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Sample records for 86rb vblizi porogov

  1. Altered agonist-activated sup 86 Rb+ efflux from arteries in canine renal hypertension

    SciTech Connect

    Cox, R.H.; Bagshaw, R.J. )

    1989-07-01

    Basal rate constants for {sup 86}Rb+ efflux from renal arteries of renal hypertensive dogs were lower than those of control animals whereas no differences were found for coronary arteries. Norepinephrine produced parallel increases in efflux rate constants for hypertensive and control renal arteries, but serotonin produced smaller responses in hypertensive compared to control coronary arteries.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance.

    PubMed

    Güven, M; Onaran, I; Ulutin, T; Sultuybek, G; Hatemi, H

    2001-04-01

    Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia. PMID:11383909

  7. Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance.

    PubMed

    Güven, M; Onaran, I; Ulutin, T; Sultuybek, G; Hatemi, H

    2001-01-01

    Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia. PMID:11508792

  8. 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.

  9. The effects of the putative potassium channel activator WAY-120,491 on 86Rb efflux from the rabbit aorta

    SciTech Connect

    Lodge, N.J.; Cohen, R.B.; Havens, C.N.; Colatsky, T.J. )

    1991-02-01

    WAY-120,491 ((-)-(3S-trans)-2-(3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one) is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.

  10. Effects of hydrochlorothiazide on contraction and 86Rb efflux in rat aorta.

    PubMed

    Wang, G S; Li, Y S; Fu, S X

    1993-09-01

    Hydrochlorothiazide (HCT) (0.1, 0.3 mmol.L-1) inhibited the contraction of rat aortic strips induced by low (< 40 mmol.L-1), not higher concentrations of KCl. HCT (0.3 mmol.L-1) did not inhibit the CaCl2-induced contraction of the aortic strips depolarized with high K+ (KCl 80 mmol.L-1). The inhibitory effect of HCT (0.1 mmol.L-1) on KCl (20 mmol.L-1)-induced contraction was markedly antagonized by BaCl2 (0.1 mmol.L-1) and tetraethylammonium (TEA) (0.3 mmol.L-1), but not by glibenclamide (Gli, 0.01 mmol.L-1). With norepinephrine (NE) or 5-HT as agonists, HCT (0.3 mmol.L-1) also inhibited the contractions of rat aortic strips. In the 2 components of NE-induced contraction, HCT inhibited only the tonic component depending on Ca2+ influx, but not the phasic component elicited by the release of intracellular Ca2+. The inhibitory action of HCT was endothelium-independent. That the HCT (3 mmol.L-1) increased the 86Rb efflux rate coefficient was antagonized by BaCl2 (0.1 mmol.L-1), but not by Gli (0.01 mmol.L-1). The results indicated that the inhibitory effect of HCT on the contraction of rat aorta was attributable to the opening of membrane potassium channels. PMID:8010028

  11. /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.

  12. 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.

  13. 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.

  14. Effect of glucose intake on human leucocyte /sup 86/Rb influx and (/sup 3/H)-ouabain binding

    SciTech Connect

    Turaihi, K.; Baron, D.N.; Dandona, P.

    1988-02-01

    /sup 86/Rb influx and (/sup 3/H) ouabain binding by human leucocytes were measured in eight normal nonobese fasting subjects before and after a challenge with 75 g glucose orally. The mean ouabain-sensitive /sup 86/Rb influx increased significantly from 194 to 283 mmol/kg protein/h (P less than .01), and (/sup 3/H)-ouabain binding increased from 236 to 403 fmol/mg protein. The mean plasma potassium concentration fell from 4.2 to 3.9 mmol/L (P less than .05). Following intravenous glucose infusion, the median /sup 86/Rb transport increased from 186 to 267 mmol/kg protein/h, while median plasma potassium concentration fell from 4.3 to 3.9 mmol/L. Therefore, glucose intake acutely increases Na-K ATPase units, stimulates potassium (Rb) transport, and causes a concomitant fall in plasma potassium concentrations. Nutritional intake is probably an important determinant of Na-K ATPase units and activity in the human leucocyte.

  15. Aging: ouabain-sensitive /sup 86/Rb+ uptake rate and responsiveness to digoxin in rat left atrial muscle

    SciTech Connect

    Kennedy, R.H.; Seifen, E.

    1989-01-01

    Previous work in anesthetized rats has demonstrated that the sensitivity to cardiotoxic actions of cardiotonic steroids is increased in senescence, and studies in crude homogenates and partially purified membrane preparations have suggested that this altered responsiveness is related to an aging-associated reduction in the sarcolemmal content of Na,K-adenosine triphosphatase. This decrease in Na,K-adenosine triphosphatase could enhance the sensitivity to digitalis-like compounds by reducing the reserve capacity of the Na+-pump and thus the extent of digitalis-induced pump inhibition required before the onset of toxicity. Current experiments examined dose-dependent actions of digoxin in atrial muscle isolated from 3-, 12- and 24- to 25-month-old rats and determined if alterations in responsiveness correlated with changes in ouabain-sensitive 86Rb+ uptake rate, an estimate of Na+-pump activity. Atrial preparations from aged rats were more sensitive to the cardiotoxic actions of digoxin; however, the inotropic efficacy before the onset of toxicity was not affected by age. Both 1) the maximum attainable ouabain-sensitive 86Rb+ uptake rate and 2) the difference between maximum uptake rate and that monitored in preparations stimulated at 4.0 Hz decreased progressively with age. These results indicate that atrial muscle from aged rats is more sensitive to direct toxic effects of digoxin and suggest that this lower tolerance is mediated, at least in part, by a reduction in Na+-pump reserve capacity.

  16. Characterization of Ca(2+)-activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa-channel toxins.

    PubMed Central

    de-Allie, F. A.; Bolsover, S. R.; Nowicky, A. V.; Strong, P. N.

    1996-01-01

    1. The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+. 2. The resting 86Rb+ influx into C6 cells was 318 +/- 20 pmol s-1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx was proportional to ionophore concentration. Ionomycin-activated 86Rb+ flux was saturable (EC50 = 0.62 +/- 0.03 microM) and was not inhibited by ouabain. 3. Intracellular Ca2+ increased within 30 s from a basal level of 42 +/- 2 nM to 233 +/- 17 nM, after addition of 2 microM ionomycin. During this period, intracellular pH fell from 7.03 +/- 0.04 to 6.87 +/- 0.03 and the cell hyperpolarized from -34 +/- 10 mV to -76 +/- 2 mV. 4. Single channel conductance measurements on inside-out patches in physiological K+ solutions identified a 14 +/- 3 pS CA(2+)-activated K+ current between -25 mV and +50 mV. In symmetrical (100 mM) K+, the single channel conductance was 26 pS. 5. Externally applied quinine (IC50 = 0.12 +/- 0.34 mM) and tetraethylammonium chloride (IC50 = 10 +/- 1.9 mM) inhibited 86Rb+ influx into C6 cells in a concentration-dependent manner. Charybdotoxin (IC50 = 0.5 +/- 0.02 nM) and iberiotoxin (IC50 = 800 +/- 150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited 86Rb+ influx. In contrast, apamin and toxin I had no inhibitory effects on 86Rb+ flux. A screen of fractions from cation exchange h.p.l.c. of Mesob. tamulus venom revealed the presence of at least four charybdotoxin-like peptides. One of these was iberiotoxin; the other three are novel toxins. 6. The ionomycin-activated 86Rb+ influx into rat C6 glioma cells has proved to be a valuable pharmacological assay for the screening of toxins and crude

  17. 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.

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

    SciTech Connect

    Cox, R.H.; Tulenko, T.N. )

    1989-08-01

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

  19. 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.

  20. Evidence that pinacidil may promote the opening of ATP-sensitive K+ channels yet inhibit the opening of Ca2(+)-activated K+ channels in K(+)-contracted canine mesenteric artery.

    PubMed Central

    Masuzawa, K.; Matsuda, T.; Asano, M.

    1990-01-01

    1. The effects of cromakalim and pinacidil on contraction and 86Rb efflux were investigated in strips of canine mesenteric artery. 2. Cromakalim and pinacidil relaxed arterial strips precontracted with 20.9 mM K+ with pD2 values of 6.56 and 5.88, respectively. 3. High (above 10 microM) concentrations of pinacidil, but not cromakalim, relaxed arterial strips bathed by a medium containing 65.9 mM K+, and inhibited Ca2(+)-induced contractions in strips bathed by a medium containing 80 mM K+. These findings suggested that pinacidil may act as an inhibitor of Ca2+ influx. 4. In arterial strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux. 5. When the effects of cromakalim and pinacidil on 86Rb efflux were determined in arterial strips contracted with 65.9 mM K+, both drugs increased 86Rb efflux. The increase in 86Rb efflux induced by pinacidil was much smaller than that induced by cromakalim. Under the same conditions, nifedipine decreased 86Rb efflux. 6. After the addition of nifedipine to arterial strips contracted with 65.9 mM K+, pinacidil produced a greater increase in 86Rb efflux than in the absence of nifedipine, whereas the effects of cromakalim were the same for the two conditions. Therefore, the effects of pinacidil on 86Rb efflux may be the resultant of two opposing effects: an increased 86Rb efflux due to the opening of ATP-sensitive K+ channels, and a decreased efflux due to the closing of Ca2(+)-activated K+ channels. 7. In causing relaxation, cromakalim was competitively antagonized by glibenclamide with a pA2 value of 7.16.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2115387

  1. 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.

  2. Potentiation of P1075-induced K+ channel opening by stimulation of adenylate cyclase in rat isolated aorta.

    PubMed Central

    Linde, C.; Quast, U.

    1995-01-01

    1. The effects of analogues and stimulators of cyclic AMP on the 86Rb+ efflux-stimulating and binding properties of P1075, an opener of ATP-dependent potassium channels, were studied in rat aortic rings. The increase in 86Rb+ efflux stimulated by P1075 was taken as a qualitative measure of K+ channel opening. 2. Forskolin, a direct activator of adenylate cyclase, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and dibutyryl-cyclic AMP (db-cyclic AMP), a membrane permeant cyclic AMP-analogue, relaxed rat aortic rings contracted by noradrenaline with EC50 values of 0.06, 2 and 10 microM, respectively. 3. Forskolin, IBMX and db-cyclic AMP produced concentration-dependent increases of the 86Rb+ efflux induced by P1075 (50 nM) by up to twofold with EC50 values of about 0.1, 1.7 and 81 microM. At these concentrations the agents had little effect on the basal rate of 86Rb+ efflux. 4. The 86Rb+ efflux produced by P1075 in the presence of the cyclic AMP stimulators was inhibited by glibenclamide, a blocker of ATP-sensitive potassium channels. 5. IBMX (100 microM) induced a leftward shift of the concentration-86Rb+ efflux curve of P1075 without increasing the maximum. The enhancements of P1075-stimulated 86Rb+ efflux produced by combinations of forskolin and IBMX were either additive or less than additive. 6. The protein kinase A inhibitor, H-89, inhibited P1075-stimulated 86Rb+ efflux in the presence of IBMX significantly more than in the absence of IBMX, suggesting that the effect of increased cyclic AMP levels is mediated by protein kinase A.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582466

  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. Effects of Boron Deficiency on 86Rubidium Uptake and Photosynthesis in the Diatom Cylindrotheca fusiformis

    PubMed Central

    Smyth, Douglas A.; Dugger, W. Mack

    1980-01-01

    Culturing the diatom Cylindrotheca fusiformis under boron-deficient conditions leads to changes in 86Rb uptake and photosynthesis prior to any effect on the rate of cell division. The influx rate of 86Rb into boron-deficient cells was 79% of the control rate after 5 to 5.5 hours culture. Despite lowered 86Rb influx, however, boron-deficient diatoms accumulated more 86Rb than did control cells; this was due to the deficient cells' lower efflux rate. After 24 hours culture, boron-deficient cells had accumulated 30% more 86Rb than had control cells, while releasing 86Rb at only one-half the control rate. Increased photosynthetic rates were another effect of boron deficiency during this early stage of culture. Prior to 20 hours boron-deficient culture, diatoms had photosynthetic rates 37% greater than those of control cells. Corresponding to the increase in photosynthesis, boron-deficient diatoms had 12% more carbohydrate than control cells after 16 hours culture. PMID:16661504

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

    SciTech Connect

    Ticku, M.K.; Delgado, A.

    1989-01-01

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

  6. Increased leucocyte Na-K ATPase in obesity: reversal following weight loss

    SciTech Connect

    Turaihi, K.; Baron, D.N.; Dandona, P.

    1987-09-01

    Ouabain-sensitive /sup 86/Rb influx and (/sup 3/H) ouabain binding capacity were investigated in the leucocytes of 17 obese patients and 15 control subjects. Both were significantly increased in the obese when compared with controls. Following dietary restriction and a 4% to 5% weight reduction in the obese over 2 weeks, (/sup 3/H) ouabain binding and ouabain-sensitive /sup 86/Rb influx (a model for K+ influx) decreased to levels similar to those in controls. This shows that the number of Na-K ATPase sites on leucocyte membranes of the obese are significantly increased and that this is associated with accelerated /sup 86/Rb transport. Since both of these indices decreased following 4% to 5% reduction in body weight while the patients were still obese, increased Na-K ATPase is neither a marker of nor cardinal to the pathogenesis of obesity. We conclude that (1) increase in Na-K ATPase units and /sup 86/Rb influx are not characteristic of obesity itself and (2) dietary restriction over the short-term with limited weight reduction restores Na-K ATPase units and /sup 86/Rb influx to normal.

  7. Red Light and Auxin Effects on 86Rubidium Uptake by Oat Coleoptile and Pea Epicotyl Segments 1

    PubMed Central

    Pike, Carl S.; Richardson, Alice E.

    1979-01-01

    Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [86Rb+] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10−7 molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [86Rb+] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [86Rb+] flux is not a general feature of phytochrome action. PMID:16660665

  8. 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.

  9. Excitation- and β2-agonist-induced activation of the Na+−K+ pump in rat soleus muscle

    PubMed Central

    Buchanan, Rasmus; Nielsen, Ole Bækgaard; Clausen, Torben

    2002-01-01

    In rat skeletal muscle, Na+–K+ pump activity increases dramatically in response to excitation (up to 20-fold) or β2-agonists (2-fold), leading to a reduction in intracellular Na+. This study examines the time course of these effects and whether they are due to an increased affinity of the Na+–K+ pump for intracellular Na+. Isolated rat soleus muscles were incubated at 30 oC in Krebs-Ringer bicarbonate buffer. The effects of direct electrical stimulation on 86Rb+ uptake rate and intracellular Na+ concentration ([Na+]i) were characterized in the subsequent recovery phase. [Na+]i was varied using monensin or buffers with low Na+. In the [Na+]i range 21–69 mm, both the β2-agonist salbutamol and electrical stimulation produced a left shift of the curves relating 86Rb+ uptake rate to [Na+]i. In the first 10 s after 1 or 10 s pulse trains of 60 Hz, [Na+]i showed no increase, but 86Rb+ uptake rate increased by 22 and 86 %, respectively. Muscles excited in Na+-free Li+-substituted buffer and subsequently allowed to rest in standard buffer also showed a significant increase in 86Rb+ uptake rate and decrease in [Na+]i. Na+ loading induced by monensin or electroporation also stimulated 86Rb+ uptake rate but, contrary to excitation, increased [Na+]i. The increase in the rate of 86Rb+ uptake elicited by electrical stimulation was abolished by ouabain, but not by bumetanide. The results indicate that excitation (like salbutamol) induces a rapid increase in the affinity of the Na+–K+ pump for intracellular Na+. This leads to a Na+–K+ pump activation that does not require Na+ influx, but possibly the generation of action potentials. This improves restoration of the Na+–K+ homeostasis during work and optimizes excitability and contractile performance of the working muscle. PMID:12433963

  10. 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.

  11. Ion Fluxes and Phytochrome Protons in Mung Bean Hypocotyl Segments

    PubMed Central

    Brownlee, Colin; Kendrick, Richard E.

    1979-01-01

    K+ [86Rb+] uptake by Phaseolus aureus Roxb. hypocotyl segments cut immediately below the hook is inhibited by the active form of phytochrome (Pfr). Short load-short wash experiments indicate that the inhibition of uptake occurs across the plasmalemma. A maximal inhibition of short term uptake occurs in 10 to 50 millimolar KCI. Low temperature had only a small effect on influx and the inhibition of influx from 50 millimolar KCI. A consideration of the electrochemical gradient for K+ suggests that passive K+ fluxes may predominate under these conditions. Red light induces small depolarizations of membrane potential in subhook cells. Far red light antagonizes this effect. Pfr inhibits efflux of K+[86Rb+] from subhook segments. This effect is also relatively insensitive to low temperature. This inhibition of efflux may reflect inhibition of a K+ -K+ exchange process, or reduced passive permeability of the plasmalemma to K+. In contrast, Pfr enhances short term uptake of K+[86Rb+] in apical hypocotyl hook segments of Phaseolus aureus Roxb. Short load-short wash experiments indicate that fluxes across the plasmalemma are modified by Pfr. A maximal enhancement of short term influx occurs in 50 millimolar KCI. Influx and the red light enhancement of influx from 50 millimolar KCI are relatively insensitive to low temperature. Pfr also enhances efflux of K+[86Rb+] from preloaded apical hook segments. This increased influx may reflect enhancement of a K+ -K+ exchange process or increased passive permeability of the plasmalemma to K+. PMID:16660933

  12. 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.

  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. K+ transport across the lamprey erythrocyte membrane: characteristics of a Ba(2+)- and amiloride-sensitive pathway.

    PubMed

    Kirk, K

    1991-09-01

    The characteristics of K+ transport in erythrocytes from the river lamprey (Lampetra fluviatilis) were investigated using standard radioisotope flux techniques. The cells were shown to have a ouabain-sensitive transport pathway that carried 43K+ and 86Rb+ into the cell at similar rates. Most of the ouabain-resistant 43K+ and 86Rb+ influx was via a pathway that was insensitive to cotransport inhibitors and to the replacement of extracellular Cl- or Na+. This pathway showed a strong selectivity for 43K+ over 86Rb+. It was inhibited fully by Ba2+ (I50 approximately 2.8 mumol l-1), amiloride (I50 approximately 150 mumol l-1) and ethylisopropylamiloride (I50 approximately 3.3 mumol l-1) and less effectively by quinine and by the tetraethylammonium ion. Inhibition by Ba2+ took full effect within a few minutes whereas the full inhibitory effect of amiloride took more than 1 h to develop. Experiments with the membrane potential probe [14C]tetraphenylphosphonium ion gave results consistent with the lamprey erythrocyte membrane having a Ba(2+)-sensitive K+ conductance that was significantly greater than the membrane Na+ conductance and which gave rise to a marked dependence of the membrane potential on the extracellular K+ concentration. The rate constants for Ba(2+)-sensitive 43K+ and 86Rb+ influx decreased (proportionally) with increasing extracellular K+ concentration in a manner that was consistent with the transport being via a conductive pathway. The decrease was attributed to a depolarisation of the membrane (in response to the increasing extracellular K+ concentration) and a consequent decrease in the driving force for the conductive movement of 43K+ and 86Rb+ into the cells. Ba(2+)-sensitive 86Rb+ influx increased significantly with decreasing cell volume and with increasing intracellular pH (at a constant extracellular pH) but increased only slightly with increasing extracellular pH. The pathway operated normally in the complete absence of extracellular Ca2+ but

  15. 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

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

    SciTech Connect

    Neumann, P.M.; Chamel, A.

    1986-06-01

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

  17. Evidence for differential action of indoleacetic acid upon ion fluxes in single cells of Petroselinum sativum.

    PubMed

    Bentrup, F W; Pfrüner, H; Wagner, G

    1973-12-01

    The apparent influx of (36)Cl(-) and (86)Rb(+)/K(+) into cells from the higher plant Petroselinum sativum has been measured during the presence and absence in the culture medium of indolacetic acid (IAA) which is an essential auxin of these cells. While 10(-5) M IAA did not significantly affect the influx of (86)Rb(+)/K(+), it substantially reduced that of (36)Cl(-), i.e. by a factor 0.25 within 30 min. This differential action of IAA, which holds for a reasonable range of external pH, is assumed to bear on current hypotheses that the primary events of auxin action involve plasmalemma functions. PMID:24474466

  18. Mineral transfer in a legume/grass association

    SciTech Connect

    Habben, J.E.; Blevins, D.G.

    1986-04-01

    Previous pasture research has indicated that in a legume/grass association the grass has a higher concentration of specific minerals than grass grown alone. The purpose of this study was to determine if a deeply rooted legume could transfer minerals to an associated shallow rooted grass plant via their root systems. A greenhouse study was conducted using alfalfa and maize plants grown in a double tube design. Plants were established such that the top tube contained both alfalfa and maize roots while the bottom tube contained only the alfalfa roots. Alfalfa roots in the lower tube were exposed to 1 mCi of one of three different isotopes (/sup 32/P, /sup 86/Rb and /sup 45/Ca) over a 40 day period. Under these conditions, radioactive analysis of maize tissue showed a significant transfer of /sup 86/Rb and /sup 32/P.

  19. Reduced sodium concentration and increased sodium-potassium pump activity of erythrocytes in human hypertension

    SciTech Connect

    Simon, G.; Engel, C.R.

    1987-06-01

    Erythrocyte Nai, Nai/Ki and ouabain-sensitive and ouabain-insensitive /sup 86/Rb uptake (K transport) were measured in whole blood of 16 normotensive and 19 hypertensive white male subjects, within seconds or minutes after withdrawal of blood. Erythrocyte Nai and Nai/Ki were reduced (p less than 0.05), and ouabain-sensitive /sup 86/Rb uptake was increased (p less than 0.01) in hypertensive subjects. In a separate group of hypertensive white male subjects, an inverse correlation was found between erythrocyte Nai/Ki and ouabain-binding sites per erythrocyte (r = 0.85, p less than 0.01, n = 9). The abnormalities of erythrocyte cation fluxes in hypertensive subjects are similar to those induced by aldosterone in vascular smooth muscle cells and by glucocorticoid administration in the erythrocytes of human subjects, suggesting similarities in pathogenesis.

  20. 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.

  1. Ba2+ inhibition of VIP- and A23187-stimulated Cl- secretion by T84 cell monolayers

    SciTech Connect

    Mandel, K.G.; McRoberts, J.A.; Beuerlein, G.; Foster, E.S.; Dharmsathaphorn, K.

    1986-03-01

    Addition of either 10(-8) M vasoactive intestinal polypeptide (VIP) or 10(-6) M A23187 to T84 cell monolayers, grown on permeable supports and mounted in Ussing chambers, stimulated net Cl- secretion. The effect of 10(-6) M A23187 on Cl- flux was consistently smaller than that observed with 10(-8) M VIP. In both cases the increase in net Cl- secretion accounted for the entire change in the observed short-circuit current (Isc). Since Cl- enters the cells through a basolaterally localized Na+-K+-Cl(-)-cotransport system (J. Clin. Invest. 75: 462, 1985), the fate of K+, which is cotransported with Cl- during VIP, and A23187-mediated Cl- secretion was explored. Unidirectional and net transepithelial 42K+ flux rates were negligible compared with 36Cl- flux rates (less than 4% of Cl- flux), indicating that little K+ was secreted along with Cl-. K+ recycling across the basolateral membrane was suggested from experiments in which 86Rb+ efflux (as a tracer for K+) was measured across the apical and basolateral membranes of 86Rb+ -preloaded monolayers under voltage-clamped conditions. In the absence of secretagogues, 86Rb+ efflux was 10-fold higher across the basolateral membrane than across the apical membrane. 86Rb+ efflux across the basolateral membrane was accelerated two- to threefold by addition of either VIP or A23187. In each case accelerated efflux was inhibited by 5 mM Ba2+. Cl- secretion induced by VIP or A23187 was also inhibited by serosal addition of Ba2+.

  2. Loss of Rubidium and Potassium from Barley Roots on Sudden Chilling

    PubMed Central

    Bange, Gerard G. J.

    1979-01-01

    Sudden chilling of roots of barley pretreated in a labeled Rb+ solution at 25 C causes a transient loss of label from the nonfree space. The half-time of the release is about 10 minutes. Within the first few hours of uptake the loss increases proportionally to the root Rb+ content but varies between experiments as a fraction of this content: values from 4 to 20% were observed. The same phenomenon is found for K+ labeled with 86Rb+ PMID:16661013

  3. Na/K ATPase inhibition by digitalis-like factors in neonates

    SciTech Connect

    Bottorff, M.B.; Songu-Mize, E.; Hoon, T.J.; Phelps, S.J.; Kamper, C.A.

    1986-03-01

    At the authors institution, 48% of neonates < 1 month of age had false-positive digoxin immunoassay determinations while not receiving digoxin, presumably due to an endogenous digoxin-like immunoreactive substance (DLIS) in the plasma. Plasma from 3 neonates positive for DLIS by fluorescence polarization immunoassay (FPIA) was evaluated for inhibitory activity on human red blood cell (RBC) Na/K ATPase. Neonatal plasma aliquots containing DLIS concentrations (conc) of 0.24, 0.37, 0.43, 0.49 and 0.61 ng/ml (3.07 - 7.81 x 10/sup -10/M) were incubated with human RBC and /sup 86/Rb in order to measure /sup 86/Rb uptake inhibition with respect to DLIS negative neonatal plasma. /sup 86/Rb uptake inhibition by digoxin-spiked human serum (1.07 x 10/sup -10/ - 4.57 x 10/sup -6/M) was also measured. Percent inhibition vs. log molar conc plots for DLIS and digoxin were compared. DLIS inhibited Na/K ATPase in a linear fashion over the range studied. Comparing the linear portions of the conc-inhibition curves for digoxin and DLIS, the molar conc of digoxin producing 40% inhibition of /sup 86/Rb uptake is 333 times greater than the molar conc of DLIS producing similar inhibition. Therefore, DLIS in neonatal serum as measured by FPIA has approximately 300 times greater inhibitory activity than digoxin. The presence of circulating DLIS may reflect an adaptive or maladaptive response to some, as yet unknown, process early in life.

  4. 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

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

    SciTech Connect

    Kamatchi, G.L.; Ticku, M.K. )

    1991-02-01

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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Partial purification of endogenous digitalis-like compound(s) in cord blood

    SciTech Connect

    Balzan, S.; Ghione, S.; Biver, P.; Gazzetti, P.; Montali, U. )

    1991-02-01

    Increasing evidence indicates the presence of endogenous digitalis-like compound(s) in human body fluids. In this preliminary report, we describe a study of the partial purification by HPLC of these compounds in the plasma of neonates (who have particularly high concentrations of this substance) and adults. Plasma samples from neonates (cord blood) and adults, lyophilized and extracted with methanol, were applied on a 300 x 3.9 mm C18 Nova Pak column and eluted with a mobile phase of acetonitrile/methanol/water (17/17/66 or 14/14/72 by vol) and, after 30 min, with 100% methanol. We assayed eluted fractions for inhibitory activity of 86Rb uptake and for digoxin-like immunoreactivity. The elution profile revealed a first peak of inhibitory activity of 86Rb uptake at the beginning of the chromatography; another peak was eluted with the 100% methanol. The two peaks also cross-reacted with antidigoxin antibodies. Because the second peak could possibly reflect the nonspecific interference of various lipophilic compounds, we focused our attention on the first peak. For these fractions dose-response curves for 86Rb uptake and for displacement of digoxin were parallel, respectively, to those of ouabain and digoxin, suggesting similarities of digoxin-like immunoreactive substance to cardiac glycosides. Similar chromatographic profiles were also obtained for plasma from adults, suggesting that the endogenous glycoside-like compound(s) in the neonate may be the same as those in the adult.

  11. Furosemide-induced airway relaxation in guinea pigs: relation to Na-K-2Cl cotransporter function.

    PubMed

    Lavallee, S L; Iwamoto, L M; Claybaugh, J R; Dressel, M V; Sato, A K; Nakamura, K T

    1997-07-01

    This study tested the hypothesis that airway relaxation to furosemide is mediated via the Na-K-2Cl cotransporter. If this mechanism exists in airway smooth muscle like in vascular smooth muscle, changes in airway relaxation should be associated with changes in Na-K-2Cl cotransporter function, and both should be substrate dependent. Tracheal rings from newborn guinea pigs were bathed in standard (STD) or varying low Cl- concentration ([Cl-]) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Isometric relaxation to 300 microM furosemide or 10(-8) to 10(-5) M salbutamol was measured. Airway segments were incubated with rubidium-86 (86Rb) in STD or varying low [Cl-] HEPES, with and without 300 microM furosemide or 25 microM salbutamol. Furosemide was unable to reduce 86Rb uptake at 10 mM [Cl-], although relaxation was still observed in 10 mM [Cl-]. Salbutamol did not affect 86Rb uptake. This study demonstrated that there is a furosemide-sensitive Na-K-2Cl cotransporter in newborn guinea pig trachea. However, the effect of furosemide on cotransporter function did not always directly correspond to differences in relaxation, suggesting that the Na-K-2Cl cotransporter may play a major, but not exclusive, role in furosemide-induced airway relaxation. PMID:9252558

  12. 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.

  13. 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.

  14. A 28,000 mol. wt toxin from Bacillus thuringiensis israelensis induces cation transport in rat muscle cultures.

    PubMed

    Cahan, R; Shainberg, A; Pechatnikov, I; Nitzan, Y

    1995-07-01

    The mechanism by which the Bacillus thuringiensis israelensis (Bti) 28,000 mol. wt toxin exerts its effect on mature muscle cultures was examined. The toxin inhibited Na+/K(+)-ATPase activity as revealed by 86Rb influx. A 50% inhibition of Na+/K(+)-ATPase activity was obtained with 0.2 microgram/ml of the toxin. The inhibition was time and dose dependent, and it was reversible with low doses of the toxin (up to 0.2 microgram/ml. A considerable release of 86Rb was obtained by doses greater than 0.2 microgram/ml. The 86Rb release was also time and dose dependent. This effect is probably non-specific, since 45Ca influx is also accelerated by toxin-treated cultures. Pre-incubation of the toxin with phosphotidylserine (PS) antagonized the toxin. It is concluded that the toxin is a hydrophobic protein which interacts with the membrane. In low doses this interaction reduces the activity of the sodium pump and in high doses it causes non-specific permeability of the sarcolemma. PMID:8588218

  15. 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

  16. DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism.

    PubMed

    Shahidullah, Mohammad; Wei, Guojun; Delamere, Nicholas A

    2013-09-01

    The anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH. PMID:23677800

  17. Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells

    SciTech Connect

    Hermans, M.P.; Schmeer, W.; Henquin, J.C.

    1987-05-01

    Mouse islets were used to test the hypothesis that the B cell membrane must be depolarized for acetylcholine to increase insulin release. The resting membrane potential of B cells (at 3 mM glucose) was slightly decreased (5 mV) by acetylcholine, but no electrical activity appeared. This depolarization was accompanied by a Ca-independent acceleration of /sup 86/Rb and /sup 45/Ca efflux but no insulin release. When the B cell membrane was depolarized by a stimulatory concentration of glucose (10 mM), acetylcholine potentiated electrical activity, accelerated /sup 86/Rb and /sup 45/Ca efflux, and increased insulin release. This latter effect, but not the acceleration of /sup 45/Ca efflux, was totally dependent on extracellular Ca. If glucose-induced depolarization of the B cell membrane was prevented by diazoxide, acetylcholine lost all effects but those produced at low glucose. In contrast, when the B cell membrane was depolarized by leucine or tolbutamide (at 3 mM glucose), acetylcholine triggered a further depolarization with appearance of electrical activity, accelerated /sup 86/Rb and /sup 45/Ca efflux, and stimulated insulin release. Acetylcholine produced similar effects (except for electrical activity) in the presence of high K or arginine which, unlike the above test agents, depolarize the B cell membrane by a mechanism other than a decrease in K+ permeability. Omission of extracellular Ca abolished the releasing effect of acetylcholine under all conditions but only partially decreased the stimulation of /sup 45/Ca efflux. The results show thus that acetylcholine stimulation of insulin release does not result from mobilization of cellular Ca but requires that the B cell membrane be sufficiently depolarized to reach the threshold potential where Ca channels are activated. This may explain why acetylcholine alone does not initiate release but becomes active in the presence of a variety of agents.

  18. Nutrient and nonnutrient renal blood flow

    SciTech Connect

    Young, J.S.; Passmore, J.C.; Hartupee, D.A.; Baker, C.H. )

    1990-06-01

    The role of prostaglandins in the distribution of total renal blood flow (TRBF) between nutrient and nonnutrient compartments was investigated in anesthetized mongrel dogs. Renal blood flow distribution was assessed by the xenon 133 freeze-dissection technique and by rubidium 86 extraction after ibuprofen treatment. Ibuprofen (13 mg/kg) significantly decreased TRBF by 16.3% +/- 1.2% (mean +/- SEM electromagnetic flow probe; p less than 0.005), but did not alter blood flows to the outer cortex (3.7 vs 4.3 ml/min per gram), the inner cortex (2.6 vs 2.7 ml/min per gram), and the other medulla (1.5 vs 1.5 ml/min per gram), which suggests a decrease in nonnutrient flow. In a separate group of animals the effect of reduced blood flow on the nutrient and nonnutrient components was determined by mechanically reducing renal arterial blood flow by 48%. Unlike the ibuprofen group, nutrient blood flows were proportionally reduced with the mechanical decrease in TRBF in the outer cortex (1.9 ml/min per gram, p less than 0.05), the inner cortex (1.4 ml/min per gram, p less than 0.05), and the outer medulla (0.8 ml/min per gram, p less than 0.01). These results indicate no shift between nutrient and nonnutrient compartments. Nutrient and nonnutrient renal blood flows of the left kidney were also determined by 86Rb extraction. After ibuprofen treatment, nonextracted 86Rb decreased to 12.1% from the control value of 15.6% (p less than 0.05). Mechanical reduction of TRBF did not significantly decrease the proportion of unextracted 86Rb (18.7%).

  19. K+ transport by rat colon: adaptation to a low potassium diet

    SciTech Connect

    Tannen, R.L.; Marino, R.; Dawson, D.C.

    1986-03-01

    Recent studies with the isolated perfused rat kidney have demonstrated the existence of an intrinsic renal adaptation to conserve K+ in response to ingestion of a low K+ diet for 3 days. To determine whether the colon alters its K+ transport properties in a similar fashion, we measured transmural 86Rb fluxes across sheets of distal colonic epithelium under short-circuit conditions. Preliminary studies using a double-isotope technique demonstrated that 86Rb and 42K fluxes were similar; therefore 86Rb flux was considered equivalent to K+ flux. The distal half of the colon from each rat was divided into two segments, referred to as early and late distal colon. Experiments were carried out using rats fed a K+ -free, control (0.15 mmol/g), and high K+ (1.13 mmol/g) powdered diet of otherwise identical electrolyte content. Net K+ secretion (Jnet) by the early distal colon was reduced from 0.45 in the controls to -0.02 mueq X cm-2 X h-1 by a low K+ diet as a result of a decrease in serosal-to-mucosal flux (Jsm), with no change in mucosal-to-serosal flux (Jms). Conductance (GT) and short-circuit current (Isc) were unchanged. Jnet by the late distal colon averaged 0.17 in the controls and 0.01 mueq X cm-2 X h-1 with a low K+ diet, but this difference was not significant statistically. In comparison with the controls, a high K+ diet had no effect on Jnet by the early distal colon (0.48 mueq X cm-2 X h-1) but increased Jnet by the late distal colon substantially (0.77 mueq X cm-2 X h-1).

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

    SciTech Connect

    Simon, G.

    1986-03-01

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

  1. 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

  2. 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.

  3. Characterization and biological activity against Naegleria fowleri of amoebicins produced by Bacillus licheniformis D-13.

    PubMed Central

    Gálvez, A; Maqueda, M; Cordovilla, P; Martínez-Bueno, M; Lebbadi, M; Valdivia, E

    1994-01-01

    The strain Bacillus licheniformis D-13 produces three hydrophobic peptides (amoebicins d13-A, d13-B, and d13-C) that elicit antiamoebic activity against human-pathogenic and nonpathogenic species of Naegleria and have a broad spectrum of antibacterial activity. The three amoebicins have the same amino acid composition (three Asp, two Glu, two Val, and nine Leu residues) and molecular weight (1,870). Amoebicin d13-B causes lysis of amoebae through disorganization of the cell membrane. It also induces permeability to 86Rb and membrane disruption in asolectin vesicles. Images PMID:8092831

  4. Toxin sensitivity of the calcium-dependent rubidium efflux in Madin-Darby canine kidney cells.

    PubMed

    Tauc, M; Gastineau, M; Poujeol, P

    1993-01-29

    86Rb+ efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeus venom and charybdotoxin was attributed to the stimulation of Ca(++)-activated maxi K+ channels. Snake venom from Dendroaspis polylepis did not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca(++)-activated maxi K+ channels were predominantly found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca(++)-activated maxi K+ channel present in MDCK cells. PMID:7678959

  5. 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.

  6. 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

  7. Basolateral K+ channels in airway epithelia. II. Role in Cl- secretion and evidence for two types of K+ channel

    SciTech Connect

    McCann, J.D.; Welsh, M.J. )

    1990-06-01

    We previously described a Ca2(+)-activated K+ channel (KCLIC) in airway epithelial cells. To determine whether the KCLIC channel is a basolateral membrane channel and to understand its role in Cl- secretion, we studied airway epithelial cells grown on permeable supports. When cells were stimulated with A23187, charybdotoxin (ChTX) inhibited Cl- secretion and 86Rb efflux at the same concentrations, indicating that the KCLIC channel is required for Ca2(+)-stimulated Cl- secretion. We also investigated the function of K+ channels in adenosine 3',5'-cyclic monophosphate-stimulated secretion. Addition of isoproterenol caused a biphasic increase in Cl- secretion; the time course of the transient component correlated with the time course of the isoproterenol-induced increase in Ca2+ concentration (( Ca2+)c). ChTX inhibited the transient component, but not the prolonged component of secretion; Ba2+ inhibited the sustained component. These results suggest that when cells are grown on permeable supports isoproterenol-induced secretion depends on activation of two types of K+ channel: the KCLIC channel that is stimulated initially and a ChTX-insensitive K+ channel that is stimulated during sustained secretion. This conclusion was supported by measurement of 86Rb efflux from cell monolayers.

  8. 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

  9. 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.

  10. 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

  11. Effect of Low Temperature and Calcium on Survival and Membrane Properties of Isolated Winter Wheat Cells 1

    PubMed Central

    Pomeroy, M. Keith; Andrews, Chris J.

    1985-01-01

    Isolated cells obtained by enzymic digestion of young primary leaves of cold-hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of 86Rb was generally decreased by increasing concentrations of Ca2+, but after longer periods of incubation, the inhibiting effect of high Ca2+ levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at −1°C, while in the presence of 10 millimolar Ca2+ viability declined only gradually over a 5-week period. Ice encasement markedly reduced 86Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from −1 to −2 and −3°C, but the presence of Ca2+ in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from −10 to −30°C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at −1°C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane. PMID:16664270

  12. Effect of low temperature and calcium on survival and membrane properties of isolated winter wheat cells.

    PubMed

    Pomeroy, M K; Andrews, C J

    1985-07-01

    Isolated cells obtained by enzymic digestion of young primary leaves of cold-hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of (86)Rb was generally decreased by increasing concentrations of Ca(2+), but after longer periods of incubation, the inhibiting effect of high Ca(2+) levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at -1 degrees C, while in the presence of 10 millimolar Ca(2+) viability declined only gradually over a 5-week period. Ice encasement markedly reduced (86)Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from -1 to -2 and -3 degrees C, but the presence of Ca(2+) in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from -10 to -30 degrees C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at -1 degrees C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane. PMID:16664270

  13. Rubidium (Potassium) Uptake by Arabidopsis

    PubMed Central

    Polley, L. David; Hopkins, Johns W.

    1979-01-01

    Experiments are reported in which the uptake of 86Rb+, used as an analog of K+, into cultured cells of Arabidopsis thaliana is investigated. A single transport system is found with Km = 0.34 millimolar and Vmax = 14 nmoles per milligram of protein per hour. This system is blocked by the metabolic inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and by cold. At high concentrations of external K+ (above 1 millimolar), a significant fraction of total uptake is energy-independent. No evidence is found for more than one energy-dependent uptake system or for concentration-dependent modifications of a carrier as postulated in multiphasic transport models. Rb+ uptake was also examined in cultured cells derived from an “osmotic mutant” of Arabidopsis. The system closely resembles that found in wild type cells with the exception that the Michaelis-Menten constants are higher: Km = 1 millimolar and Vmax = 32 nanomoles per milligram of protein per hour. The possibility that these results are artifacts associated with use of cultured cells was checked by examining 86Rb+ uptake by roots of intact seedlings of wild type Arabidopsis. A single energy-dependent transport system is found with Km = 0.42 millimolar which is not significantly different from the Km of cultured cells. There is also energy-independent uptake at high external ion concentration. PMID:16660969

  14. 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

  15. Potassium Fluxes in Chlamydomonas reinhardtii (II. Compartmental Analysis).

    PubMed Central

    Malhotra, B.; Glass, ADM.

    1995-01-01

    42K+ and 86Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as I, II, and III) were apparent. Using 42K+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not significantly different when 86Rb+ was used to trace K+. Compartments I and II probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment III and the similarity between the [K+] of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 100 [mu]M (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+] very substantially. The mutants failed to grow at 100 [mu]M K+. PMID:12228560

  16. Action of ouabain and an amino-cardenolide on Na/sup +/-pump function and contractility of isolated canine heart cells

    SciTech Connect

    Porterfield, L.M.; Songu-Mize, E.; Chryssanthis, T.; Caldwell, R.W.

    1986-03-05

    Viable, rod-shaped, Ca/sup + +/-tolerant cells were isolated from the cardiac ventricle of adult mongrel dogs, a digitalis-sensitive species. These cells do not contract spontaneously but contractions were driven by electrical field stimulation. Changes in contractile amplitude were assessed by computer-assisted analysis of recorded phase contrast images. Addition of a polar aminocardenolide (AC), ASI-222, produced a dose-related increase in contractility with a concentration producing a 50% maximal response (RC/sub 50/) of 4 x 10/sup -8/M. For ouabain (OB) the RC/sub 50/ was 7 x 10/sup -7/M. Cellular Na/sup +/-pump (NaP) function was determined as digitalis-sensitive /sup 86/Rb/sup +/-uptake. Addition of AC and OB to these cells produced a dose-related decrease in /sup 86/Rb/sup +/-uptake; concentrations which produced a 50% inhibition (IC/sub 50/) of NaP function were of 6 x 10/sup -8/M and 1.2 x 10/sup -6/M for AC and OB, respectively. Their data indicates that in isolated dog heart cells AC is both a more potent inotropic agent and an inhibitor of NaP function by 15-20 fold than OB. The RC/sub 50/ and IC/sub 50/ for these processes correlate for each glycoside.

  17. 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+.

  18. 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

  19. 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.

  20. Potassium transport across rat alveolar epithelium: evidence for an apical Na+-K+ pump.

    PubMed

    Basset, G; Bouchonnet, F; Crone, C; Saumon, G

    1988-06-01

    1. Experiments were performed on rat lungs into which various solutions were instilled whilst the lungs were perfused with either whole blood or Ringer solution. Instillation of ion-free glucose solution led to a net flux of fluid and ions into the alveolar spaces. K+ ions entered faster than Na+ ions and reached a concentration about twice that in the perfusate. Ouabain in the perfusate (basolateral side) prevented the rise in alveolar K+ concentration above that in the perfusate, indicating a transcellular pathway. Ba2+ in the instillate (apical side) hindered the entry of K+ into alveoli, suggesting the presence of apical K+ channels. 2. When Ringer solution was instilled, K+ was continuously removed from the alveoli and the K+ concentration in the instillate remained constant or decreased slightly depending on the rate of fluid absorption. The net K+ efflux from alveoli to blood was 0.23 pmol/(cm2 s). When Ba2+ was added to the instillate the net K+ efflux increased to 0.36 pmol/(cm2 s). Apical ouabain reversed the K+ flux resulting in a net K+ flux of 0.19 pmol/(cm2 s) into the alveoli. This suggests the presence of an Na+-K+-ATPase located in the apical membrane of some alveolar cells. 3. The K+ transport from instillate (Ringer solution) to perfusate was traced by means of 86Rb which was added to the instillate. Ouabain in the instillate did not affect fluid absorption but reduced the apparent 86Rb permeability by 50% although the paracellular permeability (estimated with [3H]mannitol) was unaffected. This also indicates the presence of an apical Na+-K+-ATPase. When ouabain was added to the perfusate, the apparent 86Rb permeability doubled. These findings indicate that recirculation of 86Rb (and K+) occurs due to the activity of both apical and basolateral Na+-K+-ATPases. 4. When ouabain was placed on both sides of the epithelium, preventing transcellular transport, the passive 86Rb permeability was 10.3 x 10(-8) cm/s (assuming an alveolar surface area of

  1. Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na/sup +/ and Ca/sup 2 +/ fluxes, Na/sup +//K/sup +/ pump activity, and intracellular pH

    SciTech Connect

    Mendoza, S.A.; Schneider, J.A.; Lopez-Rivas, A.; Sinnett-Smith, J.W.; Rozengurt, E.

    1986-06-01

    The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive /sup 86/Rb/sup +/ uptake (a measure of Na/sup +//K/sup +/ pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na/sup +/ entry into the cells. The effect of bombesin on Na/sup +/ entry and Na/sup +//K/sup +/ pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive /sup 86/Rb/sup +/ uptake; the relative potencies of these peptides in stimulating the Na/sup +//K/sup +/ pump were comparable to their potencies in increasing DNA synthesis. Bombesin increased Na/sup +/ influx, at least in part, through an Na/sup +//H/sup +/ antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na/sup +/. In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of /sup 45/Ca/sup 2 +/ from quiescent Swiss 3T3 cells. This Ca/sup 2 +/ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca/sup 2 +/. The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity, greatly decreased the stimulation of /sup 86/Rb/sup +/ uptake and Na/sup +/ entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism.

  2. Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing.

    PubMed

    Bloch, E F; Schmetz, M A; Foulds, J; Hammer, C H; Frank, M M; Joiner, K A

    1987-02-01

    We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels. PMID:3100618

  3. Evaluation of blood-brain barrier permeability changes in rhesus monkeys and man using 82Rb and positron emission tomography

    SciTech Connect

    Yen, C.K.; Budinger, T.F.

    1981-12-01

    Dynamic positron tomography of the brain with /sup 82/Rb, obtained from a portable generator (/sup 82/Sr (25 days) - /sup 82/Rb (76 sec)), provides a means of studying blood-brain barrier (BBB) permeability in physiological and clinical investigations. The BBB in rhesus monkeys was opened unilaterally be intracarotid infusion of 3 M urea. This osmotic barrier opening allowed entry into the brain of intravenously administered rubidium chloride. The BBB opening was demonstrated noninvasively using /sup 82/Rb and positron emission tomography and corroborated by the accumulation of /sup 86/Rb in tissue samples. Positron emission tomography studies can be repeated every 5 min and indicate that dynamic tomography or static imaging can be used to study BBB permeability changes induced by a wide variety of noxious stimuli. Brain tumors in human subjects are readily detected because of the usual BBB permeability disruption in and around the tumors.

  4. Evaluation of blood--brain barrier permeability changes in rhesus monkeys and man using /sup 82/Rb and positron emission tomography

    SciTech Connect

    Yen, C.K.; Budinger, T.F.

    1981-12-01

    Dynamic positron tomography of the brain with /sup 82/Rb, obtained from a portable generator (/sup 82/Sr (25 days) -- /sup 82/Rb (76 sec)), provides a means of studying blood-brain barrier (BBB) permeability in physiological and clinical investigations. The BBB in rhesus monkeys was opened unilaterally by intracarotid infusion of 3 M urea. This osmotic barrier opening allowed entry into the brain of intravenously administered rubidium chloride. The BBB opening was demonstrated noninvasively using /sup 82/Rb and positron emission tomography and corroborated by the accumulation of /sup 86/Rb in tissue samples. Positron emission tomography studies can be repeated every 5 min and indicate that dynamic tomography or static imaging can be used to study BBB permeability changes induced by a wide variety of noxious stimuli. Brain tumors in human subjects are readily detected because of the usual BBB permeability disruption in and around the tumors.

  5. Na and K Dependence of the Na/K Pump in Cystic Fibrosis Fibroblasts

    NASA Astrophysics Data System (ADS)

    Reznik, Vivian M.; Schneider, Jerry A.; Mendoza, Stanley A.

    1981-11-01

    The Na and K dependence of the Na/K pump was measured in skin fibroblasts from patients with cystic fibrosis and age/sex-matched controls. Under basal conditions, there was no difference between control and cystic fibrosis cells in protein per cell, intracellular Na and K content, or Na/K pump activity (measured as ouabain-sensitive 86Rb uptake). There was no difference in the Na dependence of the Na/K pump between cystic fibrosis cells and control cells. In cells from patients with cystic fibrosis, the Na/K pump had a significantly lower affinity for K (Km = 1.6 mM) when compared to normals (Km = 0.9 mM). This difference was demonstrated by using two independent experimental designs.

  6. Studies on the Mode of Action of Acifluorfen-Methyl in Nonchlorophyllous Soybean Cells 1

    PubMed Central

    Matringe, M.; Scalla, R.

    1988-01-01

    Phytotoxic effects of the herbicide acifluorfen-methyl on nonchlorophyllous soybean cells were estimated by 86Rb leakage. An action spectrum study showed maximum injury at 350 to 450 nanometers, with lesser activity between 450 and 700 nanometers. Cells treated in the dark with acifluorfen-methyl accumulated fluorescent pigments with the spectral characteristics of protoporphyrin IX. The action spectrum of acifluorfen-methyl matched the absorption spectrum of this tetrapyrrole, and the extent of cellular damage in the light was related to the degree of fluorescent pigment accumulation. We propose that the phytotoxicity of diphenyl ether herbicides could be explained by their ability to cause abnormal accumulations of tetrapyrroles, which in turn induce lethal photooxidative reactions. PMID:16665956

  7. Studies on the Mechanism of Action of Dinitramine

    PubMed Central

    Travis, Robert L.; Woods, William G.

    1977-01-01

    The effect of dinitramine, a selective herbicide, on the plasma membrane of the soybean (Glycine max L.) root was studied. Used as marker systems to observe the herbicide effect were two plasma-membrane-specific enzymes, pH 6.5 ATPase and glucan synthetase. The activity of pH 6.5 ATPase decreased significantly in membrane vesicles prepared from roots harvested 15 minutes after treatment with dinitramine. Maximum inhibition occurred in roots harvested 2 hours after treatment. Glucan synthetase activity decreased similarly within 2 hours of treatment. Membrane permeability to 86Rb was rapidly increased by dinitramine. The activity of pH 6.5 ATPase returned to the control level within 8 hours of treatment with dinitramine. These results show dinitramine's initial site of action to be the plasma membrane, producing an over-all reduction in membrane function through inactivation of membrane-associated proteins. PMID:16660043

  8. 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.

  9. Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume

    SciTech Connect

    Halperin, J.A.; Martin, A.M.; Malave, S.

    1985-08-12

    The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described. Human CSF samples, drawn before and 30 minutes after the intravenous infusion of 1 liter of either saline or glucose solutions, were assayed for digitalis-like activity by inhibition of either the /sup 86/Rb/sup +/ uptake into human erythrocytes or by the activity of a purified Na/sup +/-K/sup +/ ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading. 31 references, 2 figures, 1 table.

  10. Partial characterization of endogenous digoxinlike substance in human urine

    SciTech Connect

    Vinge, E.; Ekman, R.

    1988-01-01

    Urinary samples were collected from individuals not taking cardiac glycosides. Aliquots of 30 ml were passed through preparative octadecylsilane-bonded phase columns and eluted in fractions by stepwise increasing concentrations of acetonitrile. Eluted fractions were analysed for their contents of endogenous digoxinlike substance (EDLS) by radioimmunoassay of digoxin and by a bioassay of cardiac glycosides, which measures the uptake of rubidium (/sup 86/Rb) by erythrocytes as an index of Na+, K+-ATPase activity. In both assays, digoxinlike activity was found in several fractions, but the highest values were consistently measured in the fractions eluted with 40% acetonitrile. Greater amounts of EDLS were recovered from the urine of pregnant women than from the urine of men and nonpregnant women.

  11. Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae

    PubMed Central

    D’Avanzo, Nazzareno; Cheng, Wayland W.L.; Xia, Xiaobing; Dong, Liang; Savitsky, Pavel; Nichols, Colin G.; Doyle, Declan A.

    2011-01-01

    The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter’s, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, 86Rb+ flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels. PMID:20064617

  12. Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron

    SciTech Connect

    Cheval, L.; Doucet, A. )

    1990-07-01

    To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

  13. 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

  14. 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

  15. D-glucose Stimulates the Na+/K+ Pump in Mouse Pancreatic Islet Cells

    PubMed Central

    Elmi, Adrian; Idahl, Lars-ÅKe; Sandström, Per-Erik

    2000-01-01

    To determine the effect of D-glucose on the β-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Umeå-ob/ob mice in the absence or presence of lmM ouabain. D-glucose (20 mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Umeå-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3–20 mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135 mM Na+ and 20 mM K+). Neither D-glucose (3–20 mM) nor L-glucose or 3-O-methyl-D-glucose (20 mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7mM K+). Diphenylhydantoin (150 μM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 μM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of β-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity. PMID:11469399

  16. 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.

  17. Influence of amphotericin B on the sodium pump of porcine lens epithelium.

    PubMed

    Delamere, N A; Dean, W L; Stidam, J M; Moseley, A E

    1996-02-01

    Active transport by Na(+)-K(+)-ATPase in the monolayer of lens epithelium is vital for the regulation of sodium and potassium levels within the mass of fiber cells that make up the bulk of the lens. In this study, experiments were conducted using porcine lenses to test whether Na(+)-K(+)-ATPase activity in the epithelium is altered when the permeability of lens cell plasma membranes is increased by the ionophore amphotericin B. After 24 h, sodium was significantly (P < 0.01) elevated in lenses exposed to 5 or 10 microM amphotericin B. Amphotericin B stimulated 86Rb uptake, probably through an increase of cytoplasmic sodium concentration due to increased inward sodium leak; the rate of ouabain-sensitive potassium (86Rb) uptake by intact lenses was significantly increased by amphotericin B at 5 microM (P < 0.05) and 10 microM (P < 0.01). After 24 h, the epithelium from lenses exposed to amphotericin B had an Na(+)-K(+)-ATPase activity that was more than twofold higher (P < 0.01) than the Na(+)-K(+)-ATPase activity in control lenses. By immunoblot, there was an increase in Na(+)-K(+)-ATPase catalytic (alpha) subunit immunoreactive polypeptide in the epithelium of lenses exposed to amphotericin B. The increase stemmed from a marked increase of Na(+)-K(+)-ATPase alpha 2-immunoreactive polypeptide but little change in the amount of alpha 1-immunoreactive protein. As judged by immunoblot experiments, the amount of Na(+)-K(+)-ATPase beta 1-immunoreactive polypeptide also appeared to be higher in the epithelium of amphotericin B-treated lenses compared with control lenses. In summary, these results suggest that in response to a permeability challenge with amphotericin B, the porcine lens epithelium is able to increase the activity of Na(+)-K(+)-ATPase. The same permeability challenge also appears to stimulate the biosynthesis of Na(+)-K(+)-ATPase catalytic subunit as well as glycoprotein subunit polypeptides. PMID:8779908

  18. Red blood cells do not contribute to removal of K+ released from exhaustively working forearm muscle.

    PubMed

    Maassen, N; Foerster, M; Mairbäurl, H

    1998-07-01

    K+ released from exercising muscle via K+ channels needs to be removed from the interstitium into the blood to maintain high muscle cell membrane potential and allow normal muscle contractility. Uptake by red blood cells has been discussed as one mechanism that would also serve to regulate red blood cell volume, which was found to be constant despite increased plasma osmolality and K+ concentration ([K+pl]). We evaluated exercise-related changes in [K+pl], pH, osmolality, mean cellular Hb concentration, cell water, and red blood cell K+ concentration during exhaustive handgrip exercise. Unidirectional 86Rb+ (K+) uptake by red blood cells was measured in media with elevated extracellular K+, osmolarity, and catecholamines to simulate particularly those exercise-related changes in plasma composition that are known to stimulate K+ uptake. During exercise [K+pl] increased from 4.4 +/- 0.7 to 7.1 +/- 0.5 mmol/l plasma water and red blood cell K+ concentration increased from 137.2 +/- 6.0 to 144.6 +/- 4.6 mmol/l cell water (P 86Rb+ uptake by red blood cells was increased by approximately 20% on stimulation, caused by activation of the Na+-K+ pump and Na+-K+-2Cl- cotransport. Results indicate the K+ content of red blood cells did not change as cells passed the exhaustively exercising forearm muscle despite the elevated [K+pl]. The tendency for an increase in intracellular K+ concentration was due to a slight, although statistically not significant, decrease in red blood cell volume. K+ uptake, although elevated, was too small to move significant amounts of K+ into red blood cells. Our results suggest that red blood cells do not contribute to the removal of K+ released from muscle and do not regulate their volume by K+ uptake during exhaustive forearm exercise. PMID:9655793

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Vascular physiology and protein disposition in a preclinical model of neurodegeneration.

    PubMed

    Boswell, C Andrew; Mundo, Eduardo E; Johnstone, Bernadette; Ulufatu, Sheila; Schweiger, Michelle G; Bumbaca, Daniela; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A

    2013-05-01

    The development of clinically relevant preclinical models that mimic the hallmarks of neurodegenerative disease is an ongoing pursuit in early drug development. In particular, robust physiological characterization of central nervous system (CNS) disease models is necessary to predict drug delivery to target tissues and to correctly interpret pharmacodynamic responses to disease-modifying therapeutic candidates. Efficient drug delivery across the blood-CNS barrier is a particularly daunting task, prompting our strategy to evaluate the biodistribution of five distinct molecular probes in a well-characterized mouse model of neurodegeneration. A transgenic mouse model of amyotrophic lateral sclerosis was selected based on a phenotype resembling clinical symptoms, including loss of motor neurons from the spinal cord and paralysis in one or more limbs, due to expression of a G93A mutant form of human superoxide dismutase (SOD1). The tissue distributions of two proteins, albumin and a representative immunoglobulin G antibody, as well as two blood flow markers, the lipophilic blood flow marker Ceretec (i.e., (99m)Tc-HMPAO) and the polar ionic tracer, rubidium-86 chloride ((86)RbCl), were measured following intravenous injection in SOD1(G93A) and age-matched control mice. The radiopharmaceutical TechneScan PYP was also used to measure the distribution of (99m)Tc-labeled red blood cells as a blood pool marker. Both the antibody and (86)Rb were able to cross the blood-spinal cord barrier in SOD1(G93A) mice to a greater extent than in control mice. Although the biodistribution patterns of antibody, albumin, and RBCs were largely similar, notable differences were detected in muscle and skin. Moreover, vastly different biodistribution patterns were observed for a lipophilic and polar perfusion agent, with SOD1(G93A) mutation resulting in reduced renal filtration rates for the former but not the latter. Overall, the multiprobe strategy provided an opportunity to efficiently

  7. CHX14 is a plasma membrane K-efflux transporter that regulates K(+) redistribution in Arabidopsis thaliana.

    PubMed

    Zhao, Jian; Li, Penghui; Motes, Christy M; Park, Sunghun; Hirschi, Kendal D

    2015-11-01

    Potassium (K(+) ) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. CHX14 expression was induced by elevated K(+) and histochemical analysis of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K(+) stress as compared with wild-type seedlings. Roots of mutant seedlings displayed higher K(+) uptake rates than wild-type roots. CHX14 expression in yeast cells deficient in K(+) uptake renders the mutant cells more sensitive to deficiencies of K(+) in the medium. CHX14 mediates K(+) efflux in yeast cells loaded with high K(+) . Uptake experiments using (86) Rb(+) as a tracer for K(+) with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low-affinity K(+) efflux. Functional green fluorescent protein (GFP)-tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K(+) efflux transporter involved in K(+) homeostasis and K(+) recirculation. PMID:25754420

  8. 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.

  9. Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations.

    PubMed

    Rottenberg, H

    1984-01-01

    The uptake and binding of the lipophilic cations ethidium+, tetraphenylphosphonium+ (TPP+), triphenylmethylphosphonium+ (TPMP+), and tetraphenylarsonium+ (TPA+) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K'o) and the internal matrix volume and matrix face of the inner membrane (K'i) were determined and were utilized to estimate the membrane potential delta psi from the cation accumulation factor Rc according to the relation delta psi = RT/ZF ln [(RcVo - K'o)/(Vi + K'i)] where Vo and Vi are the volume of the external medium and the mitochondrial matrix, respectively, and Rc is the ratio of the cation content of the mitochondria and the medium. The values of delta psi estimated from this equation are in remarkably good agreement with those estimated from the distribution of 86Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations. PMID:6492133

  10. Diffusion and isotope effects in the diffusion of homovalent cations in cesium iodide

    NASA Astrophysics Data System (ADS)

    Klotsman, S. M.; Polikarpova, I. P.; Tatarinova, G. N.; Timofeev, A. N.

    1988-10-01

    Preexponentials D0 and activation energies Q for self-diffusion and diffusion of homovalent cations of small radius in single crystals of CsI measured by the tracer sectioning method are as follows: for 137Cs, 19+/-1.5 cm2 s-1 and 35.59+/-0.18 kcal/mol; for 86Rb, 30.7+/-3.0 cm2 s-1 and 35.13+/-0.22 kcal/mol; for 42K, 47+/-6 cm2 s-1 and 35.30+/-0.30 kcal/mol; for 22Na, 34.94+/-0.20 kcal/mol, respectively. The temperature dependence of the isotope effect for diffusion of 22,24Na in CsI is described by a simple exponent: E=126 exp[-(13.3+/-1.5 kcal/mol)/RT]. Calculations based on these experimental results and the ``five-frequency model'' of diffusion are provided to obtain the main diffusion parameters for all homovalent cations in CsI. The diffusion behavior of these cations in CsI is in good agreement with the model of ``displaced homovalent cations of small radius.''

  11. Interactions of the neurotoxin apamin with a Ca2+-activated K+ channel in primary neuronal cultures.

    PubMed

    Seagar, M J; Granier, C; Couraud, F

    1984-02-10

    Mono[125I]iodoapamin bound to specific sites on cultured rat embryonic neurons. The dissociation constant for the receptor-neurotoxin complex measured at equilibrium was 60-120 pM at pH 7.2 and 4 degrees C, with a maximal binding capacity of 3-8 fmol/mg of cell protein. Apamin inhibited calcium ionophore-induced 86Rb+ release from cell cultures. The dose effect curve for this pharmacological test corresponded closely to the displacement of 125I-apamin by native apamin in binding experiments. Formation of the 125I-apamin receptor complex requires exogenous K+. Reduced binding in the absence of K+ was due to diminished binding capacity rather than a lower affinity. The apamin receptor seems to be associated with a cell surface K+ site which shows 50% occupancy at 1.6 mM, and which could be involved in the regulation of channel activity. Apamin sites were present at the earliest developmental stage tested and their number did not evolve during 8 days in culture. In the same period, however, alpha-scorpion toxin binding increased by a factor of 10. The ontogenesis of Ca2+-activated K+ channels does not seem to occur in parallel with that of voltage-sensitive Na+ channels. PMID:6319399

  12. 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

  13. 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.

  14. CD63 Interacts with the Carboxy-Terminus of the Colonic H+,K+-ATPase to Increase Plasma Membrane Localization and Rb+-Uptake

    PubMed Central

    Codina, Juan; Li, Jian; DuBose, Thomas D.

    2005-01-01

    The carboxy-terminus of the colonic H+,K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the carboxy-terminus of the α-subunit of mouse colonic H+,K+-ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize interaction of CT-HKα2 with CD63, recombinant CT-HKα2 and CD63 were synthesized in vitro, incubated, and complexes immunoprecipitated. CT-HKα 2 protein (but not CT-HKα1) co-precipitated with CD63, confirming stable assembly of HKα2 with CD63. In HEK-293 transfected with HKα2 plus β1-Na+,K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1 and 86Rb+-uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2/NKβ1 complex in the cell membrane. PMID:15647390

  15. Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

    SciTech Connect

    Palma, A.L.; Wang, H.H. )

    1991-06-01

    Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclidine, 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits 86Rb flux (IC50 = 6.6 microM), and (3H)phencyclidine binding to both resting and desensitized acetylcholine receptor (IC50 = 17 microM and 0.22 microM, respectively). From an indirect Hill plot of the inhibition of (3H)phencyclidine binding by PPT, a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in alpha-bungarotoxin-treated preparations. Taken together, these results indicate that PPT mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2-3 units.

  16. Independent yields from the photofission of sup 232 Th, and the Z sub p and statistical-dynamic models

    SciTech Connect

    Smith, J.R.; Richardson, A.E. )

    1991-09-01

    Independent fission yields were measured for {sup 82}Br, {sup 96}Nb, {sup 124}Sb, and {sup 126}Sb produced by photofission of {sup 232}Th with 27-MeV peak bremsstrahlung and for {sup 136}Cs at 11, 15, and 27 MeV. Upper limits for the independent yields for {sup 86}Rb and {sup 134}Cs and mass yields for mass chains 125 and 127 were also measured for {sup 232}Th photofission at 27 MeV. Various extensions of the {ital Z}{sub {ital p}} charge-distribution model were found to give generally good agreement with experimental measurements in the asymmetric mass regions, but less satisfactory agreement in the symmetric region. A statistical charge-distribution model incorporating post-fission dynamics correlated well with experimental values in both symmetric and asymmetric regions. The statistical-dynamic model naturally predicted pairing and shell effects which were in good agreement with experimentally observed effects. One important outcome of the statistical-dynamic model calculations was the production of a linear shape on the wings of the charge-distribution curve when proximity proton transfer after scission was incorporated into the model. Such linear shapes have previously been experimentally observed without explanation.

  17. 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.

  18. Regulation of the Potassium to Sodium Ratio and of the Osmotic Potential in Relation to Salt Tolerance in Yeasts

    PubMed Central

    Norkrans, Birgitta; Kylin, Anders

    1969-01-01

    By using the isotope pairs 22Na-24Na and 42K-86Rb, the uptake and retention of Na and K was studied in the salt-tolerant Debaryomyces hansenii and in the less tolerant Saccharomyces cerevisiae at NaCl levels of 4 mm and 0.68, 1.35, and 2.7 m in the medium. The ratio of K to Na is much higher in the cells than in the media, and higher in D. hansenii than in S. cerevisiae under comparable conditions. The difference between the two species is due to a better Na extrusion and a better uptake of K in D. hansenii. The kinetics of ion transport show that at about the time when extrusion of Na could be demonstrated in D. hansenii, K-Rb previously lost to an easily washable compartment of the cells was reabsorbed in both organisms. More H+ was given off from S. cerevisiae than from D. hansenii in the course of these events. The findings fit the working hypothesis tested, which regards salt tolerance as partly dependent on the ability to mobilize energy to extrude Na from the cells and to take up K. The volume changes in S. cerevisiae are greater and are more slowly overcome than those in D. hansenii. The total salt level of the cells is not sufficient to counteract the osmotic potential of the medium, so that additional osmoregulatory mechanisms must be involved in determining halotolerance. PMID:5354950

  19. Membrane Properties of Isolated Winter Wheat Cells in Relation to Icing Stress 1

    PubMed Central

    Pomeroy, M. Keith; Pihakaski, Seppo J.; Andrews, Chris J.

    1983-01-01

    Isolated cell preparations of winter wheat (Triticum aestivum L.) were utilized to examine the effect of ice encasement at −1°C and exposure to ethanol on metabolic and biochemical properties of cells. Following icing and ethanol treatments, passive efflux of amino acids increased gradually with duration of exposure to the stress, and closely paralleled the decline in viability of cells. In contrast, uptake of 86Rb declined much more rapidly than viability following exposure to icing or ethanol. Electron spin resonance spectroscopy studies revealed no significant change in molecular ordering within the cell membranes following icing or exposure to ethanol, whereas a small but significant increase in order was detected in the noniced controls. O2 consumption by isolated cells declined only gradually due to icing and ethanol treatments, and remained relatively high even when cell viability was severely reduced. These results indicate that the plasma membrane is a primary site of injury during ice encasement and that damage to the ion transport system is the earliest manifestation of this injury. PMID:16663038

  20. Interactions among Flooding, Freezing, and Ice Encasement in Winter Wheat 1

    PubMed Central

    Gao, Ji-Yin; Andrews, Chris J.; Pomeroy, M. Keith

    1983-01-01

    Exposure of winter wheat (Triticum aestivum L.) to various combinations of flooding and freezing stresses induces much greater damage than the individual stresses. Cold-hardened plants flooded for 1 week or exposed to −6°C for 1 week show 100% survival, while survival of plants exposed to both stresses simultaneously is reduced by 20 to 30%, and cold hardiness decreases by several degrees. The level of nonstructural carbohydrates increases in crown tissue during cold acclimation, but decreases when the plants are exposed to flooding or to −6°C for 1 week. The respiratory capacity of crown tissue segments declines when the plants are stressed. Uptake of 86Rb by the roots of intact seedlings declines after exposure to either freezing or flooding, whereas passive efflux of amino acids is observed after freezing but not following flooding. This study has shown that detectable stress-induced metabolic changes occur in winter wheat before the applied stress is severe enough to reduce survival. PMID:16662997

  1. Development of NIRS models to predict protein and amylose content of brown rice and proximate compositions of rice bran.

    PubMed

    Bagchi, Torit Baran; Sharma, Srigopal; Chattopadhyay, Krishnendu

    2016-01-15

    With the escalating persuasion of economic and nutritional importance of rice grain protein and nutritional components of rice bran (RB), NIRS can be an effective tool for high throughput screening in rice breeding programme. Optimization of NIRS is prerequisite for accurate prediction of grain quality parameters. In the present study, 173 brown rice (BR) and 86 RB samples with a wide range of values were used to compare the calibration models generated by different chemometrics for grain protein (GPC) and amylose content (AC) of BR and proximate compositions (protein, crude oil, moisture, ash and fiber content) of RB. Various modified partial least square (mPLSs) models corresponding with the best mathematical treatments were identified for all components. Another set of 29 genotypes derived from the breeding programme were employed for the external validation of these calibration models. High accuracy of all these calibration and prediction models was ensured through pair t-test and correlation regression analysis between reference and predicted values. PMID:26258697

  2. Cortical Air Spaces (Aerenchyma) in Roots of Corn Subjected to Oxygen Stress

    PubMed Central

    Drew, Malcolm C.; Chamel, André; Garrec, Jean-Paul; Fourcy, André

    1980-01-01

    When the seminal root system of 14-day-old corn (Zea mays cv. Dekalb 202) was subjected to O2 stress, nodal roots with well developed cortical air spaces (aerenchyma) grew into the deoxygenated solution. Microscopic examination showed that there was extensive breakdown of cells in the midcortex of these roots, while the stele, endodermis, and inner layer of cortical cells remained complete, as did the outer layers of the cortex and the epidermis. Occasional files of intact cells, and the wall residues of collapsed cells, crossed the space between inner and outer cortex. Experiments with short, intact root segments with and without air spaces showed that in the presence of O2 the ability to absorb and translocate 86Rb+, per unit volume or length of root, was little affected by cortical degeneration. The distribution across root sections of recently supplied strontium and rubidium, determined by electron microprobe analysis, indicated that in roots with air spaces the strands of wall residues bridging the cortex could be involved in maintaining the conduction of ions from the outer cortex up to the endodermis. Images PMID:16661224

  3. Mechanism for Selectivity-inactivation Coupling in KcsA Potassium Channels

    SciTech Connect

    W Cheng; J McCoy; A Thompson; C Nichols; C Nimigean

    2011-12-31

    Structures of the prokaryotic K{sup +} channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K{sup +} channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K{sup +} selectivity. In E71A channels, Na{sup +} permeates at higher rates as seen with {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux measurements and analysis of intracellular Na{sup +} block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the 'collapsed,' presumed inactivated, conformation in low K{sup +}, but a 'flipped' conformation, that is also observed in high K{sup +}, high Na{sup +}, and even Na{sup +} only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K{sup +} selectivity. We propose a molecular mechanism by which inactivation and K{sup +} selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.

  4. Depletion of intracellular polyamines relieves inward rectification of potassium channels.

    PubMed Central

    Shyng, S L; Sha, Q; Ferrigni, T; Lopatin, A N; Nichols, C G

    1996-01-01

    Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches. PMID:8876254

  5. 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.

  6. 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.

  7. [The pathophysiologic basis for the incorporation of 99mTc-pyrophosphate into myocardial infarct].

    PubMed

    Duska, F; Hadas, L; Volenec, K; Palicka, V; Mazurová, Y; Vizd'a, J; Kafka, P; Urbanová, E; Kuba, M; Veverková, O

    1989-08-01

    The investigation studied the relation of 99mTc-pyrophosphate incorporation (99mTc-PYP) into experimental, 48-hours old myocardial infarction in dogs to tissue vascular supply and to the extent of necrotic tissue. The experimental myocardial infarction was induced in five animals during an operation by the ligation RIVA. The myocardial blood supply was measured in tissue samples, taken from transverse sections of the infarction by means of 86Rb captation. The extent of myocytolysis was measured by depletion of tissue creatine kinase (CK). In the subendocardial layer of the infarction it became obvious that 99mTc-PYP incorporation was proportional to the decrease of tissue blood supply. No relation between the extent of necrosis and the incorporation of the radioactive chemical was demonstrated. The accumulation of 99mTc-PYP requires the necrosis to be present, but its extent apparently does not influence the intensity of incorporation. In the subepicardial infarction layer there was neither a relation of the radioactive chemical cumulation to the blood flow, nor to the extent of the necrosis proved. PMID:2551518

  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. 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.

  10. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    PubMed

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. PMID:26171817

  11. Photoinactivation of sodium-potassium-chloride cotransport in LLC-PK1/Cl 4 cells by bumetanide

    SciTech Connect

    Amsler, K.; Kinne, R.

    1986-05-01

    Rb+ uptake into LLC-PK1/Cl 4 cells can be subdivided into three components: 1) ouabain-sensitive uptake, 2) bumetanide-sensitive uptake, and 3) ouabain- and bumetanide-insensitive uptake. Exposure of cells to near-UV light in the presence of low concentrations of bumetanide produces a specific, irreversible inhibition of the bumetanide-sensitive uptake component, while not affecting the other two uptake components. Irreversible inhibition of bumetanide-sensitive transport is observed when measuring either cellular uptake or efflux and also when measuring /sup 86/Rb+ uptake into membrane vesicles. The irreversible inhibition is both concentration and time dependent and is blocked under conditions where the interaction of bumetanide with the Na+-K+-Cl- cotransporter is disturbed. We conclude that bumetanide, at low concentrations, can specifically and irreversibly inhibit the Na+-K+-Cl- cotransporter of LLC-PK1/Cl 4 cells. We suggest that this irreversible inhibition is the result of the photoactivation of an ether linkage in the bumetanide molecule, leading to a covalent binding of bumetanide to the Na+-K+-Cl- cotransporter.

  12. 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.

  13. Endogenous inotropic substance from heart tissue has digitalis-like properties

    SciTech Connect

    Khatter, J.C.; Agbanyo, M.; Navaratnam, S. )

    1991-01-01

    In the past few years, we developed an extraction procedure which we successfully used to isolate a crude fraction containing digitalis-like substance (DLS) from porcine left ventricular tissue. In this study, the crude fraction was found to cross-react with digoxin antibodies and showed immunoreactivity of 4.25 {plus minus} 0.6 ng digoxin equivalent/ml. On further purification of the crude fraction using silica gel G column chromatography, a fraction C was obtained, which was highly positive inotropic on canine trabeculae and it dose-dependently inhibited ouabain sensitive {sup 86}Rb{sup +} uptake in rate heart slices. A 50% inhibition of uptake was obtained by 25 ul of fraction C. Fraction C also inhibited canine kidney Na{sup +}, K{sup +}-ATPase dose-dependently and a 50% inhibition of this enzyme required 17 ul of fraction C. Ashing of the fraction C at 500{degree}C resulted in loss of inotropic and enzyme inhibitory activities, indicating an organic nature of the unknown digitalis-like substance.

  14. 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.

  15. Characteristics of a (Na/sup +/K/sup +/)-ATPase inhibitor in extracts of tea

    SciTech Connect

    Sagnella, G.A.; MacGregor, G.A.

    1984-07-01

    Extracts of tea were examined for inhibitors of the sodium-potassium pump by investigating the effect of the extracts on 1) isolated preparations of (Na/sup +/-K/sup +/)-ATPase from hog brain and human blood cells; 2) the displacement of radioactive ouabain from its specific receptor on red blood cells, and 3) the uptake of radioactive rubidium in intact red blood cells. It has been found that extracts of tea were potent inhibitors of the purified hog brain (Na/sup +/-K/sup +/)-ATPase. However, the inhibition was not specific for the (Na/sup +/-K/sup +/)-ATPase and the extract of tea did not displace /sup 3/H-ouabain in a specific ouabain-receptor assay. Additionally, the tea extracts displayed only a small inhibitory effect on the uptake of /sup 86/Rb in intact red blood cells. These observations suggest that the material is not like digitalis and that, unlike cardiac glycosides, it may inhibit the activity of the (Na/sup +/-K/sup +/)-ATPase by interacting with the enzyme at intracellular sites.

  16. 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.

  17. 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).

  18. Potassium metabolism in seawater teleosts: II. Evidence for active potassium extrusion across the gill.

    PubMed

    Sanders, M J; Kirschner, L B

    1983-05-01

    Unidirectional K-fluxes were estimated in unanaesthetized trout and sculpin and in anaesthetized sculpin from observed 86Rb movement (JK = 1.3 JRb). In all three groups efflux exceeded influx; Jo/Jin was 2-3. The values predicted by the flux ratio equation were 0.5 for trout and 1.0 for sculpin, so active K-extrusion is indicated. The results also show that more than one half of the total influx must be ingested with food rather than passing across the gills. Flux data show that the gills are more permeable to K+ than to Na+, PK/PNa was 5.4 in trout and 2.6 in sculpin. Changes in K-concentration in the external medium did not appear to affect efflux; there was no exchange component in the total fluxes. When both Na+ and K+ were omitted from the bathing solution, efflux decreased to about 15% of the normal seawater value. This is more than would be expected if the flux were purely diffusive and supports the conclusion that extrusion contains an active component. Repletion of the ion-deficient medium with K+ (alone) increased K-efflux. However, it also repolarized the gill and increased plasma [K+], and the flux change could be accounted for by the augmented driving force; i.e. it was diffusive. The additional plasma K came from the intracellular compartment, rather than an augmented influx from the medium. PMID:6875471

  19. 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.

  20. 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.

  1. SOS1, a Genetic Locus Essential for Salt Tolerance and Potassium Acquisition.

    PubMed Central

    Wu, S. J.; Ding, L.; Zhu, J. K.

    1996-01-01

    To begin to determine which genes are essential for salt tolerance in higher plants, we identified four salt-hypersensitive mutants of Arabidopsis by using a root-bending assay on NaCl-containing agar plates. These mutants (sos1-1, sos1-2, sos1-3, and sos1-4) are allelic to each other and were caused by single recessive nuclear mutations. The SOS1 gene was mapped to chromosome 2 at 29.5 [plusmn] 6.1 centimorgans. The mutants showed no phenotypic changes except that their growth was >20 times more sensitive to inhibition by NaCl. Salt hypersensitivity is a basic cellular trait exhibited by the mutants at all developmental stages. The sos1 mutants are specifically hypersensitive to Na+ and Li+. The mutants were unable to grow on media containing low levels (below ~1 mM) of potassium. Uptake experiments using 86Rb showed that sos1 mutants are defective in high-affinity potassium uptake. sos1 plants became deficient in potassium when treated with NaCl. The results demonstrate that potassium acquisition is a critical process for salt tolerance in glycophytic plants. PMID:12239394

  2. Atrial natriuretic peptide(31-67) inhibits Na+ transport in rabbit inner medullary collecting duct cells. Role of prostaglandin E2.

    PubMed Central

    Gunning, M E; Brady, H R; Otuechere, G; Brenner, B M; Zeidel, M L

    1992-01-01

    Atrial natriuretic peptide (ANP)(31-67), a portion of the atrial peptide prohormone, circulates in humans, and its plasma level varies with atrial pressure. Like the more widely studied carboxy-terminal fragment ANP(99-126), ANP(31-67) stimulates natriuresis and diuresis. We examined the mechanism of this natriuresis by measuring the effects of ANP(31-67) on Na+ transport in cells of the rabbit inner medullary collecting duct (IMCD). ANP(31-67) (10(-8) M) caused a 26 +/- 4% inhibition of oxygen consumption (QO2); half-maximal inhibition occurred at 10(-11) M, suggesting a physiologic effect. This effect was not additive with either ouabain or amiloride, suggesting that it reflected inhibition of Na+ transport-dependent QO2. ANP(31-67) reduced the amphotericin-induced stimulation of QO2 consistent with inhibition by this peptide of the Na(+)-K(+)-ATPase. In addition, ANP(31-67) reduced ouabain-sensitive 86Rb+ uptake under Vmax conditions. Several lines of evidence indicated that PGE2, a known endogenous IMCD Na(+)-K(+)-ATPase inhibitor, mediates pump inhibition by ANP(31-67). Thus, ANP(31-67) inhibits Na+ transport by inhibiting the Na(+)-K(+)-ATPase of IMCD cells, an effect mediated by the generation of PGE2. PMID:1533229

  3. Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells.

    PubMed Central

    Kemp, P J; Roberts, G C; Boyd, C A

    1994-01-01

    86Rb+ was used to study potassium uptake and efflux in type II pneumocytes freshly isolated from adult guinea-pig and rat lung. Both species exhibited a substantial ouabain-sensitive component of potassium influx. In rats, most of the ouabain-resistant influx was abolished by bumetanide and removal of extracellular chloride elicited no further effect. In contrast, only a proportion of the ouabain-insensitive uptake was inhibitable by bumetanide in guinea-pigs and this species showed an additional component of influx, which was chloride dependent and which was reduced by either the K(+)-H(+)-ATPase inhibitor, omeprazole, or by the stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). The chloride-dependent component was also apparent in efflux experiments in guinea-pigs, but was absent in rats. Ouabain-insensitive ATPase activity was assayed in highly purified apical membranes from guinea-pig type II pneumocytes. This activity was inhibitable by omeprazole (apparent inhibition constant, Ki, was approximately 40 microM), was potassium dependent (apparent activation constant, Ka, was approximately 200 microM) and was doubled by the addition of nigericin. While potassium transport in rat type II cells is adequately accounted for by Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, our data suggest the additional presence of K(+)-Cl- cotransport and K(+)-H(+)-ATPase in guinea-pig type II pneumocytes. A model of how alveolar subphase acidification may occur is proposed. PMID:8046636

  4. Rubidium-86 uptake by red blood cells of breast cancer patients

    SciTech Connect

    Telfer, N.; Lee, Y.T.N.; Merrill, Q.; Bauer, F.K.

    1981-02-01

    The red blood cell (RBC) uptake of rubidium-86 (Rb-86), a sensitive in vitro test of RBC membrane transport, was measured in 22 controls, 8 patients with benign breast lesions, and 30 stage I/II, 13 stage III/IV untreated breast cancers. Total Rb-86 transport was measured. Following ouabain block, passive transport was determined, allowing calculation of active Rb-86 uptake by difference. There was a significant decrease in total and passive uptake by RBCs from patients with breast cancer when compared to controls and those with benign lesions. The Rb-86 uptake returned towards normal in successfully treated stages-II to IV patients. There were no correlations with the patient's age, hematocrit, or incubation hematocrit with RBC RB-86 uptakes. Results from incubation in Krebs-Ringer solution were not consistent with a plasma blocking factor. The RBC potassium content was slightly increased in the cancer patients. This, along with decreased passive transport, eliminates the nonspecific malfunction of the Na-K-pump as an explanation, and suggests that the decreased uptake is a cancer-related phenomenon.

  5. Insulin regulation of Na/K pump activity in rat hepatoma cells

    SciTech Connect

    Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

    1984-05-01

    Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by /sup 3/H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of /sup 22/Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes.

  6. 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.

  7. Seizure sensitivity is ameliorated by targeted expression of K+-Cl- cotransporter function in the mushroom body of the Drosophila brain.

    PubMed

    Hekmat-Scafe, Daria S; Mercado, Adriana; Fajilan, Adriel A; Lee, Ann W; Hsu, Richard; Mount, David B; Tanouye, Mark A

    2010-01-01

    The kcc(DHS1) allele of kazachoc (kcc) was identified as a seizure-enhancer mutation exacerbating the bang-sensitive (BS) paralytic behavioral phenotypes of several seizure-sensitive Drosophila mutants. On their own, young kcc(DHS1) flies also display seizure-like behavior and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The product of kcc shows substantial homology to KCC2, the mammalian neuronal K(+)-Cl(-) cotransporter. The kcc(DHS1) allele is a hypomorph, and its seizure-like phenotype reflects reduced expression of the kcc gene. We report here that kcc functions as a K(+)-Cl(-) cotransporter when expressed heterologously in Xenopus laevis oocytes: under hypotonic conditions that induce oocyte swelling, oocytes that express Drosophila kcc display robust ion transport activity observed as a Cl(-)-dependent uptake of the K(+) congener (86)Rb(+). Ectopic, spatially restricted expression of a UAS-kcc(+) transgene was used to determine where cotransporter function is required in order to rescue the kcc(DHS1) BS paralytic phenotype. Interestingly, phenotypic rescue is largely accounted for by targeted, circumscribed expression in the mushroom bodies (MBs) and the ellipsoid body (EB) of the central complex. Intriguingly, we observed that MB induction of kcc(+) functioned as a general seizure suppressor in Drosophila. Drosophila MBs have generated considerable interest especially for their role as the neural substrate for olfactory learning and memory; they have not been previously implicated in seizure susceptibility. We show that kcc(DHS1) seizure sensitivity in MB neurons acts via a weakening of chemical synaptic inhibition by GABAergic transmission and suggest that this is due to disruption of intracellular Cl(-) gradients in MB neurons. PMID:19884312

  8. Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.

    PubMed

    Sheerin, H E; Snyder, L M; Fairbanks, G

    1989-07-24

    Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter. PMID:2758051

  9. WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters.

    PubMed

    Rinehart, Jesse; Vázquez, Norma; Kahle, Kristopher T; Hodson, Caleb A; Ring, Aaron M; Gulcicek, Erol E; Louvi, Angeliki; Bobadilla, Norma A; Gamba, Gerardo; Lifton, Richard P

    2011-08-26

    NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling. PMID:21733846

  10. Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti).

    PubMed

    Piermarini, Peter M; Hine, Rebecca M; Schepel, Matthew; Miyauchi, Jeremy; Beyenbach, Klaus W

    2011-11-01

    The K,Cl cotransporters (KCCs) of the SLC12 superfamily play critical roles in the regulation of cell volume, concentrations of intracellular Cl(-), and epithelial transport in vertebrate tissues. To date, the role(s) of KCCs in the renal functions of mosquitoes and other insects is less clear. In the present study, we sought molecular and functional evidence for the presence of a KCC in renal (Malpighian) tubules of the mosquito Aedes aegypti. Using RT-PCR on Aedes Malpighian tubules, we identified five alternatively spliced partial cDNAs that encode putative SLC12-like KCCs. The majority transcript is AeKCC1-A(1); its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl(-)-dependent uptake of (86)Rb(+) is 1) activated by 1 mM N-ethylmaleimide and cell swelling, 2) blocked by 100 μM dihydroindenyloxyalkanoic acid (DIOA), and 3) dependent upon N-glycosylation of AeKCC1-A. In Aedes Malpighian tubules, AeKCC1 immunoreactivity localizes to the apical brush border of principal cells, which are the predominant cell type in the epithelium. In vitro physiological assays of Malpighian tubules show that peritubular DIOA (10 μM): 1) significantly reduces both the control and diuretic rates of transepithelial fluid secretion and 2) has negligible effects on the membrane voltage and input resistance of principal cells. Taken together, the above observations indicate the presence of a KCC in the apical membrane of principal cells where it participates in a major electroneutral transport pathway for the transepithelial secretion of fluid in this highly electrogenic epithelium. PMID:21813871

  11. Effects of atrial and brain natriuretic peptides upon cyclic GMP levels, potassium transport, and receptor binding in rat astrocytes

    SciTech Connect

    Beaumont, K.; Tan, P.K. )

    1990-02-01

    The ability of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) to alter cyclic GMP levels and NaKCl cotransport in rat neocortical astrocytes was determined. At concentrations of 10(-9)-10(-6) M, rat ANP99-126 (rANF), rat ANP102-126 (auriculin B), and rat ANP103-126 (atriopeptin III) stimulated 6- to 100-fold increases in cyclic GMP levels. Porcine BNP (pBNP) and rat BNP (rBNP) were 20%-90% as effective as rANF over most of this concentration range, although 10(-6) M pBNP produced a greater effect than rANF. NaKCl cotransport as measured by bumetanide-sensitive 86Rb+ influx was not altered by exposure of astrocytes to 10(-6)M rANF, pBNP, or rBNP. Both pBNP and rBNP, as well as rat ANP103-123 (atriopeptin I) and des(gl18, ser19, gly20, leu21, gly22) ANF4-23-NH2 (C-ANF4-23) strongly competed for specific 125I-rANF binding sites in astrocyte membranes with affinities ranging from 0.03 to 0.4 nM, suggesting that virtually all binding sites measured at subnanomolar concentrations of 125I-rANF were of the ANP-C (ANF-R2) receptor subtype. These receptors are thought to serve a clearance function and may be linked to a guanylate cyclase activity that is chemically and pharmacologically distinct from that coupled to ANP-A (ANF-R1) receptors. ANP receptors on astrocytes may function in limiting the access of ANP and BNP to neurons involved in body fluid and cardiovascular regulation.

  12. Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na+/K+ ATPase on EDHF relaxations in the rat hepatic artery

    PubMed Central

    Andersson, David A; Zygmunt, Peter M; Movahed, Pouya; Andersson, Tomas L G; Högestätt, Edward D

    2000-01-01

    In the rat hepatic artery, the SKCa inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 μM) and d-tubocurarine (100 μM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM).The IKCa inhibitors clotrimazole (3 μM) and 2-chlorophenyl-bisphenyl-methanol (3 μM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 μM) had no effect in the presence of apamin.Ciclazindol (10 μM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated 86Rb+ influx via the Gardos channel (IKCa) in human erythrocytes.The Na+/K+ ATPase inhibitor ouabain (500 μM) and the KIR blocker Ba2+ (30 μM) neither alone nor in combination inhibited EDHF relaxations. Ba2+ was also without effect in the presence of either apamin or charybdotoxin.In contrast to EDHF, an increase in extracellular [K+] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K+-free physiological salt solution, re-admission of K+ always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba2+.The present study provides pharmacological evidence for the involvement of SKCa and IKCa in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K+ activating Na+/K+ ATPase and KIR in this blood vessel. PMID:10742306

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

    PubMed

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

    2015-08-01

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action. PMID:25964258

  14. 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

  15. Grapevine and Arabidopsis Cation-Chloride Cotransporters Localize to the Golgi and Trans-Golgi Network and Indirectly Influence Long-Distance Ion Transport and Plant Salt Tolerance.

    PubMed

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

    2015-11-01

    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 (36)Cl(-), (22)Na(+), and (86)Rb(+) 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

  16. Cellular effects of beta-adrenergic and of cAMP stimulation on potassium transport in rat alveolar epithelium.

    PubMed

    Saumon, G; Basset, G; Bouchonnet, F; Crone, C

    1989-07-01

    Alveolar fluid absorption is greatly enhanced by cAMP and by beta-adrenergic agonists via an increase in Na+ transport. Little is known about K+ homeostasis under these circumstances. We studied K+ transport across alveolar epithelium in isolated perfused rat lungs stimulated either by dibutyryl-cAMP or isoproterenol. K+ fluxes and the apparent permeability of 86Rb across the epithelium (alveoli to plasma) were interpreted according to a model involving two types of cells, B and L, distinguished by the location of Na+-K+-ATPases (basal and luminal). Water is considered to be absorbed by B cells in a solute-coupled process energized by a basolateral Na+-K+-ATPase that is stimulated by isoproterenol and cAMP. K+ transport out of the alveoli is due to the activity of a Na+-K+-ATPase located in the apical membrane of L cells. In the present study net transport rate of K+ was -0.5 +/- 0.15 nmol/s, n = 20 (out of alveoli) in control conditions. When the epithelium was stimulated by dibutyryl-cAMP (10(-4) mol/l) net absorption of K+ reversed to net 'secretion' into alveoli (3.2 +/- 0.31 nmol/s), fluid absorption was not stimulated. K+ 'secretion' was abolished by apical Ba2+, indicating it was due to opening of apical K+ channels. Basolateral ouabain reversed net K+ 'secretion' to net absorption indicating that K+ entry into alveoli was dependent on activity of B cell basolateral Na+-K+-ATPase (masking simultaneous K+ removal by apical L cell Na+-K+-pump). When larger concentrations of dibutyryl-cAMP (10(-3) mol/l) or when isoproterenol were used to stimulate the epithelium there was a tripling of fluid absorption.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2571117

  17. 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

  18. Cotransport of water by Na⁺-K⁺-2Cl⁻ cotransporters expressed in Xenopus oocytes: NKCC1 versus NKCC2.

    PubMed

    Zeuthen, Thomas; Macaulay, Nanna

    2012-03-01

    The NKCC1 and NKCC2 isoforms of the mammalian Na⁺–K⁺–2Cl⁻ cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic increases in external K⁺ concentration elicited abrupt inward water fluxes in NKCC1; the K⁺ dependence obeyed one-site kinetics with a K₀.₅ of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l⁻¹. A comparison between ion and water fluxes indicates that 460 water molecules are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes.Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the notion of interaction between ions and water in NKCC1 and allows for an estimate of around 600 water molecules transported per turnover of the protein. Osmotic gradients did not induce water transport in NKCC2. We conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfils its role in the kidney of transporting ions but not water. The different behaviour of NKCC1 and NKCC2 is discussed on the basis of recent molecular models based on studies of structural and molecular dynamics. PMID:22250214

  19. 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.

  20. Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes.

    PubMed

    Orlov, S N; Pokudin, N I; Kotelevtsev, Y V; Gulak, P V

    1989-02-01

    Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na+/H+ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of 22Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited 22Na influx and a two-fold increase of furosemide-inhibited 86Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of 45Ca influx, which is completely blocked by amiloride. The dependence of volume-induced 22Na influx in rat erythrocytes and 45Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of 45Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Cao2+/(Nai+, Ki+) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the 32P incorporation in diphosphoinositides. The effect of shrinkage on the 32P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements. PMID:2541247

  1. Membrane changes induced by exposure of Escherichia coli to human serum.

    PubMed Central

    Kroll, H P; Bhakdi, S; Taylor, P W

    1983-01-01

    The effect of bactericidal concentrations of lysozyme-free human serum on parameters of membrane integrity has been studied in serum-susceptible and serum-resistant Escherichia coli strains. Serum treatment released all of the alkaline phosphatase from the periplasmic space of two rapidly serum-susceptible strains but did so at different rates. In contrast, no periplasmic enzyme was released from two serum-resistant strains or from one moderately susceptible smooth strain. Lysozyme-free serum and heat-inactivated serum released comparable amounts of 86Rb+ from preloaded cells at comparable rates, regardless of serum susceptibility. Serum decreased the rate of phospholipid biosynthesis in both serum-susceptible and serum-resistant strains. In susceptible but not in resistant strains, intracellular ATP pools were depleted after serum exposure. Outer membranes and cytoplasmic membranes were prepared from serum-treated E. coli, and assays for C3 and C5b-9(m) were performed. With rapidly susceptible strains, C3 deposition on the outer membrane without attachment of C5b-9(m) occurred during the short prekilling phase. Subsequent bacterial killing was accompanied by deposition of C5b-9(m), which was recovered with C3 exclusively in outer membrane fractions with increased density and by eventual total loss of recoverable cytoplasmic membranes. Minimal deposition of complement components, without accompanying cytoplasmic membrane loss, occurred with serum-resistant strains. Loss of recoverable cytoplasmic membrane was not due to the action of either serum or bacterial phospholipase A. The results raise the possibilities that C5b-9(m) primarily damages the outer membrane and that the bacteria themselves actively participate in the ensuing, as yet unclarified, metabolic reactions that finally lead to their death. Images PMID:6358036

  2. Transmembrane flux and receptor desensitization measured with membrane vesicles. Homogeneity of vesicles investigated by computer simulation.

    PubMed Central

    Cash, D J; Langer, R M; Subbarao, K; Bradbury, J R

    1988-01-01

    The use of membrane vesicles to make quantitative studies of transmembrane transport and exchange processes involves an assumption of homogeneity of the membrane vesicles. In studies of 86Rb+ exchange mediated by acetylcholine receptor from the electric organ of Electrophorus electricus and of 36Cl- exchange mediated by GABA receptor from rat brain, measurements of ion exchange and receptor desensitization precisely followed first order kinetics in support of this assumption. In other measurements a biphasic decay of receptor activity was seen. To elucidate the molecular properties of receptors from such measurements it is important to appreciate what the requirements of vesicle monodispersity are for meaningful results and what the effect of vesicle heterogeneity would be. The experiments were simulated with single vesicle populations with variable defined size distributions as well as with mixtures of different populations of vesicles. The properties of the receptors and their density in the membrane could be varied. Different receptors could be present on the same or different membrane vesicles. The simulated measurements were not very sensitive to size dispersity. A very broad size distribution of a single vesicle population was necessary to give rise to detectable deviations from first order kinetics or errors in the determined kinetic constants. Errors could become significant with mixtures of different vesicle populations, where the dispersity in initial ion exchange rate constant, proportional to the receptor concentration per internal volume, became large. In this case the apparent rate of receptor desensitization would diverge in opposite directions from the input value when measured by two different methods, suggesting an experimental test for such kinetic heterogeneity. A biphasic decrease of receptor activity could not be attributed to vesicle heterogeneity and must be due to desensitization processes with different rates. Significant errors would not

  3. Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase

    PubMed Central

    Wallace, Breanna K.; Foroutan, Shahin

    2011-01-01

    Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive 86Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O2), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK. PMID:21562306

  4. Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase.

    PubMed

    Wallace, Breanna K; Foroutan, Shahin; O'Donnell, Martha E

    2011-08-01

    Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK. PMID:21562306

  5. Adrenergic and histaminergic neural interactions in dog paws

    SciTech Connect

    Baker, C.H.; Davis, D.L.; Sutton, E.T.; Lindsey, B.G. )

    1988-09-01

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

  6. Mechanism of Action of the Diphenyl Ether Herbicide Acifluorfen-Methyl in Excised Cucumber (Cucumis sativus L.) Cotyledons 1

    PubMed Central

    Orr, Gregory L.; Hess, F. Dana

    1982-01-01

    Cucumber (Cucumis sativus L.) cotyledons were sensitive to the diphenyl ether herbicide acifluorfen-methyl (AFM); methyl 5-[2-chloro-4-(trifluoro-methyl)phenoxyl-2-nitrobenzoate. Injury was detected by monitoring the efflux of 86Rb+ from treated tissues after exposure to light (600 micro einsteins per meter2 per second; photosynthetically active radiation). AFM exhibited activity in green and etiolated tissues in the presence of both 1 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 1 micromolar 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), inhibitors of photosynthetic electron transport. Protection against injury could be obtained by pretreating the seedlings with a carotenoid biosynthesis inhibitor, 10 micromolar fluridone {1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4 (H)-pyridinone}. After a 4-hour dark pretreatment with 1 and 10 micromolar AFM, cotyledons were exposed to light (600 micro einsteins per meter2 per second; photosynthetically active radiation). Within 1 to 2 hours after light treatment, significant increases in the level of thiobarbituric acid-reacting materials could be detected. Electron microscopic observations of treated tissues revealed significant structural damage to the chloroplast envelope, tonoplast, and plasma membrane. Etiolated cucumber cotyledons treated with 1 micromolar AFM and exposed to light were less susceptible to injury when maintained in an O2-deficient atmosphere. Protection against injury could be obtained with 50 micromolar α-tocopherol. These results suggest AFM is activated in light by yellow plant pigments and then is involved in the initiation of a free radical chain reaction with polyunsaturated fatty acid moieties of phospholipid molecules making up cellular membranes. The perturbations that follow result in a loss of the membrane's selective permeability characteristics, thereby leading to cellular death. Images PMID:16662237

  7. Potassium Transport in Corn Roots 1

    PubMed Central

    Kochian, Leon V.; Lucas, William J.

    1982-01-01

    Influx isotherms were obtained for 86Rb+ uptake into 2-cm corn (Zea mays [A632 × (C3640 × Oh43)] root segments for both low- (0.2 millimolar CaSO4) and high-salt (0.2 millimolar CaSO4 + 5 millimolar KCl) grown roots. Unlike the discontinuous curves usually presented for K+ influx, our isotherms were smooth, nonsaturating curves that approached linearity at K+ (Rb+) concentrations above 1 millimolar. The kinetics for K+ transport could be resolved into saturable and linear components. The saturable components yielded Km values of 16 and 86 micromolar for low- and high-salt roots, respectively, while Vmax values were 5.62 and 1.85 moles per gram fresh weight per hour. Results of experiments with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS) indicated that the saturable and linear components were independent mechanisms of K+ transport. Short-term NEM exposures (30 seconds to 5 minutes) selectively inhibited the saturable system, but had little effect on the linear component. Increasing NEM exposures resulted in further inhibition and subsequent abolition of the saturable component; the linear component exhibited limited NEM sensitivity. PCMBS elicited the same general inhibitory trends, although it was less effective as a saturable component inhibitor. The effects of NEM and PCMBS on K+ efflux were also studied. Short NEM exposures had no effect on cytoplasmic efflux, while inhibiting vacuolar efflux significantly. From these data, it is unclear at which site(s) NEM is acting. A more complex response was obtained with PCMBS, where a monophasic efflux curve was observed. Analysis indicated that the vacuolar efflux was stimulated, while the cytoplasmic component was abolished. The nature of the linear component is discussed, and it is proposed that the mechanism may be more complex than simple facilitated diffusion. Images Fig. 1 PMID:16662752

  8. 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.

  9. Enhanced K(+) secretion in dextran sulfate-induced colitis reflects upregulation of large conductance apical K(+) channels (BK; Kcnma1).

    PubMed

    Kanthesh, Basalingappa M; Sandle, Geoffrey I; Rajendran, Vazhaikkurichi M

    2013-11-01

    Defective colonic Na(+) and Cl(-) absorption is a feature of active ulcerative colitis (UC), but little is known about changes in colonic K(+) transport. We therefore investigated colonic K(+) transport in a rat model of dextran sulfate-induced colitis. Colitis was induced in rat distal colon using 5% dextran sulfate sodium (DSS). Short-circuit current (Isc, indicating electrogenic ion transport) and (86)Rb (K(+) surrogate) fluxes were measured in colonic mucosa mounted in Ussing chambers under voltage-clamp conditions in the presence of mucosal orthovanadate (a P-type ATPase inhibitor). Serum aldosterone was measured by immunoassay. Control animals exhibited zero net K(+) flux. By contrast, DSS-treated animals exhibited active K(+) secretion, which was inhibited by 98, 76, and 22% by Ba(2+) (nonspecific K(+) channel blocker), iberiotoxin (IbTX; BK channel blocker), and TRAM-34 (IK channel blocker), respectively. Apical BK channel α-subunit mRNA abundance and protein expression, and serum aldosterone levels in DSS-treated animals, were enhanced 6-, 3-, and 6-fold respectively, compared with controls. Increasing intracellular Ca(2+) with carbachol (CCH), or intracellular cAMP with forskolin (FSK), stimulated both active Cl(-) secretion and active K(+) secretion in controls but had no or little effect in DSS-treated animals. In DSS-induced colitis, active K(+) secretion involves upregulation of apical BK channel expression, which may be aldosterone-dependent, whereas Cl(-) secretion is diminished. Since similar ion transport abnormalities occur in patients with UC, diarrhea in this disease may reflect increased colonic K(+) secretion (rather than increased Cl(-) secretion), as well as defective Na(+) and Cl(-) absorption. PMID:23986198

  10. 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.

  11. Binding of Sulfonylurea by AtMRP5, an Arabidopsis Multidrug Resistance-Related Protein That Functions in Salt Tolerance1

    PubMed Central

    Lee, Eun Kyung; Kwon, Minjae; Ko, Jae-Heung; Yi, Hochul; Hwang, Moo Gak; Chang, Soochul; Cho, Myeon Haeng

    2004-01-01

    Recently, a new member of the ABC transporter superfamily of Arabidopsis, AtMRP5, was identified and characterized. In the present work, we found that AtMRP5 can bind specifically to sulfonurea when it is expressed in HEK293 cells. We also present evidence for a new role of AtMRP5 in the salt stress response of Arabidopsis. We used reverse genetics to identify an Arabidopsis mutant (atmrp5-2) in which the AtMRP5 gene was disrupted by transferred DNA insertion. In root-bending assays using Murashige and Skoog medium supplemented with 100 mm NaCl, root growth of atmrp5-2 was substantially inhibited in contrast to the almost normal growth of wild-type seedlings. This hypersensitive response of the atmrp5-2 mutant was not observed during mannitol treatment. The root growth of the wild-type plant grown in Murashige and Skoog medium supplemented with the MRP inhibitor glibenclamide and NaCl was inhibited to a very similar extent as the root growth of atmrp5-2 grown in NaCl alone. The Na+-dependent reduction of root growth of the wild-type plant in the presence of glibenclamide was partially restored by diazoxide, a known K+ channel opener that reverses the inhibitory effects of sulfonylureas in animal cells. Moreover, the atmrp5-2 mutant was defective in 86Rb+ uptake. When seedlings were treated with 100 mm NaCl, atmrp5-2 seedlings accumulated less K+ and more Na+ than those of the wild type. These observations suggest that AtMRP5 is a putative sulfonylurea receptor that is involved in K+ homeostasis and, thus, also participates in the NaCl stress response. PMID:14684837

  12. 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.

  13. Potassium transmembrane fluxes in anoxic hepatocytes from goldfish (Carassius auratus L.).

    PubMed

    Mut, P N; Espelt, M V; Krumschnabel, G; Schwarzbaum, P J

    2006-01-01

    Despite the fact that anoxic goldfish hepatocytes can maintain the transmembrane gradients of Na(+), H(+) and Ca(2+), cyanide (CN) intoxication leads to a rapid breakdown of K(+) homeostasis. In this study, [(86)Rb(+)] K(+) fluxes across the plasma membrane of goldfish hepatocytes were studied in order to identify the possible causes of this imbalance. Four minutes of cyanide incubation induced an acute and stable 61% decrease of K(+) influx (mostly driven by Na,K-ATPase activity), whereas K(+) efflux increased by 24.3%, this imbalance yielding a net K(+) efflux of 0.279+/-0.024 nmol 10(-6) cells(-1) min(-1). This uncoupling was not observed when glycolytic ATP production was inhibited with iodoacetic acid. Although the CN-induced decrease of K(+) influx was fully reversible upon washout of the inhibitor, it could not be prevented by any of the following treatments: (1) addition of 2% bovine serum albumin, which binds extracellular fatty acids known to activate specific K(+) channels; (2) addition of ascorbate, which acts as a radical scavenger; (3) inclusion of 5 mM glucose as an extracellular carbon source; and (4) removal of medium oxygen (obtained by nitrogen bubbling). Regarding the elevation of K(+) efflux in the presence of CN, neither ATP-dependent K(+) channels nor the KCl cotransporter appeared to be activated, whereas BaCl(2), an inhibitor of voltage-gated K(+) channels, decreased K(+) efflux of CN-intoxicated cells to control levels. In summary, these results indicate that, in goldfish hepatocytes, the CN-induced K(+) imbalance results from acute Na,K-ATPase inhibition together with the activation of voltage-dependent K(+) channels, the latter probably resulting from transient membrane depolarization. PMID:16298170

  14. Effects of verapamil and alcohol on blood flow, melphalan uptake and cytotoxicity, in murine fibrosarcomas and human melanoma xenografts.

    PubMed

    Robinson, B A; Clutterbuck, R D; Millar, J L; McElwain, T J

    1986-05-01

    Verapamil had previously been shown to increase cellular melphalan uptake and cytotoxicity in fibrosarcomas, and increased the area under the blood concentration versus time curve (AUC) for melphalan in CBA mice. Verapamil (10 mg kg-1 i.p.) had no effect on the fractional distribution of cardiac output (FDCO), measured with 86Rb-rubidium chloride, to subcutaneous fibrosarcomas. 14C-Melphalan uptake by FS13 fibrosarcomas was increased 60 min after verapamil (10 mg kg-1 i.p.), but not after lower doses which did not affect the AUC. Flunarizine (5 mg kg-1 i.p.) also had no effect on FDCO to FS13 fibrosarcomas, and tended to increase 14C-melphalan content of blood and the fibrosarcomas and to promote growth delay by melphalan. Alcohol increased FDCO to FS13 fibrosarcomas, maximally at a 1:20 dilution in saline, but had no effect on 14C-melphalan uptake or growth delay. Thus, melphalan cytotoxicity correlated with tumour melphalan uptake, and both followed changes in the AUC for melphalan but not changes in FDCO. In these murine fibrosarcomas melphalan uptake and cytotoxicity were not limited by blood flow. In subcutaneous human melanoma HX46 xenografts, verapamil had no effect on the FDCO, nor on 14C-melphalan uptake, and did not affect blood 14C-melphalan levels, suggesting absence of effects on the AUC and on cellular uptake. Alcohol did not increase the FDCO to HX46 xenografts, providing evidence for a different vascular supply. PMID:3718818

  15. De Novo Synthesis of Plasma Membrane and Tonoplast Polypeptides of Barley Roots during Short-Term K+ Deprivation 1

    PubMed Central

    Fernando, Mala; Mehroke, Jarnail; Glass, Anthony D. M.

    1992-01-01

    [35S]Methionine labeling of intact barley roots (Hordeum vulgare cv Klondike) after short (6-12 h) and longer (18-24 and 90-96 h) periods of K+ deprivation revealed that several membrane polypeptides were synthesized in significantly increased amounts following withdrawal of K+ from nutrient solutions. One of these, a 43-kD polypeptide localized in plasma membrane- and tonoplast-enriched fractions, accounted for a large part of 35S incorporation into membranes when [35S]methionine was administered for 6 h following 6 h of K+ deprivation. With increasing duration of K+ deprivation, 35S incorporation into this 43-kD polypeptide decreased. This polypeptide, referred to as KR43, was not synthesized when NO3− or inorganic phosphate was removed or when Rb+ was substituted for K+. However, it was synthesized when K+ was removed and replaced by an equivalent concentration of Na+. The intrinsic nature of this polypeptide and the time course of changes in its expression, which correspond with changes of K+(86Rb) influx associated with K+ deprivation, provide evidence that this polypeptide may form part of the high-affinity K+ transport system in barley roots. A possible role for this polypeptide is discussed in the context of changes in the subcellular distribution of K+ in barley roots following interruption of K+ supply. A 45-kD microsomal polypeptide, identified in earlier studies as a response to K+ deprivation, is suggested to be an extrinsic protein, readily displaced from membranes by exposure to ethylenediaminetetraacetate. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16653116

  16. 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.

  17. 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.

  18. Properties of the Ca-activated K+ channel in pancreatic beta-cells.

    PubMed

    Atwater, I; Rosario, L; Rojas, E

    1983-12-01

    The existence of [Ca2+]i-activated K+-channels in the pancreatic beta-cell membrane is based in two observations: quinine inhibits K+-permeability and, increasing intracellular Ca2+ stimulates it. The changes in K+-permeability of the beta-cell have been monitored electrically by combining measurements of the dependence of the membrane potential on external K+ concentration and input resistance. The changes in the passive 42K and 86Rb efflux from the whole islet have been measured directly. Intracellular Ca2+ has been increased by various means, including increasing extracellular Ca2+, addition of the Ca2+-ionophore A23187 or noradrenaline and application of mitochondrial uncouplers and blockers. In addition to quinine, many other substances have been found to inhibit or modulate the [Ca2+]i-activated K+-channel. The most important of these is the natural stimulus for insulin secretion, glucose. Glucose may inhibit K+-permeability by lowering intracellular Ca2+. Glibenclamide, a hypoglycaemic sulphonylurea, is about 25 times more active than quinine in blocking the K+-channel in beta-cells. The methylxanthines, c-AMP, various calmodulin inhibitors and Ba2+ also inhibit K+-permeability. Genetically diabetic mice have been studied and show an alteration in the [Ca2+]i-activated K+-channel. It is concluded that the [Ca2+]i-activated K+-channel plays a major role in the normal function of the pancreatic beta-cell. The study of its properties should prove valuable for the understanding and treatment of diabetes. PMID:6323007

  19. High-Affinity K+ Transport in Arabidopsis: AtHAK5 and AKT1 Are Vital for Seedling Establishment and Postgermination Growth under Low-Potassium Conditions1[C][W][OA

    PubMed Central

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

    2010-01-01

    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 NH4+-free growth conditions, athak5 mutant plants exhibited growth defects at 10 μm K+, but at K+ concentrations of 20 μm 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 μm K+ and growth was impaired at concentrations up to 450 μm K+. Moreover, transfer of 3-d-old plants from high to low K+ concentrations led to growth defects and leaf chlorosis at 10 μm K+ in athak5 akt1 double mutant plants. Determination of Rb+(K+) uptake kinetics in wild-type and mutant roots using rubidium (86Rb+) 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

  20. 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

  1. 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

  2. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  3. 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

  4. Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles.

    PubMed

    Bhattacharyya, P; Shapiro, S A; Barnes, E M

    1977-02-01

    Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell. PMID:838687

  5. 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

  6. Rat hepatic (Na/sup +/, K/sup +/)-ATPase:. cap alpha. =subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping

    SciTech Connect

    Hubert, J.J.; Schenk, D.B.; Skelly, H.; Leffert, H.L.

    1986-07-01

    The catalytic ..cap alpha..-subunit of rat hepatic (Na/sup +/, K/sup +/)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody (9-A5) covalently linked to Sepharose 4B that specifically block phosphorylation of the sodium pump's ..cap alpha..-subunit from (..gamma..-/sup 32/P)ATP. The hepatic subunit is virtually identical with purified rat, dog, and human renal ..cap alpha..-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (M/sub r/ 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal ..beta..-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic ..beta..-subunits (M/sub r/ 50K and 55K) eluted from 9-A5-Spharose. Additional studies of ouabain-sensitive /sup 86/Rb/sup +/ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two know structurally conserved forms of catalytic subunit (the renallike ..cap alpha.. form) and, if at all, structurally divergent forms of the sodium pump's ..beta..-subunit.

  7. 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

  8. Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes

    PubMed Central

    Cash, Derek J.; Hess, George P.

    1980-01-01

    Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine

  9. 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.

  10. 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.

  11. Evidence for two different types of P2 receptors stimulating insulin secretion from pancreatic B cell.

    PubMed

    Petit, P; Hillaire-Buys, D; Manteghetti, M; Debrus, S; Chapal, J; Loubatières-Mariani, M M

    1998-11-01

    Adenine nucleotides have been shown to stimulate insulin secretion by acting on P2 receptors of the P2Y type. Since there have been some discrepancies in the insulin response of different analogues of ATP and ADP, we investigated whether two different types of P2 receptors exist on pancreatic B cells. The effects of alpha,beta-methylene ATP, which is more specific for the P2X subtype, were studied in vitro in pancreatic islets and isolated perfused pancreas from rats, in comparison with the potent P2Y receptor agonist ADPbetaS. In isolated islets, incubated with a slightly stimulating glucose concentration (8.3 mM), alpha,beta-me ATP (200 microM) and ADPbetaS (50 microM) similarly stimulated insulin secretion; by contrast, under a non stimulating glucose concentration (3 mM), alpha,beta-me ATP was still effective whereas ADPbetaS was not. In the same way, in islets perifused with 3 mM glucose, alpha,beta-me ATP but not ADPbetaS induced a partial but significant reduction in the peak 86Rb efflux induced by the ATP-dependent potassium channel opener diazoxide. In the isolated pancreas, perfused with a non stimulating glucose concentration (4.2 mM), ADPbetaS and alpha,beta-me ATP (5-50 microM), administered for 10 min, induced an immediate, transient and concentration-dependent increase in the insulin secretion; their relative potency was not significantly different. In contrast, with a slightly stimulating glucose concentration (8.3 mM), ADPbetaS was previously shown to be 100 fold more potent than alpha,beta-me ATP. Furthermore, at 4.2 mM glucose a second administration of alpha,beta-me ATP was ineffective. In the same way, ADPbetaS was also able to desensitize its own insulin response. At 3 mM glucose, alpha,beta-me ATP as well as ADPbetaS (50 microM) induced a transient stimulation of insulin secretion and down regulated the action of each other. These results give evidence that pancreatic B cells, in addition to P2Y receptors, which potentiate glucose

  12. ‘And then there were three’: highly efficient uptake of potassium by foliar trichomes of epiphytic bromeliads

    PubMed Central

    Winkler, Uwe; Zotz, Gerhard

    2010-01-01

    Background and Aims Vascular epiphytes have to acquire nutrients from atmospheric wash out, stem-flow, canopy soils and trapped litter. Physiological studies on the adaptations to nutrient acquisition and plant utilization of nutrients have focused on phosphorus and nitrogen; potassium, as a third highly abundant nutrient element, has received minor attention. In the present study, potassium uptake kinetics by leaves, within-plant distribution and nutrient accumulation were analysed to gain an improved understanding of physiological adaptations to non-terrestrial nutrient supply of plants. Methods Radioactively labelled 86RbCl was used as an analogue to study uptake kinetics of potassium absorbed from tanks of epiphytes, its plant distribution and the correlation between uptake efficiency and abundance of trichomes, functioning as uptake organs of leaves. Potassium in leaves was additionally analysed by atomic absorption spectroscopy to assess plant responses to potassium deficiency. Key Results Labelled rubidium was taken up from tanks over a wide range of concentrations, 0·01–90 mm, which was achieved by two uptake systems. In four tank epiphytes, the high-affinity transporters had average Km values of 41·2 µm, and the low-affinity transporters average Km values of 44·8 mm. Further analysis in Vriesea splenriet showed that high-affinity uptake of rubidium was an ATP-dependent process, while low-affinity uptake was mediated by a K+-channel. The kinetic properties of both types of transporters are comparable with those of potassium transporters in roots of terrestrial plants. Specific differences in uptake velocities of epiphytes are correlated with the abundance of trichomes on their leaf surfaces. The main sinks for potassium were fully grown leaves. These leaves thus function as internal potassium sources, which allow growth to be maintained during periods of low external potassium availability. Conclusions Vascular epiphytes possess effective mechanisms

  13. The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide.

    PubMed

    Petrunyaka, V V; Panyushkina, E A; Severina, E P; Orlov, S N

    1990-12-14

    The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes. PMID:2175654

  14. Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site

    PubMed Central

    Abe, Kazuhiro; Tani, Kazutoshi; Friedrich, Thomas; Fujiyoshi, Yoshinori

    2012-01-01

    Gastric H+,K+-ATPase is responsible for gastric acid secretion. ATP-driven H+ uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K+, resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H+/2K+ to 1H+/1K+ per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K+ congener (Rb+), the 8-Å resolution structure of H+,K+-ATPase in the transition state of dephosphorylation, (Rb+)E2∼AlF, which is distinct from the preceding Rb+-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb+)E2∼AlF highly likely represents a single bound Rb+ ion, which is clearly different from the Rb+-free E2AlF or K+-bound (K+)E2∼AlF structures. Measurement of radioactive 86Rb+ binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb+ is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H+/1K+/1ATP transport mode of H+,K+-ATPase, which is a prerequisite for generation of the 106-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit’s N terminus and the P domain observed in the (Rb+)E2∼AlF structure, we propose a refined vectorial transport model of H+,K+-ATPase, which must prevail against the highly acidic state of the gastric lumen. PMID:23091039

  15. Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site.

    PubMed

    Abe, Kazuhiro; Tani, Kazutoshi; Friedrich, Thomas; Fujiyoshi, Yoshinori

    2012-11-01

    Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K(+) congener (Rb(+)), the 8-Å resolution structure of H(+),K(+)-ATPase in the transition state of dephosphorylation, (Rb(+))E2~AlF, which is distinct from the preceding Rb(+)-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb(+))E2~AlF highly likely represents a single bound Rb(+) ion, which is clearly different from the Rb(+)-free E2AlF or K(+)-bound (K(+))E2~AlF structures. Measurement of radioactive (86)Rb(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb(+) is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H(+),K(+)-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb(+))E2~AlF structure, we propose a refined vectorial transport model of H(+),K(+)-ATPase, which must prevail against the highly acidic state of the gastric lumen. PMID:23091039

  16. Tumor necrosis factor-alpha induces Cl- and K+ secretion in human distal colon driven by prostaglandin E2.

    PubMed

    Schmitz, H; Fromm, M; Bode, H; Scholz, P; Riecken, E O; Schulzke, J D

    1996-10-01

    Increased levels of tumor necrosis factor-alpha (TNF-alpha) have been found in, for example, inflammatory bowel disease (IBD) and human immunodeficiency virus (HIV) infection. To investigate a possible contribution of TNF-alpha to the pathogenesis of diarrhea in these diseases, ion transport of human distal colon was studied in the Ussing chamber in vitro. Serosal addition of TNF-alpha increased short-circuit current (Isc) of partially stripped tissues in a dose-dependent manner. Maximum Isc increase of 1.8 +/- 0.2 mumol.h-1.cm-2 was reached after 60 +/- 9 min at 200 ng/ml TNF-alpha. Bidirectional tracer flux measurements revealed that TNF-alpha induced an increase in 36 Cl serosal-to-mucosal flux, a decrease in 36Cl- mucosal-to-serosal flux, and a slight increase in K+ secretion indicated by an increased secretory 86Rb net flux. In the highly differentiated colonic epithelial cell line HT-29/B6, TNF-alpha had no effect on Isc, suggesting a mediation step located in the subepithelium. This supposition was supported by measurements on totally stripped human tissues, since removal of subepithelial layers by total stripping reduced the TNF-alpha effect by 40%. Experiments with tetrodotoxin (10(-6)M) indicated that the TNF-alpha effect was not mediated by the enteric nervous system. The specific 5-lipoxygenase blocker ICI-230487 (5 x 10(-8)M) also had no effect on TNF-alpha action. In contrast, inhibition of cyclooxygenase by indomethacin (10(-6)M inhibited the effect of TNF-alpha. Radioimmunoassay of prostaglandin E2 (PGE2) in the serosal bathing solution revealed an increase in PGE2 production/release after addition of TNF-alpha, which paralleled the Isc response. We conclude that TNF-alpha changed Cl- and K+ transport toward secretion in human colon. This effect was mediated by PGE2 produced by subepithelial cells. Thus TNF-alpha could be a mediator of diarrhea during intestinal inflammation, e.g., in IBD and HIV infection. PMID:8897887

  17. 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.

  18. 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.

  19. 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.

  20. Aldosterone induces active K+ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon

    PubMed Central

    Singh, Satish K.; O'Hara, Bryan; Talukder, Jamilur R.

    2012-01-01

    Although both Kcnn4c and Kcnma1 channels are present on colonic mucosal membranes, only Kcnma1 has been suggested to mediate K+ secretion in the colon. Therefore, studies were initiated to investigate the relative roles of Kcnn4c and Kcnma1 in mediating aldosterone (Na-free diet)-induced K+ secretion. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net 86Rb+ (K+ surrogate) fluxes as well as short circuit currents (Isc; measure of net ion movement) were measured under voltage clamp condition in rat distal colon. Active K+ absorption, but not K+ secretion, is present in normal, while aldosterone induces active K+ secretion (1.04 ± 0.26 vs. −1.21 ± 0.15 μeq·h−1·cm−2; P < 0.001) in rat distal colon. Mucosal VO4 (a P-type ATPase inhibitor) inhibited the net K+ absorption in normal, while it significantly enhanced net K+ secretion in aldosterone animals. The aldosterone-induced K+ secretion was inhibited by the mucosal addition of 1) either Ba2+ (a nonspecific K+ channel blocker) or charybdotoxin (CTX; a common Kcnn4 and Kcnma1 channel blocker) by 89%; 2) tetraethyl ammonium (TEA) or iberiotoxin (IbTX; a Kcnma1 channel blocker) by 64%; and 3) TRAM-34 (a Kcnn4 channel blocker) by 29%. TRAM-34, but not TEA, in the presence of IbTX further significantly inhibited the aldosterone-induced K+ secretion. Thus the aldosterone-induced Ba2+/CTX-sensitive K+ secretion consists of IbTX/TEA-sensitive (Kcnma1) and IbTX/TEA-insensitive fractions. TRAM-34 inhibition of the IbTX-insensitive fraction is consistent with the aldosterone-induced K+ secretion being mediated partially via Kcnn4c. Western and quantitative PCR analyses indicated that aldosterone enhanced both Kcnn4c and Kcnma1α protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone also enhanced Kcnn4c and Kcnma1α mRNA levels, and this was prevented by exposure to actinomycin D (an RNA synthesis inhibitor). These observations indicate that aldosterone

  1. Hypotonicity and cell volume regulation in shark rectal gland: role of organic osmolytes and F-actin.

    PubMed

    Ziyadeh, F N; Mills, J W; Kleinzeller, A

    1992-03-01

    Hypotonic stress (reduction of external tonicity from approximately 900 mosM and 295 mM NaCl to approximately 600 mosM and 135 mM NaCl) produced a relatively slow regulatory volume decrease (RVD) in dogfish shark (Squalus acanthias) rectal gland cells. During the 5-h experiment, cell K+ content remained unchanged; cell content of Na+ and Cl- dropped in the initial swelling phase by some 50% (reflecting the corresponding reduction in medium NaCl), and then remained unchanged during volume recovery phase. Also, cellular fluxes of 86Rb+ and urea were not affected by hypotonic stress. However, hypotonicity enhanced 10- to 20-fold the efflux of organic cell osmolytes taurine, betaine, and trimethyloxamine, and this accounted for the loss of osmotically obliged water during RVD. Enhancement of osmolyte efflux by hypotonic stress was abolished by readjusting the low-Na+ saline to isotonicity (approximately 900 mosM) with innocuous cations (choline+, Li+, or N-methylglucamine+). The results suggest that reduction of medium tonicity may be the determinant for the RVD response to hypotonic stress. The above properties of the observed RVD were also displayed when studying changes on cell F-actin at the basolateral cell face; hypotonic stress (medium with 135 mM NaCl) produced a rapid disappearance of fluorescence related to this cytoskeletal component, whereas no such changes were seen in low-Na+ salines made isotonic with choline or N-methylglucamine chloride nor in a saline made hyposmolar by omitting urea. Hence, hypotonicity is required to affect F-actin organization (depolymerization?). These changes of F-actin fluorescence are transient; they were completed within 5-10 min of hypotonic stress, and afterwards a gradual reconstitution of cell F-actin organization was seen. The above observations are consistent with the assumption that, in shark rectal gland cells, transient loss of cytoskeleton (F-actin) organization at the basolateral cell face, induced by hypotonicity

  2. 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

  3. 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

  4. The change and significance of the Na+-K+-ATPase alpha-subunit in ouabain-hypertensive rats.

    PubMed

    Tian, G; Dang, C; Lu, Z

    2001-11-01

    Ouabain has recently been identified as an endogenous Na+-K+ pump inhibitor having a close association with hypertension. However, some patients with hypertention do not show high levels of endogenous ouabain (EO), and patients with high EO levels do not necessarily suffer from hypertention. It is believed that the Na+-K+-ATPase activity in essential hypertension does not undergo homogenous change. The present study was designed, therefore, to investigate the expression and the significance of the Na+-K+-ATPase alpha-subunit isoforms in kidney tissue in ouabain-hypertensive rats. Ouabain was administered chronically to establish a model of ouabain-hypertensive rats. Biochemical analysis, cytobiology and sABC immunohistochemistry were they used to assay for expression of Na+-K+-ATPase alpha-subunit isoforms in kidney tissue. After the first week of receiving ouabain, 65% (n=13) of rats had hypertension. After the second week, the blood pressure of these 13 hypertensive rats was increased significantly compared to the baseline and control levels (p<0.05). The plasma renin activity was normal, and angiotensin II and aldosterone levels were increased significantly in these rats (p<0.05). But in the other 35% (n=7) of rats of the experimental group, there was no apparent increase in blood pressure after receiving ouabain. The plasma ouabain level in the non-hypertensive subgroup was significantly higher than that in the hypertensive subgroup, but the 86Rb intake and the number of 3H-ouabain binding sites did not decrease. The Na+-K+-ATPase activity showed non-homogeneous changes. In hypertensive rats, the expression levels of ouabain paralleled the degree of hypertension (r=0.88, p<0.05). The positive granules were mainly scattered in the cytoblastoma of the reticular zone of adrenal cortex. There were thus different levels of expression of Na+-K+-ATPase alpha-subunit isoforms in this model. In the hypertension subgroup the alpha1 was most strongly expressed, followed

  5. 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

  6. A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore.

    PubMed

    Cheng, J; Guffanti, A A; Krulwich, T A

    1997-03-01

    A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na(+)-dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3'-terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na(+)-sensitive phenotype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+ efflux, which was further stimulated by CCCP. In the absence of CCCP, NatAB-mediated Na+ efflux was stimulated by K+. Concomitant NatAB-dependent K+ uptake occurred, as monitored by 86Rb+ uptake; this uptake was inhibited by CCCP and is thus secondary to the primary, electrogenic Na+ efflux