of age (tissue to media ratio, T/M, of 2.1 +/- 0.2) and remained at this level for the first 14 days of life. Accumulation did not rise to adult levels until 21 days of age (T/M 5.5 +/- 0.6). Rubidium accumulation in the anterior uvea, a measure of Na+, K+-pump activity, was higher than adult values 6 hr after birth (T/M25.2 +/- 0.9). Activity remained elevated through day 28 and did not fall to adult levels until day 60 (T/M 13.4 +/- 0.6). Accumulation studies on isolated ciliary processes were similar to those obtained from anterior uveal tissue. Daily subcutaneous injections of penicillin (300,000 units/kg/day) for 1 week had no effect on anterior uvea PAH accumulation (penicillin T/M was 1.7 +/- 0.1 and saline control T/M was 2.0 +/- 0.2). Accumulation of either /sup 3/H-PAH or /sup 86/Rb+ by the choroid plexus was present 1 day after birth in amounts that were similar to adult values and did not change during the 90 days of testing.

  7. Glucose homeostasis in pregnant rats submitted to dietary protein restriction.

    PubMed

    de Mello, Maria Alice Rostom; Luciano, Eliete; Carneiro, Everardo Magalhães; Latorraca, Márcia Queiroz; Machado de Oliveira, Camnila Aparecida; Boschero, Antonio Carlos

    2003-01-01

    In the present work, we examined the effects of feeding a low protein diet during pregnancy on glucose-induced insulin secretion and glucose homeostasis in rats. Young (60 days), pregnant (P) or non-pregnant (NP) rats were fed during pregnancy or for 21 days (the NP) a normal (17%) or a low (6%) protein diet. Serum glucose and insulin levels and pancreas insulin content in the fed state; total area under serum glucose curve (AG) after a glucose load and serum glucose disappearance rate (Kitt) after insulin administration; as well as 86Rb outflow, 45Ca uptake and insulin secretion by isolated pancreatic islets in response to glucose were evaluated. Serum glucose was lower in 17%-P (12%) and 6%-P (27%) than in corresponding NP-rats. Serum insulin was higher in 17%-P (153%) and 6%-P (77%) compared to the corresponding NP-rats. Pancreatic insulin was higher in 6%-rats (55%) than in 17%-rats. No differences were found in AG among the groups whereas Kitt was lower in 6%-NP and higher in 6%-P than in the equivalent 17% rats. Increasing glucose concentration from 2.8 to 16.7 mmol/l, reduced 86Rb outflow from isolated islets from all groups. Increasing glucose concentration from 2.8 to 16.7 mmol/l elevated 45Ca uptake by 17%-NP (47%), 17%-P (40%) and 6%-P (214%) islets but not by 6%-NP ones. The increase in 45Ca uptake was followed by an increase in insulin release by the 17%-NP (2767%), 17%-P (2850%) and 6%-P (1200%) islets. In conclusion, 6%-P rats show impaired glucose induced insulin secretion related to reduced calcium uptake by pancreatic islets. However, the poor insulin secretion did not fully compensate the high peripheral sensitivity to the hormone, resulting in hypoglycemia. PMID:15686122

  8. Agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action

    SciTech Connect

    Forman, S.A.; Firestone, L.L.; Miller, K.W.

    1987-05-19

    Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring /sup 86/Rb/sup +/ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: (1) integrated net ion efflux (in 10 s) from untreated vesicles, (2) integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with ..cap alpha..-bungarotoxin, and (3) initial rates of efflux (5-100 ms) from vesicles that were partially blocked with ..cap alpha..-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10/sup 8/-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum /sup 86/Rb/sup +/ efflux by 50% (K/sub B/ values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, K/sub B/ values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times K/sub B/ did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. The authors conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

  9. Studies on lipids and the activity of Na,K-ATPase in lens fibre cells.

    PubMed Central

    Dean, W L; Delamere, N A; Borchman, D; Moseley, A E; Ahuja, R P

    1996-01-01

    Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase alpha1 and beta1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the NA,K-ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase alpha1 and beta1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase alpha1 beta1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase alpha1 beta1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K-ATPase alpha1 and beta1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes. PMID:8615795

  10. A-272651, a nonpeptidic blocker of large-conductance Ca2+-activated K+ channels, modulates bladder smooth muscle contractility and neuronal action potentials

    PubMed Central

    Shieh, C-C; Turner, S C; Zhang, X-F; Milicic, I; Parihar, A; Jinkerson, T; Wilkins, J; Buckner, S A; Gopalakrishnan, M

    2007-01-01

    Background and Purpose: The large-conductance Ca2+-activated K+ channel (BKCa, KCa1.1) links membrane excitability with intracellular Ca2+ signaling and plays important roles in smooth muscle contraction, neuronal firing, and neuroendocrine secretion. This study reports the characterization of a novel BKCa channel blocker, 2,4-dimethoxy-N-naphthalen-2-yl-benzamide (A-272651). Experimental Approach: 86Rb+ efflux in HEK-293 cells expressing BKCa was measured. Effects of A-272651 on BKCa α- and BKCa αβ1-mediated currents were evaluated by patch-clamp. Effects on contractility were assessed using low-frequency electrical field stimulated pig detrusor and spontaneously contracting guinea pig detrusor. Effects of A-272651 on neuronal activity were determined in rat small diameter dorsal root ganglia (DRG). Key Results: A-272651 (10 μM) inhibited 86Rb+ efflux evoked by NS-1608 in HEK-293 cells expressing BKCa currents. A-272651 concentration-dependently inhibited BKCa currents with IC50 values of 4.59 μM (Hill coefficient 1.04, measured at +40 mV), and 2.82 μM (Hill coefficient 0.89), respectively, for BKCa α and BKCa αβ1-mediated currents. Like iberiotoxin, A-272651 enhanced field stimulated twitch responses in pig detrusor and spontaneous contractions in guinea pig detrusor with EC50 values of 4.05±0.05 and 37.95±0.12 μM, respectively. In capsaicin-sensitive DRG neurons, application of A-272651 increased action potential firing and prolonged action potential duration. Conclusions and Implications: These data demonstrate that A-272651 modulates smooth muscle contractility and neuronal firing properties. Unlike previously reported peptide BKCa blockers, A-272651 represents one of the first small molecule BKCa channel blockers that could serve as a useful tool for further characterization of BKCa channels in physiological and pathological states. PMID:17519951

  11. Cytopathic action of Naegleria fowleri amoebae on rat neuroblastoma target cells.

    PubMed

    Marciano-Cabral, F; Zoghby, K L; Bradley, S G

    1990-01-01

    The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of 51Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of 51Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of 51Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either 86Rb or 51Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of 86Rb and 51Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.

  12. Biochemical analysis of SV40 small t mediated theophylline resistance in CV-1 cells

    SciTech Connect

    Renz, C.D.

    1986-01-01

    The papovavirus SV40 encodes for the two tumor antigens, large T and small t. While much is known about large T, little information is available about the role of small t in the viral life cycle. The authors have developed a system for studying small t antigen based on its ability to overcome the G/sub 0/ growth arrest induced by the methylxanthine, theophylline. Uninfected CV-1 cells, the permissive host for SV40, are arrested by 1-2mM theophylline. In contrast, Wt-infected cells are not arrested by the same concentrations of this drug. Biochemical studies were designed to analyze the effects of theophylline and the means by which small t can overcome the growth arrest of CV-1 cells. Theophylline, a cyclic AMP analogue, does not appear to arrest CV-1 cells by a cAMP-dependent mechanism. Theophylline appears to arrest CV-1 cells by inhibiting sodium influx. Both /sub 86/Rb/sup +/ and /sup 22/Na/sup +/ uptake were inhibited by theophylline. Amiloride and TMB-8, drugs which are known to inhibit the plasma membrane Na/sup +//H/sup +/ antiporter, decreased /sup 86/Rb/sup +/ and /sup 22/Na/sup +/ uptake to the same degree as theophylline. Because these drugs also arrested mock and D1- but not Wt-infected cells it is possible that theophylline inhibits sodium uptake by inhibiting this antiporter. Furthermore, because Wt-infected cells are resistant to the growth arrest induced by these drugs, it is possible that small t acts either by directly altering this antiporter or by bypassing the step which requires the activity of the antiporter.

  13. Intracellular mediators of Na -K pump activity in guinea pig pancreatic acinar cells

    SciTech Connect

    Hootman, S.R.; Ochs, D.L.; Williams, J.A.

    1985-10-01

    The involvement of CaS and cyclic nucleotides in neurohormonal regulation of Na -K -ATPase (Na -K pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by (3H)ouabain binding and by ouabain-sensitive YWRb uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of (TH)ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in CaS -free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular CaS , while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free CaS levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent CaS chelator. Basal intracellular CaS concentration ((CaS )i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively.

  14. Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives

    SciTech Connect

    Rottenberg, H.; Koeppe, R.E. II )

    1989-05-16

    Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb{sup +} transport was measured directly from the uptake of {sup 86}Rb. The truncated gramicidins show enhanced selectivity for K{sup +} and Rb{sup +} when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that {sup 86}Rb exchange is stimulated by desfor and desval almost to the same extend at gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded {beta}{sup 6.3}-helical dimer (channel) in its conductance characteristic and its structure. On the basis of the secondary structure of the truncated derivatives, the authors suggest that the antiparallel double-stranded helix dimer (pore) is a likely alternative structure for this novel channel.

  15. Dihydroxy bile salt-induced secretion of rubidium ion across the rabbit distal colon

    SciTech Connect

    Freel, R.W.

    1987-04-01

    Possible mechanisms of dihydroxy bile salt-induced K/sup +/ secretion by the mammalian colon were evaluated by studying the effects of taurochenodeoxycholate (TCDC) on /sup 86/Rb/sup +/ transport across the isolated, short-circuited rabbit distal colon. Simultaneous measurements of /sup 86/Rb/sup +/ and /sup 42/K/sup +/ unidirectional fluxes were highly correlated, indicating that Rb/sup +/ is a suitable tracer for K/sup +/ transport across the colon. Furthermore, mucosal Ba/sup 2 +/ (4 mM) or serosal ouabain (0.1 mM) decreased serosal to mucosal rubidium flux (J/sub s ..-->.. m//sup Rb/) without affecting J/sub m ..-->.. s//sup Rb/. Dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.5 mM serosal) specifically increased J/sub s ..-->.. m//sup Rb of controls through a barium- (4 mM, mucosal) sensitive pathway without affecting J/sub s ..-->.. m//Rb/. Mucosal addition of 2 mM TCDC increased tissue conductance (G/sub T/), reduced short-circuit (I/sub sc/) slightly, and reversed J/sub net//sup Rb/ principally by increasing J/sub s ..-->.. m//sup Rb/. The TCDC-induced increases in J/sub s ..-->.. m//sup Rb/ were reduced by 0.1 mM serosal ouabain or 4 mM mucosal Ba/sup 2 +/. Pretreatment with the putative calcium-calmodulin antagonist trifluoperazine blocked dBcAMP- and TCDC-induced increases in J/sub s ..-->.. m//sup Rb/ but did not prevent changes in G/sub T/ or I/sub sc/ produced by either secretagogue. It is concluded that TCDC stimulates Rb/sup +/ or K/sup +/ secretion by primarily increasing the efflux of these ions across Ba/sup 2 +/-sensitive K/sup +/ channels in the apical membrane and that this increase in permeability may be mediated intracellularly by the calcium-calmodulin complex.

  16. Transcapillary exchange in the cat salivary gland during secretion, bradykinin infusion and after chronic duct ligation.

    PubMed Central

    Mann, G E; Smaje, L H; Yudilevich, D L

    1979-01-01

    1. Capillary permeability-surface area products for 86Rb, [51Cr]EDTA (mol. wt. 357), [57Co]cyanocobalamin (mol. wt. 1353) and [125I]insulin (approximate mol. wt. 6000) have been measured using the single-circulation, multiple-tracer dilution technique in the in situ perfused submandibular salivary gland during parasympathetic nerve stimulation, close-arterial bradykinin infusion and following chronic duct ligation. 2. In glands with a natural blood supply, permeability-surface area for 86Rb and [51Cr]EDTA increased during parasympathetic stimulation, but this was shown to be related to the concomitant increase in blood flow rather than to a change in capillary permeability or in surface area. 3. In glands perfused at constant flow, parasympathetic stimulation led to a decrease in permeability-surface area for EDTA (-19.1 +/- 5.2%, mean +/- S.E., n = 5, P less than 0.05) cyanocobalamin (-12.3 +/- 6.0, n = 12, P less than 0.05), and insulin (-15.3 +/- 4.8, n = 11, P less than 0.02). It is suggested that this may be the result of a redistribution of flow from the acinar microcirculation to a less permeable ductal vasculature. 4. Bradykinin infusion had no significant effect on permeability-surface area for EDTA and cyanocobalamin in perfused glands. 5. In perfused glands, ligation of the submandibular duct for 3--12 days reduced permeability-surface area (ml.min-1.g-1) for [51Cr]EDTA from 5.26 +/- 0.60 (mean +/- S.E., n = 9) to 4.20 +/- 0.12 (n = 4, P less than 0.30), [57Co]cyanocobalamin from 3.22 +/- 0.12 (n = 48) to 2.02 +/- 0.08 (n = 15, P less than 0.001) and [125I]insulin from 1.52 +/- 0.07 (n = 39) to 0.72 +/- 0.23 (n = 11, P less than 0.001). PMID:119844

  17. Evidence for increased permeability of rat liver plasma membrane vesicles after CCl/sub 4/ in vivo

    SciTech Connect

    Tsokos-Kuhn, J.O.; Hughes, H.; Mitchell, J.R.

    1986-03-01

    Plasma membrane vesicles (PMs) of CCl/sub 4/-dosed rats show nearly total loss of ATP-driven Ca2+ uptake, more than produced by acetaminophen or bromobenzene. The authors postulated that CCl/sub 4/ PMs are more permeable or have less Ca2+ binding capacity and now report studies to test these hypotheses. After passive loading in 1 mM Ca2+, biphasic Ca2+ efflux was much more rapid in CCl/sub 4/ PMs; kefflux = 0.272 and 0.0516 min-1 for control, and 1.78 and 0.171 min-1 for CCl/sub 4/, based on semilog plots of averaged efflux data (N = 6). CCl/sub 4/ PMs lost 81% of zero time Ca2+ (corrected for Ca2+ not releasable by A23187) in 5 min, while controls lost 45%. /sup 86/Rb+ uptake in the presence of valinomycin measured the ability of PMs to maintain a K+ gradient when 100 mM KCl-loaded PMs were stripped of external K+. CCl/sub 4/ PMs took up half as much /sup 86/Rb+, suggesting the K+ gradient was half that maintained by controls (N =3). Ca content of CCl/sub 4/ PMs was decreased: zero time (12.3 +/- 0.8 vs 8.3 +/- 0.8 nmols/mg; p < .01) and post-A23187 (3.3 +/- 0.3 vs 1.6 +/- 0.3 nmols/mg; p<.01) and post-A23187 (3.3 +/- 0.3 nmols/mg; p < .005) Ca2+ content were less in CCl/sub 4/ PMs in passive loading/efflux studies. Total Ca in CCl/sub 4/ PMs, by atomic absorption, was 10.9 +/- 1.2 vs 17.4 +/- 2.0 nmols/mg in controls; p < .025. Inhibition of the Ca2+ pump cannot be ruled out, but it seems clear that membrane structural changes influencing permeability occur after CCl/sub 4/ administration.

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    Hannen, V.; Aprile, E.; 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.

    2011-10-01

    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 isotope 83Rb(T½ = 86.2 d) 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 83Rbγ-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 the production of 83Rb at the Rez cyclotron.

  20. Potassium channel conductance as a control mechanism in hair follicles.

    PubMed

    Buhl, A E; Conrad, S J; Waldon, D J; Brunden, M N

    1993-07-01

    The opening of intracellular potassium channels is a common mechanism of action for a set of anti-hypertensive drugs that includes the hair-growth-inducing agent minoxidil. Recent work suggests potassium channel openers (PCOs) also influence hair growth. Correlative studies demonstrate that a series of PCOs including minoxidil, pinacidil, P-1075, an active pinacidil analog, RP-49,356, cromakalim, and nicorandil maintain hair growth in cultured vibrissa follicles. Studies using balding stumptail macaques verify that minoxidil, P-1075, and cromakalim but not RP-49,356 stimulate hair growth. The definition of potassium channels and documentation of drug effects on these channels is classically done using electrophysiologic techniques. Such studies require the identification and isolation of target cells. Both these are among the unsolved problems in the area of hair biology. Estimating K+ flux using 86Rb+ as a K+ tracer is an accepted method of assessing potassium channel conductance in other organ systems. Both pinacidil and RP-49,356 induce measurable Rb+ flux in isolated vibrissa follicles and a hair epithelial cell line whereas neither minoxidil nor minoxidil sulfate had measurable effects. Potassium channels have been studied successfully in other organ systems using specific pharmacologic blockers for the various channel subtypes. Blockers including glyburide, tetraethylammonium, and procaine failed to inhibit minoxidil stimulation of cultured follicles. The current explosion of knowledge on potassium channel biology, cloning of channels, and continued progress in hair biology promise to clarify the role of K+ ions in the control of hair follicles.

  1. Discovery of Highly Potent and Selective α4β2-Nicotinic Acetylcholine Receptor (nAChR) Partial Agonists Containing an Isoxazolylpyridine Ether Scaffold that Demonstrate Antidepressant-like Activity. Part II

    PubMed Central

    Yu, Li-Fang; Eaton, J. Brek; Fedolak, Allison; Zhang, Han-Kun; Hanania, Taleen; Brunner, Dani; Lukas, Ronald J.; Kozikowski, Alan P.

    2012-01-01

    In our continued efforts to develop α4β2-nicotinic acetylcholine receptor (nAChR) partial agonists as novel antidepressants having a unique mechanism of action, structure activity relationship (SAR) exploration of certain isoxazolylpyridine ethers is presented. In particular, modifications to both the azetidine ring present in the starting structure 4 and its metabolically liable hydroxyl side chain substituent have been explored to improve compound druggability. The pharmacological characterization of all new compounds has been carried out using [3H]epibatidine binding studies together with functional assays based on 86Rb+ ion flux measurements. We found that the deletion of the metabolically liable hydroxyl group or its replacement by a fluoromethyl group not only maintained potency and selectivity, but also resulted in compounds showing antidepressant-like properties in the mouse forced swim test. These isoxazolylpyridine ethers appear to represent promising lead candidates in the design of innovative chemical tools containing reporter groups for imaging purposes and of possible therapeutics. PMID:23092294

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

    SciTech Connect

    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.

  3. Short-Term Experiments on Ion Transport by Seedlings and Excised Roots 1

    PubMed Central

    Huang, Zhang-Zhi; Yan, Xiaolong; Jalil, Abdul; Norlyn, Jack D.; Epstein, Emanuel

    1992-01-01

    The absorption of K+ by excised roots of barley (Hordeum vulgare L. cv California Mariout) has been systematically compared with that of entire, undisturbed seedlings. Some experiments have also been done with wheat (Triticum aestivum L.) and an amphiploid obtained from a cross between it and salt-tolerant tall wheatgrass (Lophopyrum elongatum Host Löve [syn. Agropyron elongatum Host]). For all three genotypes, the rate of K+ absorption measured in a 20-min period was identical for entire 8-d-old seedlings and their excised roots within the experimental error. Manipulation gentler than root excision, viz. careful transfer of seedlings from one experimental solution to another, was also without effect on the rate of K+ absorption. Absorption of K+ measured by assay of its 86Rb label in the tissue was identical with that measured by K+ depletion of the experimental solutions assayed chemically. For the plant materials and conditions of these experiments, the excised root technique for studying ion transport into roots is validated. The advantages of the technique, and findings differing from the present ones, are discussed. Images Figure 2 PMID:16653217

  4. Interaction of the alpha subunit of Na,K-ATPase with cofilin.

    PubMed Central

    Lee, K; Jung, J; Kim, M; Guidotti, G

    2001-01-01

    The alpha1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the alpha subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive (86)Rb(+) uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo. PMID:11139403

  5. Displacement of Ca2+ by Na+ from the Plasmalemma of Root Cells 1

    PubMed Central

    Cramer, Grant R.; Läuchli, André; Polito, Vito S.

    1985-01-01

    A microfluorometric assay using chlorotetracycline (CTC) as a probe for membrane-associated Ca2+ in intact cotton (Gossypium hirsutum L. cv Acala SJ-2) root hairs indicated displacement of Ca2+ by Na+ from membrane sites with increasing levels of NaCl (0 to 250 millimolar). K+(86Rb) efflux increased dramatically at high salinity. An increase in external Ca2+ concentration (10 millimolar) mitigated both responses. Other cations and mannitol, which did not affect Ca2+-CTC chelation properties, were found to have no effect on Ca2+-CTC fluorescence, indicating a Na+-specific effect. Reduction of Ca2+-CTC fluorescence by ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, which does not cross membranes, provided an indication that reduction by Na+ of Ca2+-CTC fluorescence may be occurring primarily at the plasmalemma. The findings support prior proposals that Ca2+ protects membranes from adverse effects of Na+ thereby maintaining membrane integrity and minimizing leakage of cytosolic K+. PMID:16664372

  6. Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

    SciTech Connect

    Ciapa, B.; Payan, P. ); Allemand, D. )

    1989-12-01

    The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive {sup 86}Rb uptake and amiloride-sensitive {sup 24}Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na{sup +}/H{sup +} and Na{sup +}/K{sup +} exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na{sup +}/H{sup +} activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na{sup +}/H{sup +} exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 minutes. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na{sup +} stat, pH stat).

  7. Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components

    SciTech Connect

    Iida, K.; Whitlow, M.B.; Nussenzweig, V.

    1989-03-01

    We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated /sup 86/Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

  8. Pancreatic blood flow in experimental acute pancreatitis

    SciTech Connect

    Berry, A.R.; Millar, A.M.; Taylor, T.V.

    1982-05-01

    The etiology and pathogenesis of acute necrotizing hemorrhagic pancreatitis remain controversial. Recent work has suggested that an early fall in pancreatic blood flow, causing ischemia, may be the initiating factor. Using an established rat model of hemorrhagic pancreatitis and the fractional indicator distribution technique with /sup 86/RbCl, pancreatic blood flow and tissue perfusion have been measured at various times in the condition. Six groups of ten rats were studied: control sham operation and pancreatitis groups were sacrificed at 1, 6, and 24 hr. Pancreatic blood flow (% of cardiac output) and perfusion (blood flow/g tissue) were measured. Blood flow was increased by a maximum of 53% at 1 hr (P less than 0.001) and remained elevated for 24 hr, and perfusion was increased by a maximum of 70% (P less than 0.001) at 1 hr and remained elevated at 6 hr. Pancreatic perfusion declines after 6 hr due to increasing gland edema. The results demonstrate a significant increase in pancreatic blood flow and perfusion in experimentally induced acute pancreatitis, suggesting a primary inflammatory response, and refute the ischemic etiological theory.

  9. Nuclear Data Sheets for A = 86

    SciTech Connect

    Negret, Alexandru; Singh, Balraj

    2015-02-15

    The experimental nuclear spectroscopic data for known nuclides of mass number 86 (Ga, Ge, As, Se, Br, Kr, Rb, Sr, Y, Zr, Nb, Mo, Tc) have been evaluated and presented together with Adopted properties for levels and γ rays. New high-spin data are available for {sup 86}Se, {sup 86}Br, {sup 86}Kr, {sup 86}Sr, {sup 86}Y, {sup 86}Zr and {sup 86}Mo; and lifetime data for high-spin states in {sup 86}Y and {sup 86}Zr. No significant new data since the 2001 NDS for A=86 have been reported for {sup 86}Rb and {sup 86}Nb. No data are yet available for excited states in {sup 86}Ga and {sup 86}As. The decay scheme of radioactive {sup 86}Ge is unknown, while those for {sup 86}Ga, {sup 86}As, 47.4 min isomer of {sup 86}Y, {sup 86}Nb and {sup 86}Tc are deemed as incomplete. Isomerism in {sup 86}Nb remains unconfirmed. This work supersedes the data presented in the previous NDS evaluation of A=86 published by 2001Si43.

  10. Protective effect of eicosapentaenoic acid on ouabain toxicity in neonatal rat cardiac myocytes

    SciTech Connect

    Hallaq, H.; Leaf, A. ); Sellmayer, A. ); Smith, T.W. )

    1990-10-01

    Isolated neonatal cardiac myocytes have been utilized as a model for the study of cardiac arrhythmogenic factors. The myocytes respond to the toxic effects of a potent cardiac glycoside, ouabain at 0.1 mM, by an increase in their spontaneous beating rate and a reduction in amplitude of contractions resulting within minutes in a lethal state of contracture. Incubating the isolated myocytes for 3{endash}5 days in culture medium enriched with 5 {mu}M arachidonic acid had no effect on the development of lethal contracture after subsequent exposure to 0.1 mM ouabain. By contrast, incubating the myocytes for 3{endash}5 days with 5 {mu}M eicosapentaenoic acid completely prevented the toxic effects of ouabain at 0.1 mM. No differences in bumetanide-inhibitable {sup 86}Rb flux were observed between the three preparations. However, measurements with fura-2 of cytosolic free calcium levels indicated that control and arachidonic acid-enriched myocytes developed toxic cytosolic calcium concentrations of 845 {plus minus} 29 and 757 {plus minus} 64 nM, respectively, on exposure to 0.1 mM ouabain, whereas in eicosapentaenoic acid-enriched myocytes, physiologic calcium levels were preserved. Incubating the myocytes with eicosapentaenoic acid for 3{endash}5 days resulted in a small reduction of arachidonic acid and a small but significant increase of eicosapentaenoic acid in membrane phospolipids of the myocytes.

  11. Intestinal circulation during inhalation anesthesia

    SciTech Connect

    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.

  12. Reduction in vitro of red cell glutathione reproduces defects of cellular sodium transport seen in oedematous malnutrition.

    PubMed

    Forrester, T; Golden, M; Brand, S; Swales, J

    1990-05-01

    Red cells in oedematous malnutrition (kwashiorkor) have an increased sodium content, 'leakiness' to sodium and enhanced sodium pumping. In non-oedematous malnutrition (marasmus) there is a reduction in the sodium pump activity. The explanation has hitherto been unknown but the glutathione content of red cells is low in kwashiorkor and normal in marasmus. We artificially lowered the glutathione content of normal red cells to values characteristic of mild oedematous malnutrition, using the enzyme inhibitors bischloronitrosourea (BCNU) and buthionine sulfoximine (BSOX). After preincubation, the cells were washed to remove the inhibitors and oxidized glutathione. Cellular content of sodium and potassium, and 86Rb influx were then measured. The reduction in glutathione reproduced the abnormalities of sodium content and flux observed in kwashiorkor. We suggest that oxidant stress in kwashiorkor, by reducing cellular glutathione, may affect cell membrane electrolyte transport. This may act through alterations in membrane sulfhydryl groups. Glutathione depletion may therefore play an important role in the clinical picture and natural history of oedematous malnutrition and may have relevance to other conditions where oxidant stress occurs.

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

    PubMed

    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.

  14. Effects of temperature on blood flow in facial tissues

    SciTech Connect

    Kwon, H.J.; Rhee, J.G.; Song, C.W.; Waite, D.E.

    1986-10-01

    This study evaluated the effect of temperature on blood flow by using rubidium 86 to determine the fraction of cardiac output going to specific facial tissues of rats. Seventy-seven male Holtzman rats were divided into two groups and various subgroups. The intraoral subgroups were categorized according to the temperature of the water irrigating the oral cavity (11, 39, 41, 43, or 45/sup 0/C). The extraoral subgroups were categorized according to whether heat or ice was applied to the cheek of the rats and the duration of application (10 or 20 minutes). Rats were killed within 60 seconds after injection of 86Rb, and tissue samples were taken from the buccal mucosa, facial skin, masseter muscle, mandible, tongue, and maxilla. The fraction of isotope uptake in each gram of these tissues (FU/g) was then calculated. For the intraoral group, the bath temperature of 39/sup 0/C increased the FU/g in all tissues except masseter muscle and tongue. Higher bath temperature did not further alter the FU/g. Lowering the temperature to 11 degrees C changed the FU/g significantly only in the tongue. For the extraoral group, 20-minute application of ice was required to reduce FU/g significantly in all tissues but masseter muscle and skin. Extraoral heat application for 10 minutes increased FU/g significantly in skin and tongue, but no further change was seen after 20 minutes of application.

  15. Effect of changes in dietary sodium on active electrolyte transport by erythrocytes at different stages of human pregnancy.

    PubMed

    Gallery, E D; Rowe, J; Brown, M A; Ross, M

    1988-02-01

    1. Active electrolyte transport was examined in erythrocytes from women in the second and third trimesters of pregnancy and post partum, and compared with that in ovulating women. 2. There was a significant reduction in intracellular sodium ([Na]i) and increase in intracellular potassium ([K]i) in pregnancy with a return towards normal values in the post-partum period. 3. Maximum specific ouabain binding [number of Na+,K+-adenosine triphosphatase (Na+, K+-ATPase) units] was increased by 70% in pregnancy and returned slowly towards normal values post partum. 4. Na+,K+-ATPase activity as determined by ouabain-sensitive 86Rb influx in artificial media was also increased in pregnancy by 13%. It returned towards normal post partum. 5. The increases in Na+,K+-ATPase in pregnancy were not closely related to the concomitant increases in aldosterone or cholesterol nor to reticulocytosis and were not affected by 7 days of high (greater than 250 mmol/day) or low (less than 50 mmol/day) sodium intake.

  16. Effects of dietary sodium deprivation on erythrocyte sodium concentration and cation transport in normotensive and untreated hypertensive subjects.

    PubMed

    Stokes, G S; Monaghan, J C; Middleton, A T; Shirlow, M; Marwood, J F

    1986-02-01

    There is controversy about the effects of dietary sodium deprivation on cellular cation transport. Using washed erythrocytes for in vitro 22Na and 86Rb uptake studies, we studied the effects of a strict low-salt diet (20 mmol/day) for 4 days in 14 normotensive and 13 hypertensive subjects. Urinary sodium excretion fell from 147 +/- 13 to 18 +/- 3 mmol/24 h in the normotensive group and from 155 +/- 16 to 20 +/- 2 mmol/24 h in the hypertensive group. In both groups, there was a fall in plasma sodium concentration and activation of the renin-aldosterone axis. Both systolic and diastolic blood pressures fell in the hypertensive, but not the normotensive group. There were small but significant (P less than 0.025) decreases in cell cation concentrations and passive cation transport in the normotensive, but not the hypertensive group. No significant change in sodium pump activity or in Na+K+ cotransport was seen in either group. These observations provide no support for the concept that a decrease in dietary sodium intake can induce changes in cell cation transport, detectable in vitro, to which reduction in blood pressure may be attributed.

  17. Lack of interaction between digoxin and quinidine in cultured heart cells

    SciTech Connect

    Horowitz, J.D.; Barry, W.H.; Smith, T.W.

    1982-03-01

    Previous investigations have raised the possibility that the digoxin-quinidine interaction is associated with a reduction in the positive inotropic effect of digoxin due to displacement of digoxin from cardiac as well as skeletal muscle. To circumvent some of the complexities presented by intact animal models, this interaction was investigated in cultured chick embryo ventricular cells. Quinidine, even at relatively high concentrations (10(-4)--2 x 10(-3) M), did not significantly affect positive inotropic effects of digoxin and did not protect against cellular contracture induced by toxic digoxin concentrations, despite preincubation of cells with quinidine for 60 min. The effects of digoxin on monovalent cation transport, as judged by active uptake of the K analog 86Rb, were also not altered by 10(-4) M to 2 x 10(-3) M quinidine. These data suggest that quinidine does not displace digoxin from Na, K adenosine triphosphatase binding sites in this preparation. Although these data must be extrapolated to the intact animal with caution, our findings suggest that changes in digoxin clearance are more likely of primary importance in the digoxin-quinidine interaction, and indicate that the approximately 2-fold increase in serum digoxin concentration observed after addition of quinidine would be expected to have direct effects on myocardial cells comparable with those seen with increased digoxin concentration in the absence of quinidine.

  18. Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells

    SciTech Connect

    Allen, J.C.; Navran, S.S.; Seidel, C.L.; Dennison, D.K.; Amann, J.M.; Jemelka, S.K.

    1989-04-01

    Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number (( /sup 3/H) ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited /sup 86/Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of (/sup 3/H)ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.

  19. Propionate induces cell swelling and K+ accumulation in shark rectal gland

    SciTech Connect

    Feldman, G.M.; Ziyadeh, F.N.; Mills, J.W.; Booz, G.W.; Kleinzeller, A. )

    1989-08-01

    Small organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter. The Na+-H+ exchange process tends to correct intracellular pH (pHi), and thus it maintains a favorable gradient for propionic acid diffusion and allows propionate to accumulate. Activation of the Na+-H+ antiport also facilitates Na+ entry into the cell and Nai accumulation. At the same time Na+-K+-ATPase activity, unaffected by propionate, replaces Nai with Ki, whereas the K+ leak rate, decreased by propionate, allows Ki to accumulate. As judged by {sup 86}Rb+ efflux, the reduction in K+ leak was not due to propionate-induced cell acidification or reduction in Cli concentration. Despite inducing cell swelling, propionate did not disrupt cell structural elements and F actin distribution along cell membranes.

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

    SciTech Connect

    Soszynski, P.; Slowinska-Srzednicka, J.; Zgliczynski, S. )

    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.

  1. Effect of osmolarity on potassium transport in isolated cerebral microvessels

    SciTech Connect

    Lin, J.D.

    1988-01-01

    Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of /sup 86/Rb into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity of K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.

  2. Elenoside, a new cytotoxic drug, with cardiac and extracardiac activity.

    PubMed

    Navarro, Eduardo; Alonso, Simeona Josefina; Trujillo, Juan; Jorge, Elena; Pérez, Cirilo; Hernández-Calzadilla, Carlos

    2002-08-01

    This paper deals with the effects of elenoside, (3-hidroxymethyl-1-methoxy-5,6-methylene-dioxy-4-(3,4-methylenedioxyphenyl)-2-naftoic acid lactone-beta-D-glucoside) an arylnaphthalene lignan with broad spectrum cytotoxicity in a human tumor cell line panel, isolated from Justicia hyssopifolia (Acanthaceae) grown in the Canary Islands (Spain), on isolated cardiac auricle of rabbits, urinary excretion of rats, and on isolated rat ileum. These effects, using a vehicle (propylene glycol-ethanol-plant oil-Tween 80 (40:10:50:2) as a standard, are presented. Elenoside at concentrations of 3.2x10(-4), 6.4 x 10(-4), and 1.2 x 10(-3) M produced an increase in the contraction force of auricles in a concentration-dependent way. At doses of 25 and 50 mg/kg, an antidiuretic effect and a decrease in sodium excretion were observed. Elenoside at concentrations of 3.2 x 10(-4), 6.4 x 10(-4) and 1.2 x 10(-3) M produced an increase in the contraction force of ileum in a concentration-dependent manner. Elenoside produced the concentration dependent inhibition of 86Rb uptake. These results indicate that elenoside has digitalis-like activity similar to mammalian lignans. Moreover, this lignan has an irritant effect on the gastrointestinal tract.

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

    SciTech Connect

    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.

  4. Effect of ATPase Inhibitors on Cell Potential and K+ Influx in Corn Roots 1

    PubMed Central

    Cheeseman, John M.; Lafayette, Peter R.; Gronewald, John W.; Hanson, John B.

    1980-01-01

    Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Ψ) and K+ (86Rb) influx of corn root tissue over a wide range of K+ activity. N,N′Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K+ influx and depolarized Ψ at low, but not at high, K+ activity (K°). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K+ influx over a wide range of K°, with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Ψp) above 0.2 millimolar K°. Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K+ influx and Ψp at low K° can be attributed to inhibition of the plasmalemma ATPase. It is concluded that by DCCD treatment, the energy-linked electrogenic system at high K° is separated from the energy-linked K+ influx system at low K°. The results are analyzed in terms of electrical analogue models of the membrane. The presence of two, algebraically additive electrogenic components is indicated; one is better modeled as a current source (system I) and one as a voltage source (system II). No K+ stimulation of system II is required to produce the observed K° dependence of Ψp. PMID:16661348

  5. The toxicity of the mutagen 'MX' and its analogue, mucochloric acid, to rainbow trout hepatocytes and gill epithelial cells and to Daphnia magna.

    PubMed

    Isomaa, B; Holmström, T H; Lilius, H; Franzén, R; Kronberg, L

    1995-06-26

    The cytotoxicity of the, in Salmonella, potent mutagenic compound, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and its structural analogue 3,4-dichloro-5-hydroxy-2[5H]-furanone (mucochloric acid, MCA), was studied in freshly isolated rainbow trout hepatocytes and gill epithelial cells by determining 86Rb-leakage and decrease in fluorescence intensity in calcein AM-loaded cells. The acute toxicity of the compounds to Daphnia magna was studied by determining the concentration causing immobilization of the organism. MX proved to be more toxic than MCA both in the cellular assays and in the acute toxicity test with D. magna. MX was more toxic to hepatocytes than to gill epithelial cells. The uptake of [14C]MX was also much more efficient in hepatocytes than in gill epithelial cells. The uptake of [14C]MX in hepatocytes was not inhibited by taurocholic acid in excess, indicating that MX is not taken up by the carrier complex responsible for the uptake of taurocholate in the hepatocytes. Both the acute toxicity to D. magna and cytotoxicity of MX and MCA was rather low (EC50 values > 0.1 mM) and we conclude that it is very unlikely that MX and MCA at concentrations occurring in recipients receiving chlorination effluents from pulp mills or chlorinated domestic sewage, as regards their acute toxicity, implies a risk for aquatic animals.

  6. Serum factor induces selective increase in Na-channel expression in cultured skeletal muscle

    SciTech Connect

    Brodie, C.; Sampson, S.R. )

    1991-07-01

    The authors have examined effects of horse serum (HS) and various fractions (1 million-1M, 300K, 100K, and 30K nominal molecular weight limit) obtained by ultrafiltration on expression of TTX-sensitive Na-channels and on activities of the Na-K pump and glucose transport systems in cultured myotubes obtained from 1-2-day-old neonatal rat pups. Five-day-old cells were transferred to serum-free medium with no hormone or growth factor supplements (DMEM) for 24 hr and then treated with the various serum fractions for 48 hr. Measurements were made of specific (3H)-saxitoxin (STX) binding, action potential properties, 86Rb-uptake and 2-deoxyglucose (2-DG) uptake. HS significantly increased all parameters compared to DMEM (increases in STX-binding, 69%; Rb-uptake, 65%; 2-DG uptake, 93%). Results of treatment with the separate fractions showed that the 300K fraction caused a significantly greater increase in STX-binding than either HS or the other fractions. In contrast, the increases in Rb and 2-DG uptakes induced by the different fractions were not different from that obtained with HS. They conclude that serum contains a factor that selectively increases expression of TTX-sensitive Na-channels in skeletal muscle.

  7. Morphological and physiological differences between aeroponically and hydroponically grown sunflower plants.

    PubMed

    Szabó-Nagy, A; Abdulai, M D; Erdei, L

    1994-01-01

    The effects of aeroponic (AP) and hydroponic (HP) conditions on growth rate, morphological traits, potassium uptake and redox activities were compared in sunflower seedlings. Higher growth rate was found under HP than AP conditions and morphological traits were also different. The thickness of AP grown roots increased and new lateral roots with thickened root hairs were formed while the length of AP roots was small. Microscopical studies on cross-sections of the embedded root segments showed that the diameter of cross-section, the diameter of stele and the width of cortex of AP grown roots were significantly higher than those of HP plants. The element composition of AP or HP grown plants also differed, due to the different ion uptake processes. Potassium (86Rb+) uptake of AP grown plants was low and it seemed to be a passive process, while in case of HP grown plants it was decreased by both dinitrophenol and ferricyanide suggesting that the uptake process was coupled to the proton gradient. Roots of AP grown plants showed high ferricyanide reductase activity and it was accompanied by an increased acidification of the medium.

  8. Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

    SciTech Connect

    Antoine, M.H.; Hermann, M.; Herchuelz, A.; Lebrun, P. )

    1991-07-01

    Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the K+ channel openers. The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.

  9. The relationship between the hypokalaemic response to adrenaline, beta-adrenoceptors, and Na(+)-K+ pumps in skeletal and cardiac muscle membranes in the rabbit

    SciTech Connect

    Elfellah, M.S.; Reid, J.L. )

    1990-01-01

    The hypokalaemic response to adrenaline and the involvement of beta-adrenoceptors and Na(+)-K+ pumps were investigated in control rabbits and animals chronically pretreated with adrenaline. The hypokalaemic response to acute intravenous infusion of adrenaline was significantly reduced when rabbits were chronically pretreated with adrenaline for 10 days. Chronic pretreatment of rabbits with adrenaline significantly reduced the densities for (125I)cyanopindolol and (3H)ouabain binding sites in skeletal muscle and heart. Furthermore, there was a strong positive correlation (r = 0.97, p less than 0.001) between the Bmax for ICYP and (3H)ouabain, in the rabbit heart. Ouabain-sensitive 86Rb uptake and the activity of 3-O-methylfluorescein phosphate phosphatase were used to assess the function of the Na(+)-K+ pump in skeletal and cardiac muscle. There was no significant difference in these functional indices of the Na(+)-K+ pump between the control and adrenaline-pretreated animals, in skeletal or cardiac muscle. Thus, downregulation of the (3H)ouabain binding sites did not appear to be accompanied by reduced function of the Na(+)-K+ pump. Additional investigations are required to confirm further the dissociation between the function of the pump and the ouabain binding sites.

  10. Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

    SciTech Connect

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-06-14

    In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

  11. Nonradioactive rubidium ion efflux assay and its applications in drug discovery and development.

    PubMed

    Terstappen, Georg C

    2004-10-01

    The recent sequencing of the human genome has created comprehensive information of all potential drug targets. Based on current estimations for the total number of genes, around 400 poreforming ion channel genes can be expected corresponding to about 1.3% of the human genome. Since many ion channels are involved in diseases and the currently marketed drugs act only on a small fraction of these pore-forming membrane proteins, there is a big opportunity for innovative ion channel drug discovery. In fact, recent advances in the development of functional ion channel assays are currently enabling a more systematic exploitation of this important target class. In particular, fluorescence-based methods, automated electrophysiology, and ion flux assays are most important in this regard. This article will briefly describe these methods focusing on the nonradioactive Rb(+) efflux assay that I developed in the early 1990s since it has found widespread application in drug discovery and development and greatly displaced (86)Rb(+) assays for the analysis of K(+) and nonselective cation channels in the pharmaceutical industry.

  12. Effects of artificial sweeteners on insulin release and cationic fluxes in rat pancreatic islets.

    PubMed

    Malaisse, W J; Vanonderbergen, A; Louchami, K; Jijakli, H; Malaisse-Lagae, F

    1998-11-01

    Beta-L-glucose pentaacetate, but not alpha-D-galactose pentaacetate, was recently reported to taste bitter and to stimulate insulin release. This finding led, in the present study, to the investigation of the effects of both bitter and non-bitter artificial sweeteners on insulin release and cationic fluxes in isolated rat pancreatic islets. Sodium saccharin (1.0-10.0 mM), sodium cyclamate (5.0-10.0 mM), stevioside (1.0 mM) and acesulfame-K (1.0-15.0 mM), all of which display a bitter taste, augmented insulin release from islets incubated in the presence of 7.0 mM D-glucose. In contrast, aspartame (1.0-10.0 mM), which is devoid of bitter taste, failed to affect insulin secretion. A positive secretory response to acesulfame-K was still observed when the extracellular K+ concentration was adjusted to the same value as that in control media. No major changes in 86Rb and 45Ca outflow from pre-labelled perifused islets could be attributed to the saccharin, cyclamic or acesulfame anions. It is proposed that the insulinotropic action of some artificial sweeteners and, possibly, that of selected hexose pentaacetate esters may require G-protein-coupled receptors similar to those operative in the recognition of bitter compounds by taste buds.

  13. Regulation of the Na,K-pump by leptin in 3T3-L1 fibroblasts.

    PubMed

    Sweeney, G; Niu, W; Kanani, R; Klip, A

    2000-03-01

    Leptin, the product of the obesity (ob) gene, controls energy intake and expenditure primarily by actions on the central nervous system. However, recently it has become apparent that leptin also elicits a growing and diverse array of effects on peripheral tissues. The Na,K-pump is an electrogenic plasma membrane protein which actively extrudes 3Na+ ions and imports 2K+ ions per molecule of ATP hydrolysed. The pump is responsible for the maintenance of the electrochemical potential of all cells, which in turn drives all ion-coupled transport mechanisms. In this study we use 3T3-L1 fibroblasts to show that leptin inhibits Na,K-pump activity, as assessed by ouabain-sensitive 86Rb+ uptake. Inhibition of the Na,K-pump correlated with increased serine phosphorylation of the catalytic Na,K-pump alpha1 subunit. Upon investigation of leptin-stimulated signalling pathways using specific pharmacological inhibitors, only wortmannin prevented inhibition of the Na,K-pump by leptin. Moreover, leptin stimulated phosphotyrosine-associated PI 3-kinase activity in these cells. In summary, leptin was found to inhibit Na,K-pump activity, likely via PI 3-kinase. We propose that this effect may have wide ranging cardiovascular and metabolic implications and perhaps explain physiological effects of the hormone such as natriuresis.

  14. Inhibition of gastric H+,K+-ATPase by 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), an inhibitor of volume-regulated anion channel.

    PubMed

    Fujii, Takuto; Takahashi, Yuji; Takeshima, Hiroshi; Saitoh, Chisato; Shimizu, Takahiro; Takeguchi, Noriaki; Sakai, Hideki

    2015-10-15

    4-(2-Butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB) has been used as an inhibitor of volume-regulated anion channel (VRAC), which is expressed in almost all cells (IC50 is around 4 µM). Here, we found that DCPIB significantly inhibited the activities of gastric proton pump (H+,K+-ATPase) in isolated gastric tubulovesicles and the membrane sample of the H+,K+-ATPase-expressing cells, and their IC50 values were around 9 µM. In the tubulovesicles, no significant expression of leucine rich repeat containing 8 family member A (LRRC8A), an essential component of VRAC, was observed. The inhibitory effect of DCPIB was also found in the membrane sample obtained from the cells in which LRRC8A had been knocked down. On the other hand, DCPIB had no significant effect on the activity of Na+,K+-ATPase or Ca2+-ATPase. In the H+,K+-ATPase-expressing cells, DCPIB inhibited the 86Rb+ transport activity of H+,K+-ATPase but not that of Na+,K+-ATPase. DCPIB had no effect on the activity of Cl- channels other than VRAC in the cells. These results suggest that DCPIB directly inhibits H+,K+-ATPase activity. DCPIB may be a beneficial tool for studying the H+,K+-ATPase function in vitro.

  15. Altered erythrocyte Na-K pump in anorectic patients

    SciTech Connect

    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.

  16. Aspects of the haemolytic reaction induced by Kanagawa haemolysin of Vibrio parahaemolyticus.

    PubMed

    Huntley, J S; Hall, A C

    1994-11-01

    Vibrio parahaemolyticus, an important enteric pathogen, produces toxin (Kanagawa haemolysin, KH), the presence of which correlates well with pathogenicity. KH induced lysis of human red blood cells (HRBC); the kinetics were strongly dependent on KH concentration (0-1 HU/ml) and rather independent of target cell concentration [0.5 < or = haematocrit (%) < or = 6] and the ratio KH:HRBC. The suggestion that KH-induced haemolysis is due to colloid osmosis is supported by results indicating: (1) osmotic protection (by suspension in iso-osmotic choline chloride, D-sorbitol or L-valine, or MOPS-buffered saline with added sucrose), (2) a cell volume increase prior to lysis, and (3) an increase in HRBC cation (86Rb+) influx after KH addition, indicating raised passive cation permeation. The effect of temperature on KH-induced haemolysis indicates the importance of processes other than the action of a simple water-filled pore, because of the high activation energy [53.30 +/- 2.79 kJ (mol.)-1] involved. Although haemolytic rate was attenuated by washout after 5 min KH exposure, the KH-induced lesion itself was not susceptible to washout by either extracellular volume expansion (at constant osmolarity) or centrifugation/resuspension. This suggests that HRBC binding of KH from aqueous solution still continues after 5 min exposure at 37 degrees C. Pre-vortexing KH with dibutyl phthalate (DBP) dramatically reduced the haemolytic activity of the aqueous toxin preparation, suggesting a protein-lipid interaction, which may support the contention that KH can move from a hydrophilic to a hydrophobic environment. Two features were identified that are characteristic of highly purified TDH preparations: (1) thermostability of haemolysin, and (2) monovalent cation selectivity series of lesion: Cs+ > Li+ > K+ > Rb+ > Na+, confirming that TDH is the important leak-inducing agent of KH. PMID:7886698

  17. Physiological and biochemical studies of newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart

    SciTech Connect

    Hunter, D.D.

    1986-01-01

    Exposure of either chicken embryos in ovo or cultured embryonic chicken cardiac cells in vitro to the muscarinic agonist carbachol results in a 70-90% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed in cardiac cells. Block of agonist-receptor interactions in ovo with the antagonist atropine or removal of the agonist in vitro results in a gradual increase in mAChR number, reaching the control level in 14 hr. Measurements of physiological sensitivity of atria or cultured cells show that, even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at this time. Newly synthesized mAChR which appear following affinity alkylation in cultured cells are also poorly coupled to the stimulation of /sup 86/Rb/sup +/ efflux, indicating that decreased physiological sensitivity is not due to an unknown effect of long-term agonist exposure on general cellular function, but rather reflects an intrinsic property of newly synthesized mAChR. This increase in sensitivity is also not blocked by cycloheximide. The increase in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of newly synthesized mAChR on the surface nor to reduced agonist affinity of the mAChR. The diminished sensitivity and subsequent maturation observed in cells containing newly synthesized receptors is due either to a small change in mAChR, or to a change in an as-yet-undefined component of the mAChR transduction system; this alteration represents a novel locus for modulation of cholinergic signals in the heart.

  18. Role of α5 Nicotinic Acetylcholine Receptors in Pharmacological and Behavioral Effects of Nicotine in Mice

    PubMed Central

    Marks, M. J.; Vann, R. E.; Chen, X.; Gamage, T. F.; Warner, J. A.; Damaj, M. I.

    2010-01-01

    Incorporation of the α5 nicotinic acetylcholine receptor (nAChR) subunit can greatly influence nAChR function without altering receptor number. Although few animal studies have assessed the role of the α5 nAChR in nicotine-mediated behaviors, recent evidence suggests an association between polymorphisms in the α5 nAChR gene and nicotine dependence phenotypes in humans. Thus, additional studies are imperative to elucidate the role and function of the α5 nAChR subunit in nicotine dependence. Using α5(−/−) mice, the current study aimed to examine the role of α5 nAChRs in the initial pharmacological effects of nicotine, nicotine reward using the conditioned place preference model, and the discriminative effects of nicotine using a two-lever drug discrimination model. 86Rb+ efflux and 125I-epibatidine binding assays were conducted to examine the effect of α5 nAChR subunit deletion on expression and activity of functional nAChRs. Results show that α5(−/−) mice are less sensitive to the initial effects of nicotine in antinociception, locomotor activity, and hypothermia measures and that the α5 nAChR is involved in nicotine reward. Alternatively, α5(−/−) mice did not differ from wild-type littermates in sensitivity to the discriminative stimulus effects of nicotine. Furthermore, deletion of the α5 nAChR subunit resulted in a statistically significant decrease in function in the thalamus and hindbrain, but the decreases noted in spinal cord were not statistically significant. Receptor number was unaltered in all areas tested. Taken together, results of the study suggest that α5 nAChRs are involved in nicotine-mediated behaviors relevant to development of nicotine dependence. PMID:20400469

  19. Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

    PubMed Central

    1981-01-01

    Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed. PMID:7204489

  20. Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells

    PubMed Central

    Mettler, Irvin J.; Leonard, Robert T.

    1979-01-01

    An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K+ (86Rb), 36Cl−, H232PO4− and 45Ca2+ from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K+, Cl−, and H2PO4− measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N2 gas. In contrast, Ca2+ influx was not tightly coupled to respiratory energy production. The influx of K+ was highest between pH 6.5 and 7.5 whereas the influx of H2PO4− and Cl− was greatest between pH 4.5 and 5.5. Influx of K+ and Cl− was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K+ influx by more than 200% but had no effect on the influx of either Cl− or H2PO4−. Apparent H+ efflux, as measured by decrease in solution pH, was enhanced by K+, stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport. Images PMID:16660675

  1. Early metabolic effects and mechanism of ammonium transport in yeast

    SciTech Connect

    Pena, A.; Pardo, J.P.; Ramirez, J.

    1987-03-01

    Studies were performed to define the effects and mechanism of NH+4 transport in yeast. The following results were obtained. Glucose was a better facilitator than ethanol-H/sub 2/O/sub 2/ for ammonium transport; low concentrations of uncouplers or respiratory inhibitors could inhibit the transport with ethanol as the substrate. With glucose, respiratory inhibitors showed only small inhibitory effects, and only high concentrations of azide or trifluoromethoxy carbonylcyanide phenylhydrazone could inhibit ammonium transport. Ammonium in the free state could be concentrated approximately 200-fold by the cells. Also, the addition of ammonium produced stimulation of both respiration and fermentation; an increased rate of H+ extrusion and an alkalinization of the interior of the cell; a decrease of the membrane potential, as monitored by fluorescent cyanine; an immediate decrease of the levels of ATP and an increase of ADP, which may account for the stimulation of both fermentation and respiration; and an increase of the levels of inorganic phosphate. Ammonium was found to inhibit 86Rb+ transport much less than K+. Also, while K+ produced a competitive type of inhibition, that produced by NH4+ was of the noncompetitive type. From the distribution ratio of ammonium and the pH gradient, an electrochemical potential gradient of around -180 mV was calculated. The results indicate that ammonium is transported in yeast by a mechanism similar to that of monovalent alkaline cations, driven by a membrane potential. The immediate metabolic effects of this cation seem to be due to an increased (H+)ATPase, to which its transport is coupled. However, the carriers seem to be different. The transport system studied in this work was that of low affinity.

  2. Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells.

    PubMed

    Bookchin, R M; Etzion, Z; Sorette, M; Mohandas, N; Skepper, J N; Lew, V L

    2000-07-01

    We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance. PMID:10859357

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

    SciTech Connect

    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.

  4. In situ tracer tests to determine retention properties of a block scale fracture network in granitic rock at the Aspö Hard Rock Laboratory, Sweden.

    PubMed

    Andersson, Peter; Byegård, Johan; Tullborg, Eva-Lena; Doe, Thomas; Hermanson, Jan; Winberg, Anders

    2004-06-01

    Experiments were conducted at the Aspö Hard Rock Laboratory in order to improve the understanding of radionuclide retention properties of fractured crystalline bedrock in the 10-100 m scale (TRUE Block Scale Project, jointly funded by ANDRA, ENRESA, Nirex, JNC, Posiva and SKB). A series of tracer experiments were performed using sorbing tracers in three different flow paths. The different flow paths had Euclidian lengths of 14, 17 and 33 m, respectively, and one to three water conducting structures. Four tests were performed using different cocktails made up of radioactive sorbing tracers (22,24Na+, 42K+, 47Ca2+, 85Sr2+, 83,86Rb+, 131,133Ba2+ and 134,137Cs+). For each tracer injection, the breakthrough of sorbing tracers was compared to the breakthrough of a conservative tracer, 82Br-, 131I-, HTO and 186ReO4-, respectively. In the two longer flow paths, no breakthrough of 83Rb+ and 137Cs+ was observed after 8 months of pumping. Selected tracer tests were subject to basic modelling in which a one-dimensional (1D) advection-dispersion model, including surface sorption, and an unlimited matrix diffusion were used for the interpretation of the results. The results of the modelling indicated that there is a slightly higher mass transfer into a highly porous material in the block-scale experiment compared with in situ experiments performed over shorter distances and significantly higher than what would have been expected from laboratory data obtained from studies of the interactions in nonaltered intact rock.

  5. Influences of follicle-stimulating hormone, proteases, and antiproteases on permeability of the barrier generated by Sertoli cells in a two-chambered assembly

    SciTech Connect

    Ailenberg, M.; Fritz, I.B.

    1989-03-01

    Factors have been identified that influence the integrity of the barrier generated by Sertoli cells (SC) in culture in a two-chambered assembly. The permeability of the barrier was assessed by determining rates of equilibration of (3H)methoxyinulin or (86Rb)Cl across the Sertoli cell monolayer. The complete system consisted of a confluent monolayer of SC maintained on an extracellular matrix (Matrigel)-coated filter together with peritubular cells on the opposite side of the filter. In confirmation of previous results, levels of plasminogen activator (PA) activity secreted were increased by treatment of SC with FSH or with cAMP derivatives ((Bu)2cAMP (dbcAMP)). PA levels in the culture medium were inversely related to times required for 50% equilibration of (3H)methoxyinulin across the SC monolayer. Thus, elevated PA levels, elicited by stimulation with FSH or dbcAMP, were associated with a decreased integrity of the barrier generated by SC preparations maintained in serum-free medium in the complete system. The increase in permeability of the barrier in SC elicited by FSH dbcAMP could be prevented, however, by the addition of various antiproteases. FSH actions on barrier function were complex. Effects of FSH that favored barrier integrity were most readily detected when proteolytic activity was inhibited. The addition of intact serum increased the integrity of the barrier, but acid-treated serum depleted of antiproteases had no such effect. We advance the hypothesis that proteases are implicated in modulation of the formation and maintenance of the seminiferous tubule barrier by SC.

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

    PubMed Central

    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. Ursodeoxycholic acid choleresis: Relationship to biliary HCO sup minus sub 3 and effects of Na sup + -H sup + exchange inhibitors

    SciTech Connect

    Renner, E.L.; Lake, J.R.; Cragoe, E.J. Jr.; van Dyke, R.W.; Scharschmidt, B.F. Merck Sharp and Dohme Research Laboratories, West Point, PA )

    1988-02-01

    The authors have recently shown that substitution of Li{sup +} for perfusate Na{sup +} eliminates the HCO{sub 3}{sup {minus}}-rich choleresis produced by ursodeoxycholic acid (UDCA) in isolated perfused rat liver and that the increase in bile flow produced by both UDCA and taurocholic acid is partially inhibited by 1 mM amiloride. Although these findings are consistent with a role for Na{sup +}-H{sup +} exchange in the choleresis produced by these bile acids, both Li{sup +} substitution and amiloride affect other cellular processes, including Na{sup +}-K{sup +}-ATPase activity. They have now further explored both the relationship between UDCA-stimulated bile flow and biliary HCO{sub 3}{sup {minus}} secretion and the possible role of Na{sup +}-H{sup +} exchange in this process by comparing the effects of amiloride with two of its more potent and presumably more specific analogues, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA). None of the inhibitors significantly altered biliary UDCA output or the relationship between UDCA-induced bile flow and either biliary (HCO{sub 3}{sup {minus}}) or biliary HCO{sub 3}{sub {minus}} output. Effects of these inhibitors did not appear attributable either to nonspecific toxicity, as reflected by hepatic release of lactate dehydrogenase or K{sup +}, or to inhibition of hepatic Na{sup +}-K{sup +}-ATPase, measured as Na{sup +}-dependent uptake of {sup 86}Rb. These findings indicate that UDCA-induced but not basal bile formation is closely coupled to biliary HCO{sub 3}{sup {minus}} concentration and output, and they provide additional evidence that UDCA choleresis requires an intact Na{sup +}-H{sup +} exchange mechanism.

  8. MICE EXPRESSING THE ADNFLE VALINE 287 LEUCINE MUTATION OF THE β2 NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNIT DISPLAY INCREASED SENSITIVITY TO ACUTE NICOTINE ADMINISTRATION AND ALTERED PRESYNAPTIC NICOTINIC RECEPTOR FUNCTION

    PubMed Central

    O’Neill, Heidi C.; Laverty, Duncan C.; Patzlaff, Natalie E.; Cohen, Bruce N.; Fonck, Carlos; McKinney, Sheri; McIntosh, J. Michael; Lindstrom, Jon M.; Lester, Henry A.; Grady, Sharon R.; Marks, Michael J.

    2012-01-01

    Several mutations in α4 or β2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the β2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (β2VL). Previous studies indicated that the β2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of β2VL mice. No differences in β2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the β2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [3H]-dopamine release and 86Rb efflux were observed. Maximal nAChR-mediated [3H]-GABA release in the cortex was also decreased in the MT, but maximal [3H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30 s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression. PMID:23123803

  9. Effect of canrenone on the digitalis site of Na+/K(+)-ATPase in human placental membranes and in erythrocytes.

    PubMed

    Balzan, S; Nicolini, G; Bellitto, L; Ghione, S; Biver, P; Montali, U

    2003-07-01

    It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabain-like factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated (1) the effect of canrenone on Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain binding in placental membrane (40%) and erythrocytes (60%). Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain (5 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes. PMID:12827023

  10. Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1).

    PubMed

    Monette, Michelle Y; Forbush, Biff

    2012-01-13

    The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.

  11. The inwardly rectifying potassium channel Kir1.1: development of functional assays to identify and characterize channel inhibitors.

    PubMed

    Felix, John P; Priest, Birgit T; Solly, Kelli; Bailey, Timothy; Brochu, Richard M; Liu, Chou J; Kohler, Martin G; Kiss, Laszlo; Alonso-Galicia, Magdalena; Tang, Haifeng; Pasternak, Alexander; Kaczorowski, Gregory J; Garcia, Maria L

    2012-10-01

    The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartter's syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization. PMID:22881347

  12. Differential mechanisms of Cantú syndrome-associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel.

    PubMed

    Cooper, Paige E; Sala-Rabanal, Monica; Lee, Sun Joo; Nichols, Colin G

    2015-12-01

    Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium ((86)Rb(+)) efflux assays, we show that K(ATP) channels formed with P429L, A475V, or C1039Y mutants enhance K(ATP) activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive K(ATP) channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation. PMID:26621776

  13. Mice expressing the ADNFLE valine 287 leucine mutation of the Β2 nicotinic acetylcholine receptor subunit display increased sensitivity to acute nicotine administration and altered presynaptic nicotinic receptor function.

    PubMed

    O'Neill, Heidi C; Laverty, Duncan C; Patzlaff, Natalie E; Cohen, Bruce N; Fonck, Carlos; McKinney, Sheri; McIntosh, J Michael; Lindstrom, Jon M; Lester, Henry A; Grady, Sharon R; Marks, Michael J

    2013-01-01

    Several mutations in α4 or β2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the β2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (β2VL). Previous studies indicated that the β2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of β2VL mice. No differences in β2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the β2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.

  14. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Hirsch, R. E.; Lewis, D. R.; Qi, Z.; Sussman, M. R.; Lewis, B. D.

    1999-01-01

    A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

  15. Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

    PubMed

    Su, W; Shmukler, B E; Chernova, M N; Stuart-Tilley, A K; de Franceschi, L; Brugnara, C; Alper, S L

    1999-11-01

    Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

  16. Differential mechanisms of Cantú syndrome–associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel

    PubMed Central

    Cooper, Paige E.; Sala-Rabanal, Monica; Lee, Sun Joo

    2015-01-01

    Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium (86Rb+) efflux assays, we show that KATP channels formed with P429L, A475V, or C1039Y mutants enhance KATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive KATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation. PMID:26621776

  17. Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

    SciTech Connect

    Van Dyke, R.W.; Scharschmidt, B.F.

    1987-11-01

    Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible /sup 86/Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by /sup 36/Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

  18. Dextromethorphan and its metabolite dextrorphan block alpha3beta4 neuronal nicotinic receptors.

    PubMed

    Hernandez, S C; Bertolino, M; Xiao, Y; Pringle, K E; Caruso, F S; Kellar, K J

    2000-06-01

    Dextromethorphan (DM), a structural analog of morphine and codeine, has been widely used as a cough suppressant for more than 40 years. DM is not itself a potent analgesic, but it has been reported to enhance analgesia produced by morphine and nonsteroidal anti-inflammatory drugs. Although DM is considered to be nonaddictive, it has been reported to reduce morphine tolerance in rats and to be useful in helping addicted subjects to withdraw from heroin. Here we studied the effects of DM on neuronal nicotinic receptors stably expressed in human embryonic kidney cells. Studies were carried out to examine the effects of DM on nicotine-stimulated whole cell currents and nicotine-stimulated (86)Rb(+) efflux. We found that both DM and its metabolite dextrorphan block nicotinic receptor function in a noncompetitive but reversible manner, suggesting that both drugs block the receptor channel. Consistent with blockade of the receptor channel, neither drug competed for the nicotinic agonist binding sites labeled by [(3)H]epibatidine. Although DM is approximately 9-fold less potent than the widely used noncompetitive nicotinic antagonist mecamylamine in blocking nicotinic receptor function, the block by DM appears to reverse more slowly than that by mecamylamine. These data indicate that DM is a useful antagonist for studying nicotinic receptor function and suggest that it might prove to be a clinically useful neuronal nicotinic receptor antagonist, possibly helpful as an aid for helping people addicted to nicotine to refrain from smoking, as well as in other conditions where blockade of neuronal nicotinic receptors would be helpful. PMID:10869398

  19. Influence of fentanyl and morphine on intestinal circulation

    SciTech Connect

    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.

  20. Nuclear reactions in Rb, Sr, Y, and Zr targets

    NASA Astrophysics Data System (ADS)

    Regnier, S.; Lavielle, B.; Simonoff, M.; Simonoff, G. N.

    1982-09-01

    Excitation functions of all stable or long-lived krypton isotopes were measured or estimated for incident protons and neutrons in Rb, Sr, Y, and Zr targets. Experimental data concern mostly Y and Zr targets bombarded with 0.059 to 24 GeV protons. The products 78-86Kr, 74As, 75Se, 83,84,86Rb, 85Sr, 88Y, 88,95Zr, and 92Nbm were measured using high-sensitivity mass spectrometry and nondestructive γ counting. Lighter products such as 38,39,42Ar and 12 radioactive isotopes from 7Be to 65Zn were also measured in some cases and their cross sections are given in an appendix. Most excitation functions pass through a maximum between 0.4 and 0.8 GeV, and the peak energy could depend on the ΔA value. The results, combined with a general survey of nuclear reactions in Ga to Nb targets, permitted the development of new systematics leading to the calculation of spallation-produced Kr isotopes in the moon bombarded with galactic and solar cosmic rays. Compared to cosmogenic krypton measured in nine well-documented lunar samples, 83Kr is predicted with a precision better than 33% (1σ) and the production ratios iKr/83Kr are predicted to better than 25%. It is concluded that the cosmogenic ratios 86Kr/83Kr and 81Kr/83Kr are dependent on the main target element concentrations. This should be taken into account in strontium-rich samples when calculating exposure ages of extraterrestrial materials. NUCLEAR REACTIONS 89Y and Zr, (p, spallation) E=0.059-24 GeV; measured σ(E) for 78-86Kr and 12 radioactive products. Systematics of p- and n-induced reactions in Rb, Sr, Y, and Zr. Cosmogenic krypton.

  1. Role of intracellular calcium in cellular volume regulation

    SciTech Connect

    Wong, S.M.; Chase, H.S. Jr.

    1986-06-01

    We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of /sup 86/Rb and /sup 22/Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular (Ca) (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ((Ca)i), as measured by quin 2 fluorescence: (Ca)i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular (Ca) to less than 100 nM prevented the swelling-induced increase in (Ca)i, suggesting that the source of the increase in (Ca)i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases (Ca)i. This increase is likely to play a critical role in cellular volume regulation.

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

    PubMed

    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. PMID:27677555

  3. Grapevine and Arabidopsis Cation-Chloride Cotransporters Localize to the Golgi and Trans-Golgi Network and Indirectly Influence Long-Distance Ion Transport and Plant Salt Tolerance1[OPEN

    PubMed Central

    Henderson, Sam W.; Wege, Stefanie; Qiu, Jiaen; Blackmore, Deidre H.; Walker, Amanda R.; Tyerman, Stephen D.; Walker, Rob R.; Gilliham, Matthew

    2015-01-01

    Plant cation-chloride cotransporters (CCCs) have been implicated in conferring salt tolerance. They are predicted to improve shoot salt exclusion by directly catalyzing the retrieval of sodium (Na+) and chloride (Cl−) ions from the root xylem. We investigated whether grapevine (Vitis vinifera [Vvi]) CCC has a role in salt tolerance by cloning and functionally characterizing the gene from the cultivar Cabernet Sauvignon. Amino acid sequence analysis revealed that VviCCC shares a high degree of similarity with other plant CCCs. A VviCCC-yellow fluorescent protein translational fusion protein localized to the Golgi and the trans-Golgi network and not the plasma membrane when expressed transiently in tobacco (Nicotiana benthamiana) leaves and Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. AtCCC-green fluorescent protein from Arabidopsis also localized to the Golgi and the trans-Golgi network. In Xenopus laevis oocytes, VviCCC targeted to the plasma membrane, where it catalyzed bumetanide-sensitive 36Cl–, 22Na+, and 86Rb+ uptake, suggesting that VviCCC (like AtCCC) belongs to the Na+-K+-2Cl– cotransporter class of CCCs. Expression of VviCCC in an Arabidopsis ccc knockout mutant abolished the mutant’s stunted growth phenotypes and reduced shoot Cl– and Na+ content to wild-type levels after growing plants in 50 mm NaCl. In grapevine roots, VviCCC transcript abundance was not regulated by Cl– treatment and was present at similar levels in both the root stele and cortex of three Vitis spp. genotypes that exhibit differential shoot salt exclusion. Our findings indicate that CCC function is conserved between grapevine and Arabidopsis, but neither protein is likely to directly mediate ion transfer with the xylem or have a direct role in salt tolerance. PMID:26378102

  4. Cytomegalovirus: pathophysiological mechanisms of the cytomegalovirus-induced cellular responses

    SciTech Connect

    Nokta, M.A.

    1986-01-01

    Cytomegalovirus (CMV) infection of fibroblasts of human origin is associated with a cascade of morphologic cellular responses which in other systems have been associated with regulation of intracellular free (IF) (Ca/sup + +/). In the present study, the relationship of specific ion fluxes (Ca/sup + +/, Na/sup +/) to the development of cytomegalovirus (CMV)-induced morphologic cellular responses was investigated. An influx of Ca/sup + +/ was observed by the first hour after CMV infection (PI), and total calcium sequestered by infected cells was enhanced by 5 hr Pl. A gradual rise in intracellular free (IF) (Ca/sup + +/) was observed that continued through 48 hour postinfection (hr Pl). The IF (Ca/sup + +/) response to CMV infection was shown to be multiplicity dependent, require viable virus, and occur under conditions consistent with the expression of immediate early CMV genes. Development and progression of cytomegaly was found to be independent of CMV DNA synthesis and appeared to be dependent on the IF (Ca/sup + +/) response. Ca/sup + +/ influx blockers (e.g. verapamil) and cyclic nucleotide modulators (e.g. papaverine) inhibited both Ca/sup + +/ responses and cytomegaly. Quabain-sensitive /sup 86/Rb uptake and sequestering of Ca/sup + +/ increased in parallel with development of cytomegaly. There may be a relationship between Ca/sup + +/ influx, IF (Ca/sup + +/), activation of the Na/sup +//H/sup +/ exchanger, induction of Na/sup +/, Cl/sup -/, HCO/sub 3/ cotransport, Na/sup +/ entry, Na/sup +//K/sup +/ ATPase activity and development of CMV-induced morphologic cellular responses including cytomegaly.

  5. Cell volume regulation in human neutrophils: 2-(aminomethyl)phenols as Cl- channel inhibitors.

    PubMed

    Simchowitz, L; Textor, J A; Cragoe, E J

    1993-07-01

    When subjected to hypotonic stress, human peripheral neutrophils initially swell due to rapid water entry and thereafter recover toward the normal cell size (approximately 330 microns 3). Neutrophils do not behave as perfect osmometers: when resuspended in half-isotonic medium (150 mosM), they swell by only approximately 40% rather than doubling in size as predicted for ideal behavior. As with lymphocytes, restoration to the normal cell size involves the net loss of K+ and Cl- from the cytosol through independent conductance pathways. Volume regulation is sensitive to 0.4-1 mM of quinine, UK-5099, 3,5-diiodosalicylate (DISA), MK-473 (an indanyloxyacetate derivative), and to MK-447 [a 2-(aminomethyl)phenol]. From correlation of drug effects on the time course of cell volume recovery and the associated volume-activated 86Rb+ and 36Cl- fluxes, it was evident that quinine blocked only K+ channels, whereas MK-447 acted as a selective inhibitor of Cl- channels. In contrast, UK-5099, DISA, and MK-473 were nonspecific in that the compounds displayed comparable suppressive effects on all three parameters. Structure-activity relationships in the MK-447 series revealed the critical elements of the molecule responsible for drug potency. In particular, the importance of the neighboring ionizable 1-hydroxyl and 2-aminomethyl groups and the formation of secondary ring structures for biological activity is emphasized. The most potent derivative thus far identified, termed analogue A [inhibitor constant (Ki) approximately 16 microM], had a potency approximately sixfold greater than that of the parent compound (Ki approximately 90 microM). These findings define the mechanism of action of a relatively new class of agents that behave as inhibitors of swelling-activated Cl- channels in these cells.

  6. Killing of an encapsulated strain of Escherichia coli by human serum.

    PubMed

    Taylor, P W; Kroll, H P

    1983-01-01

    Changes in cell viability and in factors affecting metabolic integrity were examined after exposure of Escherichia coli LP1092 to human serum. Antibody-dependent classical pathway activity accounted for the rapid killing of strain LP1092 by complement. Removal of serum lysozyme by bentonite absorption or by neutralization with anti-human lysozyme immunoglobulin G resulted in a reduction in the rate of killing; optimal activity could be restored by the addition of physiological amounts of egg-white lysozyme. The pattern of 86Rb+ and alkaline phosphatase release obtained after serum treatment did not support the view that complement simultaneously disrupts cytoplasmic and outer membrane integrity. Macromolecular synthesis was affected late in the reaction sequence; complete inhibition of precursor incorporation into RNA, DNA, and protein occurred only after almost total loss of bacterial colony-forming ability. Addition of chloramphenicol, an inhibitor of protein synthesis, to the bactericidal system resulted in a marked reduction in the rate of serum killing. Killing was completely inhibited by an inhibitor (KCN) and an uncoupler (2,4-dinitrophenol) of oxidative phosphorylation. Exposure of LP1092 cells to serum was followed by a rapid and large increase in intracellular ATP levels; ATP synthesis did not occur when bacteria were exposed to dialyzed serum, which killed LP1092 cells at a much reduced rate. Addition of glucose or serum ultrafiltrate to dialyzed serum restored optimal bactericidal activity. We suggest that optimal killing of gram-negative bacteria is an energy-dependent process requiring an input of bacterially generated ATP. PMID:6185430

  7. High-affinity K(+) transport in Arabidopsis: AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions.

    PubMed

    Pyo, Young Jae; Gierth, Markus; Schroeder, Julian I; Cho, Myeon Haeng

    2010-06-01

    Potassium (K(+)) is a major plant nutrient required for growth and development. It is generally accepted that plant roots absorb K(+) through uptake systems operating at low concentrations (high-affinity transport) and/or high external concentrations (low-affinity transport). To understand the molecular basis of high-affinity K(+) uptake in Arabidopsis (Arabidopsis thaliana), we analyzed loss-of-function mutants in AtHAK5 and AKT1, two transmembrane proteins active in roots. Compared with the wild type under NH(4)(+)-free growth conditions, athak5 mutant plants exhibited growth defects at 10 mum K(+), but at K(+) concentrations of 20 mum and above, athak5 mutants were visibly indistinguishable from the wild type. While germination, scored as radicle emergence, was only slightly decreased in athak5 akt1 double mutants on low-K(+) medium, double mutants failed to grow on medium containing up to 100 mum K(+) and growth was impaired at concentrations up to 450 mum K(+). Moreover, transfer of 3-d-old plants from high to low K(+) concentrations led to growth defects and leaf chlorosis at 10 mum K(+) in athak5 akt1 double mutant plants. Determination of Rb(+)(K(+)) uptake kinetics in wild-type and mutant roots using rubidium ((86)Rb(+)) as a tracer for K(+) revealed that high-affinity Rb(+)(K(+)) uptake into roots is almost completely abolished in double mutants and impaired in single mutants. These results strongly indicate that AtHAK5 and AKT1 are the two major, physiologically relevant molecular entities mediating high-affinity K(+) uptake into roots during seedling establishment and postgermination growth and that residual Rb(+)(K(+)) uptake measured in athak5 akt1 double mutant roots is insufficient to enable plant growth. PMID:20413648

  8. Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK-293 cells

    PubMed Central

    Gopalakrishnan, Murali; Molinari, Eduardo J; Shieh, Char-Chang; Monteggia, Lisa M; Roch, Jean-Marc; Whiteaker, Kristi L; Scott, Victoria E S; Sullivan, James P; Brioni, Jorge D

    2000-01-01

    The pharmacological properties of KATP channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K+ channel Kir6.2 were characterized in HEK-293 cells.[3H]-Glyburide (glibenclamide) bound to transfected cells with a Bmax value of 18.5 pmol mg−1 protein and with a KD value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain.Functional activity of KATP channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 μM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC50=0.92 nM).Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered 86Rb+ efflux from transfected cells that was sensitive to inhibition by glyburide (IC50=3.6 nM).Diazoxide, but not levcromakalim, evoked concentration-dependent decreases in DiBAC4(3) fluorescence responses with an EC50 value of 14.1 μM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities.In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native KATP channels described in pancreatic and neuronal tissues. PMID:10742287

  9. Stimulus-secretion coupling in beta-cells: modulation by pH.

    PubMed

    Pace, C S; Tarvin, J T; Smith, J S

    1983-01-01

    We have examined the influence of changes in pH on the oscillatory pattern of electrical activity (EA) in the beta-cell by altering medium pH (pHo) and using permeable weak buffers to alter intracellular pH (pHi). A decrease in pH in the presence of glucose elicited depolarization to the active phase and constant spike activity, whereas an increase in pH elicited a decrease in spike activity or silent hyperpolarization. On inhibition of HCO3:Cl antiport by addition of DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), probenecid, or withdrawal of medium HCO-3, there was an increase in the duration of the active phase. A similar result was obtained on the inhibition of Na:H antiport by the addition of amiloride or the reduction of medium [Na+]. The influence of H+ and glucose has been proposed to decrease K+ permeability (PK). However, the influence of pH on 86Rb+ efflux was most effective at subthreshold or 4.2 mM glucose; only a moderate decrease in PK occurred at 8.3 mM glucose, and no effect was obtained at 16.7 mM glucose. Alteration of pHi, and not pHo, induces similar effects on glucose-induced electrical and secretory events. There is a clear dissociation between the influence of inhibitors of the Na:H and HCO3:Cl antiporters on the electrical and secretory events. DIDS and amiloride increased glucose-induced EA, but markedly inhibited the secretory response to glucose. It is evident that pH modulates the electrical events and cationic fluxes and ultimately influences the transduction of information to the mechanisms controlling the secretory process in the beta-cell. PMID:6295181

  10. Effects of thyroid hormone on Na sup + -K sup + transport in resting and stimulated rat skeletal muscle

    SciTech Connect

    Everts, M.E.; Clausen, T. )

    1988-11-01

    The effects of hypothyroidism and 3,5,3{prime}-triiodothyronine (T{sub 3}) treatment on passive Na{sup +}-K{sup +} fluxes and Na{sup +}-K{sup +} pump concentration were investigated in isolated rat muscle. Within 12 h after a single dose of T{sub 3} (20 {mu}g/100 g body wt), K{sup +} efflux had increased by 21% in soleus and by 20% in extensor digitorum longus muscle. In the presence of ouabain, even larger effects were observed. These changes were associated with a 12% rise in amiloride-suppressible Na{sup +} influx but no significant increase in ({sup 3}H)ouabain binding site concentration. After 3 days of T{sub 3} treatment, the stimulating effect on K{sup +} efflux and Na{sup +} influx in soleus reached a plateau {approximately}80 and 40% above control levels, respectively, whereas the maximum increase in ({sup 3}H)ouabain binding site concentration (103%) was only fully developed after 8 days. Hypothyroidism decreased {sup 86}Rb efflux by 30%. The efflux of K{sup +} and the influx of Na{sup +} per contraction (both {approximately}7 nmol/g wet wt) as well as the net loss of K{sup +} induced by electrical stimulation were unaffected by T{sub 3} treatment. The rise in resting K{sup +} efflux after 12-24 h of T{sub 3} treatment could be partly blocked by dantrolene or trifluoroperazine, indicating that an increase in the cytoplasmic Ca{sup 2+} concentration may contribute to the early rise in K{sup +} efflux. It is concluded that the early rise in the resting passive leaks of Na{sup +} and K{sup +} induced by T{sub 3} is a major driving force for Na{sup +}-K{sup +} pump synthesis.

  11. In situ tracer tests to determine retention properties of a block scale fracture network in granitic rock at the Äspö Hard Rock Laboratory, Sweden

    NASA Astrophysics Data System (ADS)

    Andersson, Peter; Byegård, Johan; Tullborg, Eva-Lena; Doe, Thomas; Hermanson, Jan; Winberg, Anders

    2004-06-01

    Experiments were conducted at the Äspö Hard Rock Laboratory in order to improve the understanding of radionuclide retention properties of fractured crystalline bedrock in the 10-100 m scale (TRUE Block Scale Project, jointly funded by ANDRA, ENRESA, Nirex, JNC, Posiva and SKB). A series of tracer experiments were performed using sorbing tracers in three different flow paths. The different flow paths had Euclidian lengths of 14, 17 and 33 m, respectively, and one to three water conducting structures. Four tests were performed using different cocktails made up of radioactive sorbing tracers ( 22,24Na +, 42K +, 47Ca 2+, 85Sr 2+, 83,86Rb +, 131,133Ba 2+ and 134,137Cs +). For each tracer injection, the breakthrough of sorbing tracers was compared to the breakthrough of a conservative tracer, 82Br -, 131I -, HTO and 186ReO 4-, respectively. In the two longer flow paths, no breakthrough of 83Rb + and 137Cs + was observed after 8 months of pumping. Selected tracer tests were subject to basic modelling in which a one-dimensional (1D) advection-dispersion model, including surface sorption, and an unlimited matrix diffusion were used for the interpretation of the results. The results of the modelling indicated that there is a slightly higher mass transfer into a highly porous material in the block-scale experiment compared with in situ experiments performed over shorter distances and significantly higher than what would have been expected from laboratory data obtained from studies of the interactions in nonaltered intact rock.

  12. Cation transport across the guinea-pig placenta perfused in situ.

    PubMed Central

    Bailey, D J; Bradbury, M W; France, V M; Hedley, R; Naik, S; Parry, H

    1979-01-01

    1. The guinea-pig placenta perfused in situ via the umbilical circulation has been used to measure unidirectional fluxes of Na from mother to fetus, and in the reverse direction, with 24Na and 22Na. There was no significant difference between the two fluxes, each being 22 mumole.min-1. 2. Ouabain 10(-5) M in the perfusion fluid had no detectable effect on radioisotopic movements of Na in either direction. 3. Unidirectional fluxes of 42K in both directions were approximately equal at 1.7 mumole.min-1 mother fetus and 1.8 mumole.min-1 in the reverse direction, despite a K concentration of 3.4 mM on the maternal side and 5.0 mM on the fetal side of the placenta. 4. Extraction of 42K and 86Rb from the perfusion fluid was inhibited by 43% by 10(-5) M-ouabain in the fluid. This effect was largely due to a reduction of isotope uptake by the placental tissue. 5. The relative permeabilities of the placenta, mother to fetus, were Rb approximately K (3.2) greater than Na (1.0) greater than Li (0.55). 6. Under the experimental conditions, the electrical potential difference between perfusion fluid and maternal blood was 6 mV (fetus negative). It was shifted towards the positive by a low Na fluid. 7. The results suggest the presence of a dominant Na-K pump (active component towards mother) sited at the maternal-facing membrane of the syncytiotrophoblast together with a subsidiary pump oriented in the opposite direction and probably sited together with a subsidiary pump oriented in the opposite direction and probably sited at the fetal-facing membrane of the syncytiotrophoblast. 8. A high proportion of Na movement particularly towards the fetus is probably passive, occurring through water-filled spaces, whilst K movement is more dependent on active transport. PMID:430428

  13. Solute transport in crystalline rocks at Aspö--II: blind predictions, inverse modelling and lessons learnt from test STT1.

    PubMed

    Jakob, Andreas; Mazurek, Martin; Heer, Walter

    2003-03-01

    Based on the results from detailed structural and petrological characterisation and on up-scaled laboratory values for sorption and diffusion, blind predictions were made for the STT1 dipole tracer test performed in the Swedish Aspö Hard Rock Laboratory. The tracers used were nonsorbing, such as uranine and tritiated water, weakly sorbing 22Na(+), 85Sr(2+), 47Ca(2+)and more strongly sorbing 86Rb(+), 133Ba(2+), 137Cs(+). Our model consists of two parts: (1) a flow part based on a 2D-streamtube formalism accounting for the natural background flow field and with an underlying homogeneous and isotropic transmissivity field and (2) a transport part in terms of the dual porosity medium approach which is linked to the flow part by the flow porosity. The calibration of the model was done using the data from one single uranine breakthrough (PDT3). The study clearly showed that matrix diffusion into a highly porous material, fault gouge, had to be included in our model evidenced by the characteristic shape of the breakthrough curve and in line with geological observations. After the disclosure of the measurements, it turned out that, in spite of the simplicity of our model, the prediction for the nonsorbing and weakly sorbing tracers was fairly good. The blind prediction for the more strongly sorbing tracers was in general less accurate. The reason for the good predictions is deemed to be the result of the choice of a model structure strongly based on geological observation. The breakthrough curves were inversely modelled to determine in situ values for the transport parameters and to draw consequences on the model structure applied. For good fits, only one additional fracture family in contact with cataclasite had to be taken into account, but no new transport mechanisms had to be invoked. The in situ values for the effective diffusion coefficient for fault gouge are a factor of 2-15 larger than the laboratory data. For cataclasite, both data sets have values comparable to

  14. Potassium transport in cortical collecting tubules from mineralocorticoid-treated rat

    SciTech Connect

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

    1987-07-01

    Unidirectional fluxes of /sup 86/Rb/sup +/ were used to examine the stimulation of K/sup +/ secretion produced by arginine vasopressin (ADH) in isolated perfused cortical collecting tubules from rats treated with desoxycorticosterone. ADH at 100 ..mu..U/ml in the bathing solution increased the bath-to-lumen flux (J/sub b..-->..1/; pmol /center dot/ min/sup -1/ /center dot/ mm/sup -1/) from 16.9 /plus minus/ 2.3 to 43.2 /plus minus/ 4.6. The lumen-to-bath flux (J/sub 1..-->..b/) fell from 3.2 /plus minus/ 0.7 to 1.3 /plus minus/ 0.4 with ADH due to hyperpolarization of the transepithelial voltage from -12.6 /plus minus/ 1.3 to /minus/39.3 /plus minus/ 2.0 mV, but the calculated Rb/sup +/ permeability was unaltered at 0.20-0.26 ..mu..m/s. Although 2 mM lumen Ba/sup 2 +/ inhibited J/sub b..-->..1/ by 55 /plus minus/ 6%, the flux ratio (J/sub b..-->..1//J/sub 1..-->..b/) of 28 /plus minus/ 8 was larger than predicted for passive exchange. In the absence of ADH 2 mM Ba/sup 2 +/ reduced J/sub b..-->..1/ to the level predicted for passive movement, but addition of ADH with Ba/sup 2 +/ still present increased J/sub 1..-->..1/ by an amount identical to that observed without Ba/sup 2 +/, although the absolute J/sub b..-->..1/ was less. Simultaneous addition of 2 mM luminal and 4 mM bath Ba/sup 2 +/ also inhibited J/sub 1..-->..b/ for /sup 22/Na/sup +/ but not to passive levels. These results indicate either that the concentrations of Ba/sup 2 +/ used were insufficient to block K/sup +/ conductances completely or that K/sup +//Rb/sup +/ secretion can occur through a Ba/sup 2 +/-insensitive pathway.

  15. Alpha Production Cross Sections for Some Target Fusion Structural Materials up to 35 MeV

    NASA Astrophysics Data System (ADS)

    Yiğit, M.; Tel, E.

    2013-08-01

    In the next century, because of the worldwide energy shortage, human life will badly be affected. Nuclear fusion energy is the remarkable solution to the rising energy challenges because it has the great potential for sustainability, economic and reliability. There have been many research and development studies to get energy from fusion. Moreover, the neutron induced reaction cross section data around 14-15 MeV are need to the design and development of nuclear fusion reactors. Thus, the working out the systematics of ( n, α) reaction cross sections is very important and necessary for the definition of the excitation curves at around 14-15 MeV energy. In this study, neutron induced reaction cross sections for structural fusion materials such as Sc ( Scandium), Co ( Cobalt), Ni ( Nickel), Cu ( Copper), Y ( Yttrium), Mo ( Molybdenum), Zr ( Zirconium) and Nb ( Niobium) have been investigated for the ( n, α) reactions. The new calculations on the excitation functions of 45 Sc( n, a) 42 K, 59 Co ( n, a) 56 Mn, 62 Ni( n, a) 59 Fe, 63 Cu( n, a) 60 Co, 65 Cu( n, a) 62 Co, 89 Y( n, a) 86 Rb, 92 Mo( n, a) 89 Zr, 98 Mo( n, a) 95 Zr, 92 Zr( n, a) 89 Sr, 94 Zr( n, a) 91 Sr and 93 Nb( n, a) 90 Y reactions have been carried out up to 35 MeV incident neutron energies. In these calculations, the pre-equilibrium and equilibrium effects have been investigated. The pre-equilibrium calculations involve the new evaluated the geometry dependent hybrid model, hybrid model and the cascade exciton model. The equilibrium effects of the excitation functions for the investigated reactions are calculated according to the Weisskopf-Ewing model. Additionaly, in the present work, the ( n, α) reaction cross sections have calculated by using evaluated empirical formulas developed by Tel et al. at 14-15 MeV energy. The calculated results have been discussed and compared with the available experimental data taken from EXFOR database.

  16. Solute transport in crystalline rocks at Aspö--II: blind predictions, inverse modelling and lessons learnt from test STT1.

    PubMed

    Jakob, Andreas; Mazurek, Martin; Heer, Walter

    2003-03-01

    Based on the results from detailed structural and petrological characterisation and on up-scaled laboratory values for sorption and diffusion, blind predictions were made for the STT1 dipole tracer test performed in the Swedish Aspö Hard Rock Laboratory. The tracers used were nonsorbing, such as uranine and tritiated water, weakly sorbing 22Na(+), 85Sr(2+), 47Ca(2+)and more strongly sorbing 86Rb(+), 133Ba(2+), 137Cs(+). Our model consists of two parts: (1) a flow part based on a 2D-streamtube formalism accounting for the natural background flow field and with an underlying homogeneous and isotropic transmissivity field and (2) a transport part in terms of the dual porosity medium approach which is linked to the flow part by the flow porosity. The calibration of the model was done using the data from one single uranine breakthrough (PDT3). The study clearly showed that matrix diffusion into a highly porous material, fault gouge, had to be included in our model evidenced by the characteristic shape of the breakthrough curve and in line with geological observations. After the disclosure of the measurements, it turned out that, in spite of the simplicity of our model, the prediction for the nonsorbing and weakly sorbing tracers was fairly good. The blind prediction for the more strongly sorbing tracers was in general less accurate. The reason for the good predictions is deemed to be the result of the choice of a model structure strongly based on geological observation. The breakthrough curves were inversely modelled to determine in situ values for the transport parameters and to draw consequences on the model structure applied. For good fits, only one additional fracture family in contact with cataclasite had to be taken into account, but no new transport mechanisms had to be invoked. The in situ values for the effective diffusion coefficient for fault gouge are a factor of 2-15 larger than the laboratory data. For cataclasite, both data sets have values comparable to

  17. Mechanisms of fusicoccin action: kinetic modification and inactivation of K(+) channels in guard cells.

    PubMed

    Blatt, M R; Clint, G M

    1989-12-01

    extracellular potassium concentration. Current decay at negative potentials was quickened, also. These results identify the outward-rectifying K(+) channels as one site of action for FC at a higher plant cell membrane; they complete the link introduced in the preceding paper between K(+) channel current, K(+)((86)Rb(+)) flux and irreversible cation uptake in the toxin. The data also offer some insights toward a kinetic description of channel gating. Finally, they provide a vehicle for interpreting FC-induced changes in K(+) and net H(+) flux, and in membrane potential without the necessity for postulating gross changes in H(+) pumping.

  18. Effects of ion substitution on bile acid-dependent and -independent bile formation by rat liver.

    PubMed Central

    Van Dyke, R W; Stephens, J E; Scharschmidt, B F

    1982-01-01

    To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by greater than 70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by greater than or equal to 50% and decreased biliary bicarbonate output by approximately 60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes. These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attributable to the role(s) of sodium in hepatic sodium-coupled taurocholate uptake and/or in maintenance of Na,K-ATPase activity. The surprising finding that bile acid-independent bile formation was substantially unaltered by complete replacement of sodium with the permeant cation lithium does not appear to be explained by Na,K-ATPase-mediated lithium transport. Although

  19. Vasopressin alters the mechanism of apical Cl- entry from Na+:Cl- to Na+:K+:2Cl- cotransport in mouse medullary thick ascending limb

    SciTech Connect

    Sun, A.; Grossman, E.B.; Lombardi, M.; Hebert, S.C. )

    1991-02-01

    Experiments were performed using in vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K+ dependence of the apical, furosemide-sensitive Na{sup +}:Cl{sup {minus}} cotransporter and on transport-related oxygen consumption (QO{sub 2}). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K+ from, and adding one mM ouabain to, the basolateral solution (ouabain(zero-K+)) provided an index to apical cotransporter activity and was used to evaluate the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na{sup +} and Cl{sup {minus}}, but not K{sup +}, while the presence of AVP the apical cotransporter required all three ions. {sup 86}Rb{sup +} uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover, {sup 22}Na{sup +} uptake was unaffected by extracellular K+ in the absence of AVP while after AVP exposure {sup 22}Na{sup +} uptake was strictly K{sup +}-dependent. The AVP-induced coupling of K{sup +} to the Na{sup +}:Cl{sup {minus}} cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular {sup 22}Na{sup +} uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na{sup +}:Cl{sup {minus}} cotransport to Na{sup +}:K{sup +}:2Cl{sup {minus}} cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K{sup +} to the apical Na{sup +}:Cl{sup {minus}} cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.

  20. HCO3(-)-coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

    SciTech Connect

    Fitz, J.G.; Lidofsky, S.D.; Weisiger, R.A.; Xie, M.H.; Cochran, M.; Grotmol, T.; Scharschmidt, B.F. )

    1991-05-01

    Recent studies in hepatocytes indicate that Na(+)-coupled HCO3- transport contributes importantly to regulation of intracellular pH and membrane HCO3- transport. However, the direction of net coupled Na+ and HCO3- movement and the effect of HCO3- on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO3- on Na+ influx and turnover were measured in primary rat hepatocyte cultures with 22Na+, and (Na+)i was measured in single hepatocytes using the Na(+)-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na(+)-dependent or ouabain-suppressible 86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and (Na+)i. In hepatocyte monolayers, HCO3- increased 22Na+ entry and turnover rates by 50-65%, without measurably altering 22Na+ pool size or cell volume, and HCO3- also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO3- produced an abrupt and sustained rise in (Na+)i from approximately 8 to 12 mM. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased congruent to 2.5-fold in the presence of HCO3-, and the rise in (Na+)i produced by inhibition of the Na+/K+ pump was similarly increased congruent to 2.5-fold in the presence of HCO3-. In intact perfused rat liver, HCO3- increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO3- movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO3- to maintain (Na+)i within the physiologic range.

  1. 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

    SciTech Connect

    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.

  2. Hormone-sensitive hepatic Na/sup +/-pump: evidence for regulation by diacylglycerol and tumor promoters

    SciTech Connect

    Lynch, C.J.; Wilson, P.B.; Blackmore, P.F.; Exton, J.H.

    1986-11-05

    Ouabain-sensitive /sup 86/Rb/sup +/ uptake by isolated rat hepatocytes was studied to elucidate how Ca/sup 2 +/-mobilizing hormones stimulate the Na/sup +/-pump. Stimulation of this uptake was observed with concentrations of vasopressin ((8-arginine)vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca/sup 2 +/ mobilization and phosphorylase activation. These results suggested that changes in cytosolic Ca/sup 2 +/, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP. However, in hepatocytes incubated in Ca/sup 2 +/-free Krebs-Henseleit buffer, Na/sup +/-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca/sup 2 +/. Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca/sup 2 +/, but only modestly attenuated AVP-stimulated Na/sup +/-pump activity. Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na/sup +//K/sup +/-ATPase-mediated transport activity. Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na/sup +/-pump in the absence of changes in cytosolic or total cellular Ca/sup 2 +/ levels. Stimulation of the Na/sup +/-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism. Stimulation of the Na/sup +/-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na/sup +//H/sup +/ exchange, but these compounds blocked the action of insulin. These data suggest that the elevated Na/sup +//K/sup +/-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca/sup 2 +/-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C.

  3. Ouabain stimulates Na-K-ATPase through a sodium/hydrogen exchanger-1 (NHE-1)-dependent mechanism in human kidney proximal tubule cells

    PubMed Central

    Holthouser, Kristine A.; Mandal, Amritlal; Merchant, Michael L.; Schelling, Jeffrey R.; Delamere, Nicholas A.; Valdes, Ronald R.; Tyagi, Suresh C.; Lederer, Eleanor D.

    2010-01-01

    Recent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPase are interdependent. To test whether treatment with picomolar ouabain regulates Na-K-ATPase through an NHE-1-dependent mechanism, we examined the role of NHE-1 in ouabain-mediated stimulation of Na-K-ATPase in kidney proximal tubule cell lines [opossum kidney (OK), HK-2, HKC-5, and HKC-11] and rat kidney basolateral membranes. Ouabain stimulated Na-K-ATPase activity and tyrosine phosphorylation in cells that express NHE-1 (OK, HKC-5, and HKC-11) but not in HK-2 cells that express very low levels of NHE-1. Inhibition of NHE-1 with 5 μM EIPA, a NHE-1-specific inhibitor, prevented ouabain-mediated stimulation of 86Rb uptake and Na-K-ATPase phosphorylation in OK, HKC-5, and HKC-11 cells. Expression of wild-type NHE-1 in HK2 cells restored regulation of Na-K-ATPase by picomolar ouabain. Treatment with picomolar ouabain increased membrane expression of Na-K-ATPase and enhanced NHE-1-Na-K-ATPase α1-subunit association. Treatment with ouabain (1 μg·kg body wt−1·day−1) increased Na-K-ATPase activity, expression, phosphorylation, and association with NHE-1 increased in rat kidney cortical basolateral membranes. Eight days' treatment with ouabain (1 μg·kg body wt−1·day−1) resulted in increased blood pressure in these rats. These results suggest that the association of NHE-1 with Na-K-ATPase is critical for ouabain-mediated regulation of Na-K-ATPase and that these effects may play a role in cardioglycoside-stimulated hypertension. PMID:20427472

  4. Ion fluxes in Acetabularia: vesicular shuttle.

    PubMed

    Mummert, H; Gradmann, D

    1991-12-01

    Ion flux relations in the unicellular marine alga Acetabularia have been investigated by uptake and washout kinetics of radioactive tracers (22Na+, 42K+, 36Cl- and 86Rb+) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K+ with influx and efflux of about 100 nmol.m-2.sec-1. K+ passes three- to sevenfold more easily than Rb+ does. Under normal conditions, Cl- (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mumol.m-2.sec-1, and a cytoplasmic as well as vacuolar [Cl-] of about 420 mM ([Cl-]o = 529 mM). At 4 degrees C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl-], are significantly reduced. Na+ ([Na+]i: about 70 mM, [Na+]o: 461 mM), which is of minor electrophysiological relevance compared to K+, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mumol.m-2.sec-1 for influx and efflux). Some results with Na+ and Cl- are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normal Acetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the

  5. A component of Premarin® enhances multiple cognitive functions and influences nicotinic receptor expression

    PubMed Central

    Talboom, Joshua S.; Engler-Chiurazzi, Elizabeth B.; Whiteaker, Paul; Simard, Alain R.; Lukas, Ronald; Acosta, Jazmin I.; Prokai, Laszlo; Bimonte-Nelson, Heather A.

    2010-01-01

    In women, ovarian hormone loss at menopause has been related to cognitive decline, and some studies suggest that estrogen-containing hormone therapy (HT) can mitigate these effects. Recently, the Women’s Health Initiative study found that conjugated equine estrogens, the most commonly prescribed HT, do not benefit cognition. Isolated components of conjugated equine estrogens (tradename Premarin®) have been evaluated in vitro, with Δ8,9-dehydroestrone (Δ8E1) and equilin showing the strongest neuroprotective profiles. It has not been evaluated whether Δ8E1 or equilin impact cognition or the cholinergic system, which is affected by other estrogens and known to modulate cognition. Here, in middle-aged, ovariectomized rats, we evaluated the effects of Δ8E1 and equilin treatments on a cognitive battery and cholinergic nicotinic receptors (nAChR). Specifically, we used 125I-labeled epibatidine binding to assay the neuronal nicotinic receptor containing 4α and 2β subunits (α4β2-nAChR), since this nicotinic receptor subtype has been shown previously to be sensitive to other estrogens. Δ8E1 enhanced spatial working, recent and reference memory. Δ8E1 also decreased hippocampal and entorhinal cortex α4β2-nAChR expression, which was related to spatial reference memory performance. Equilin treatment did not affect spatial memory or rat α4β2-nAChR expression. Neither estrogen impacted 86Rb+ efflux, indicating lack of direct action on human α4β2 nAChR function. Both estrogens influenced vaginal smear profiles, uterine weights, and serum luteinizing hormone levels, analogous to classic estrogens. The findings indicate that specific isolated Premarin® components differ in their ability to affect cognition and nAChR expression. Taken with the works of others showing Δ8E1-induced benefits on several dimensions of health-related concerns associated with menopause, this identifies Δ8E1 as a new avenue to be investigated as a potential component of HT that may

  6. The potent and selective α4β2*/α6*-nicotinic acetylcholine receptor partial agonist 2-[5-[5-((S)Azetidin-2-ylmethoxy)-3-pyridinyl]-3-isoxazolyl]ethanol demonstrates antidepressive-like behavior in animal models and a favorable ADME-tox profile

    PubMed Central

    Yu, Li-Fang; Brek Eaton, J; Zhang, Han-Kun; Sabath, Emily; Hanania, Taleen; Li, Guan-Nan; van Breemen, Richard B; Whiteaker, Paul; Liu, Qiang; Wu, Jie; Chang, Yong-Chang; Lukas, Ronald J; Brunner, Dani; Kozikowski, Alan P

    2014-01-01

    Preclinical and clinical studies demonstrated that the inhibition of cholinergic supersensitivity through nicotinic antagonists and partial agonists can be used successfully to treat depressed patients, especially those who are poor responders to selective serotonin reuptake inhibitors (SSRIs). In our effort to develop novel antidepressant drugs, LF-3-88 was identified as a potent nicotinic acetylcholine receptor (nAChR) partial agonist with subnanomolar to nanomolar affinities for β2-containing nAChRs (α2β2, α3β2, α4β2, and α4β2*) and superior selectivity away from α3β4 − (Ki > 104 nmol/L) and α7-nAChRs (Ki > 104 nmol/L) as well as 51 other central nervous system (CNS)-related neurotransmitter receptors and transporters. Functional activities at different nAChR subtypes were characterized utilizing 86Rb+ ion efflux assays, two-electrode voltage-clamp (TEVC) recording in oocytes, and whole-cell current recording measurements. In mouse models, administration of LF-3-88 resulted in antidepressive-like behavioral signatures 15 min post injection in the SmartCube® test (5 and 10 mg/kg, i.p.; about 45-min session), decreased immobility in the forced swim test (1–3 mg/kg, i.p.; 1–10 mg/kg, p.o.; 30 min pretreatment, 6-min trial), and decreased latency to approach food in the novelty-suppressed feeding test after 29 days chronic administration once daily (5 mg/kg but not 10 mg/kg, p.o.; 15-min trial). In addition, LF-3-88 exhibited a favorable profile in pharmacokinetic/ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) assays. This compound was also shown to cause no mortality in wild-type Balb/CJ mice when tested at 300 mg/kg. These results further support the potential of potent and selective nicotinic partial agonists for use in the treatment of depression. PMID:25505580

  7. 31P-nuclear magnetic resonance spectroscopy in vivo of six human melanoma xenograft lines: tumour bioenergetic status and blood supply.

    PubMed Central

    Lyng, H.; Olsen, D. R.; Southon, T. E.; Rofstad, E. K.

    1993-01-01

    Six human melanoma xenograft lines grown s.c. in BALB/c-nu/nu mice were subjected to 31P-nuclear magnetic resonance (31P-NMR) spectroscopy in vivo. The following resonances were detected: phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), phosphocreatine (PCr) and nucleoside triphosphate gamma, alpha and beta (NTP gamma, alpha and beta). The main purpose of the work was to search for possible relationships between 31P-NMR resonance ratios and tumour pH on the one hand and blood supply per viable tumour cell on the other. The latter parameter was measured by using the 86Rb uptake method. Tumour bioenergetic status [the (PCr + NTP beta)/Pi resonance ratio], tumour pH and blood supply per viable tumour cell decreased with increasing tumour volume for five of the six xenograft lines. The decrease in tumour bioenergetic status was due to a decrease in the (PCr + NTP beta)/total resonance ratio as well as an increase in the Pi/total resonance ratio. The decrease in the (PCr + NTP beta)/total resonance ratio was mainly a consequence of a decrease in the PCr/total resonance ratio for two lines and mainly a consequence of a decrease in the NTP beta/total resonance ratio for three lines. The magnitude of the decrease in the (PCr + NTP beta)/total resonance ratio and the magnitude of the decrease in tumour pH were correlated to the magnitude of the decrease in blood supply per viable tumour cell. Tumour pH decreased with decreasing tumour bioenergetic status, and the magnitude of this decrease was larger for the tumour lines showing a high than for those showing a low blood supply per viable tumour cell. No correlations across the tumour lines were found between tumour pH and tumour bioenergetic status or any other resonance ratio on the one hand and blood supply per viable tumour cell on the other. The differences in the 31P-NMR spectrum between the tumour lines were probably caused by differences in the intrinsic biochemical properties of the tumour

  8. The Na+/K(+)-pump in rat peritoneal mast cells: some aspects of regulation of activity and cellular function.

    PubMed

    Knudsen, T

    1995-11-01

    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 have important clinical implications. The aim of the present work has been to document the existence of the Na+/K(+)-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+/K(+)-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with 86Rb+ 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 for cellular degranulation. Histamine was measured by spectrofluorometry. The finding of an ouabain-sensitive uptake mechanism in the mast cell documents the presence of a functional Na+/K(+)-pump in this cell. The pump activity is inhibited by lanthanides and by the divalent cations calcium, magnesium, barium and strontium. The pump has a large reserve capacity which probably is caused by a low intracellular concentration of sodium. This enables the pump to respond to changes in the intracellular sodium concentration. The inhibitory effect of di- and trivalent ions on the pump activity is probably a result of the inhibitory effect of these ions on the cellular sodium uptake. The digitalis glycosides, ouabain and digoxin, but not the more lipophilic drug digitoxigenin, increase both IgE-directed and non-IgE-directed histamine release