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Sample records for 86rb vblizi porogov

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

    PubMed

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

    2014-04-01

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

  2. Vectorial (transcellular) transport of potassium (/sup 86/Rb+) by cultured Sertoli cells

    SciTech Connect

    Muffly, K.E.; Hall, P.F.

    1988-10-01

    Sertoli cells from rats aged 25 days were grown on Millipore filters (pore diameter 0.5 micron) for 7 days and were then used for determination of transport of 86Rb+ through the cells (base to apex); this procedure is referred to as measuring transcellular or vectorial transport. Sertoli cells were also used to measure apical efflux of 86Rb+ by loading the cells with the isotope to steady state and then incubating cells so that the apical surfaces were in contact with medium not containing 86Rb+, from which samples were taken. Basal efflux was measured in the same way except that the opposite surface of the cells was in contact with the medium. Cells grown on filters treated with collagen IV plus fibronectin showed transcellular transport of 86Rb+; t1/2 for equilibration across the cells was 9-12 min. The rate of transport was accelerated by addition of (Bu)2cAMP, forskolin, or FSH to the incubation medium. Half-maximal responses were seen with (Bu)2cAMP at 0.2 mM and with forskolin at 20 microM. Apical efflux (t1/2 9.8 +/- 2.1 min) was not influenced by the presence or absence of K+ in the medium nor by azide or (Bu)2cAMP. Basal efflux showed similar values for t1/2 in the presence of K+ (9.7 +/- 1.9 min) and values of 21.4 +/- 4.2 min in the absence of K+. Vectorial transport of 86Rb+ by these cells may account for the K+ gradient seen in the seminiferous tubule and appears to result from a basolateral potassium pump together with an apical membrane that is permeable to K+.

  3. 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.

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

    PubMed

    Yamamoto, H A; Harris, R A

    1983-04-08

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

  6. 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.

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

    SciTech Connect

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

    1984-07-30

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

  8. Endothelium-dependent relaxation of the pig aorta: relationship to stimulation of 86Rb efflux from isolated endothelial cells.

    PubMed Central

    Gordon, J. L.; Martin, W.

    1983-01-01

    Bradykinin, adenosine triphosphate (ATP) and acetylcholine each relaxed histamine-contracted strips of pig aorta in a dose-dependent manner. These relaxations were abolished when the endothelium was removed. Relaxation induced by ATP was mimicked by adenosine diphosphate (ADP) but adenosine monophosphate (AMP) and adenosine were about 120 times less potent. Relaxation induced by acetylcholine was antagonized by atropine in a competitive manner, and carbachol induced the same degree of relaxation as acetylcholine, but was about 10 times less potent. The calcium ionophore, A23187, also induced a dose-dependent relaxation of pig aortic strips provided the endothelium was present, suggesting that a rise in the level of ionized calcium within the endothelial cells is one means by which vascular smooth muscle relaxation can be triggered. Bradykinin, ATP, ADP, AMP, adenosine and A23187 each induced a dose-dependent increase in 86Rb efflux from preloaded pig aortic endothelial cells. The dose-response curves for stimulation of 86Rb efflux and for endothelium-dependent relaxation were similar for each individual compound. ADP was equipotent with ATP, but AMP and adenosine were about 120 times less potent. Neither acetylcholine nor carbachol, in concentrations that induce endothelium-dependent relaxation, had any effect on 86Rb efflux from isolated aortic endothelial cells. Lanthanum, which blocks calcium influx, abolished the increases in 86Rb efflux induced by bradykinin and ATP, and the calcium ionophore A23187 was the most effective stimulant of 86Rb efflux, suggesting that the potassium transport induced by these agents is calcium-activated. It is concluded that endothelial responses to bradykinin and ATP can be assessed by monitoring 86Rb efflux, which probably reflects a calcium-activated efflux of potassium associated with the endothelium-dependent vascular relaxation induced by these agents. This pathway is apparently not involved in endothelial responses to

  9. 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.

  10. /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.

  11. 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.

  12. 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.

  13. 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.

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

    PubMed

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

    2014-02-21

    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 (22)Na, (65)Zn, (86)Rb, (109)Cd and (137)Cs 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 (22)Na, (86)Rb and (137)Cs, and low-energy x-rays contributed significantly to (65)Zn and (109)Cd 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.

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

    PubMed Central

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

    1985-01-01

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

  16. A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro.

    PubMed

    Sangadala, S; Walters, F S; English, L H; Adang, M J

    1994-04-01

    CryIA(c) delta-endotoxin, a member of the CryI family of Bacillus thuringiensis insecticidal proteins, specifically recognizes and binds with high affinity to target proteins in the midgut of susceptible insects. Protein blots of Manduca sexta brush-border membranes probed with 125I-CryIA(c) identify a major binding protein of 120 kDa and a minor binding protein of 65 kDa. Monoclonal antibodies were raised against the 120-kDa toxin binding protein. Using isoelectric focusing and monoclonal antibodies (2B3, 8G1, and 12B8) 120- and 65-kDa brush-border proteins were isolated. Labeled CryIA(c) and monoclonal antibodies probed to blots of the affinity-selected proteins recognized the 120- and 65-kDa proteins. When reconstituted into phospholipid vesicles, antibody-selected proteins increased toxin binding (35%) and enhanced toxin-induced 86Rb+ release up to 1000-fold. The 120-kDa protein was identified as aminopeptidase N (EC 3.4.11.2). A CryIA(c)-sensitive phosphatase was also present in the 120/65-kDa protein mixture. These findings provide the first identification of B. thuringiensis toxin binding proteins, although confirmation is needed in vivo.

  17. Extraction and separation of (137)cs and (86)rb by means of 4-t-butyl-2(alpha-methylbenzyl)phenol.

    PubMed

    Rais, J; Krtil, J; Chotívka, V

    1971-02-01

    The extraction of caesium-137 and rubidium-86 by a solution of 4-t-butyl-2(alpha-methylbenzyl)phenol in n-octane and tetrachloromethane has been investigated. The separation of rubidium and caesium has been performed by the Craig method and by reversedphase extraction chromatography.

  18. 10 CFR Appendix L to Part 110 - Illustrative List of Byproduct Materials Under NRC Export/Import Licensing Authority a

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...) Rubidium 86 (Rb 86) Rubidium 87 (Rb 87) Ruthenium 97 (Ru 97) Ruthenium 103 (Ru 103) Ruthenium 105 (Ru 105) Ruthenium 106 (Ru 106) Samarium 151 (Sm 151) Samarium 153 (Sm 153) Scandium 46 (Sc 46) Scandium 47 (Sc...

  19. 10 CFR Appendix L to Part 110 - Illustrative List of Byproduct Materials Under NRC Export/Import Licensing Authority a

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...) Rubidium 86 (Rb 86) Rubidium 87 (Rb 87) Ruthenium 97 (Ru 97) Ruthenium 103 (Ru 103) Ruthenium 105 (Ru 105) Ruthenium 106 (Ru 106) Samarium 151 (Sm 151) Samarium 153 (Sm 153) Scandium 46 (Sc 46) Scandium 47 (Sc...

  20. Is activation of the Na+K+ pump necessary for NGF-mediated neuronal survival

    SciTech Connect

    Sendtner, M.; Gnahn, H.; Wakade, A.; Thoenen, H.

    1988-02-01

    The ability of nerve growth factor to cause rapid activation of the Na+K+ pump of its responsive cells was examined by measuring the uptake of /sup 86/Rb+. A significant increase in /sup 86/Rb+ uptake in E8 chick dorsal root ganglion sensory neurons after NGF treatment was seen only if the cells had been damaged during the preparation procedure. Such damaged cells could not survive in culture in the presence of NGF, and undamaged cells that did survive in response to NGF exhibited no increased /sup 86/Rb+ uptake rate. Furthermore, cultured calf adrenal medullary cells did not show an increase in /sup 86/Rb+ uptake after treatment with NGF, although these cells respond to NGF with an increased synthesis of catecholaminergic enzymes. These results are incompatible with the hypothesis that the mechanism of action of NGF that promotes neuronal survival and enzyme induction results from an initial stimulation of the Na+K+ pump.

  1. 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.

  2. Intracellular sodium, potassium and magnesium concentration, ouabain-sensitive 86rubidium-uptake and sodium-efflux and Na+, K+-cotransport activity in erythrocytes of normal male subjects studied on two occasions.

    PubMed

    Lijnen, P; Hespel, P; Lommelen, G; Laermans, M; M'Buyamba-Kabangu, J R; Amery, A

    1986-09-01

    The red cell Na+,K+-ATPase pump activity estimated by the ouabain-sensitive 86Rb-uptake or Na+-efflux, the Na+,K+-cotransport activity measured either by the furosemide-sensitive K+- or Na+- efflux or by the ethacrynic acid-sensitive 86Rb-uptake as well as the intraerythrocyte concentration of sodium, potassium and magnesium were studied in 29 normal male subjects with one to three weeks interval between the first and second blood sampling. Both the red cell sodium and potassium concentration, the erythrocyte ouabain-sensitive 86Rb-uptake and Na+-efflux, the furosemide-sensitive Na+- and K+-efflux and the ethacrynic acid-sensitive 86Rb-uptake are stable over time in the same individual. The furosemide-sensitive Na+-and K+-efflux is significantly related to the ethacrynic acid-sensitive 86Rb-uptake. The intraerythrocyte Na+ concentration was negatively related to the ouabain-sensitive 86Rb-uptake, but not to the ouabain-sensitive Na+-efflux; it was, however, negatively related to the rate constant for the ouabain-sensitive Na+-efflux.

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

    SciTech Connect

    Weston, A.H.

    1987-01-01

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

  4. 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.

  5. 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.

  6. Basolateral K sup + channels in airway epithelia. I. Regulation by Ca sup 2+ and block by charybdotoxin

    SciTech Connect

    McCann, J.D.; Matsuda, J.; Garcia, M.; Kaczorowski, G.; Welsh, M.J. )

    1990-06-01

    In airway epithelia, adenosine 3',5'-cyclic monophosphate (cAMP) stimulates Cl- secretion by activating apical membrane Cl- channels and basolateral membrane K+ channels. Cl- channels are regulated by cAMP-dependent phosphorylation, whereas K+ channels are regulated by the cytosolic Ca2+ concentration, (Ca2+)c. Our recent observation that cAMP increases (Ca2+)c suggested that cAMP might indirectly regulate K+ channels by increasing (Ca2+)c. To study regulation of K+ channels we measured 86Rb efflux, single K+ channels in membrane patches, and (Ca2+)c with the fluorescent indicator fura-2. Isoproterenol and Ca2+ ionophore, A23187, transiently increased (Ca2+)c and transiently stimulated 86Rb efflux. Stimulation of 86Rb efflux resulted from release of intracellular Ca2+ stores. 86Rb efflux was blocked by Ba2+ or charybdotoxin, but not by tetraethylammonium. Charybdotoxin prevented all of the 86Rb efflux that was stimulated by A23187 or by forskolin. Charybdotoxin also blocked the low-conductance inwardly rectifying K+ channel (KCLIC) in membrane patches. These results indicate that the KCLIC channel is responsible for the Ca2(+)-dependent increase in K+ permeability in airway epithelial cells. They also indicate that cAMP-induced release of intracellular Ca2+ is sufficient to activate K+ channels.

  7. Effects of potassium channel toxins from Leiurus quinquestriatus hebraeus venom on responses to cromakalim in rabbit blood vessels.

    PubMed Central

    Strong, P. N.; Weir, S. W.; Beech, D. J.; Hiestand, P.; Kocher, H. P.

    1989-01-01

    1. The effects of fractionated Leiurus quinquestriatus hebraeus venom on cromakalim-induced 86Rb+ efflux in rabbit aortic smooth muscle were examined. 2. Crude venom (0.1-30 micrograms ml-1) produced a concentration-dependent decrease of 1 microM cromakalim-induced 86Rb+ response. The maximum blocking activity attainable was approximately 60%. 3. Fractionation of crude venom by gel permeation chromatography and subsequent chromatography on a cation ion-exchange column, produced two fractions (X and XI), active in the 86Rb+ blocking assay. 4. Fraction XII contained charybdotoxin (approximately 85% pure). After a final high performance liquid chromatography (h.p.l.c.) purification step, the purified toxin failed to inhibit the cromakalim-stimulated 86Rb+ efflux although it was a potent inhibitor of A23187-induced K+ flux in human erythrocytes and the large conductance calcium-activated potassium channel in rabbit portal vein smooth muscle. 5. Subsequent purification of fraction X by h.p.l.c. yielded a minor peak which contained 86Rb+ blocking activity. This subfraction was also capable of inhibiting apamin-sensitive, angiotensin II-stimulated K+ flux in guinea-pig hepatocytes. 6. It is concluded that the potassium channel opened by cromakalim in rabbit aortic smooth muscle is not blocked by charybdotoxin but by another distinct toxin in the venom of Leiurus quinquestriatus hebraeus. PMID:2531622

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

    PubMed

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

    1993-04-15

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

  10. Sodium and potassium transport in the halophilic yeast Debaryomyces hansenii.

    PubMed

    González-Hernández, J C; Cárdenas-Monroy, C A; Peña, A

    2004-04-15

    Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.6 M of either NaCl or KCl, accumulating high concentrations of Na(+) or K(+). After growth in NaCl or KCl, a rapid efflux of either accumulated cation was observed if the cells were incubated in the presence of KCl or NaCl, respectively, accompanied by a slower accumulation of the cation present in the incubation medium. However, a similar, rapid efflux was observed if cells were incubated in buffer, in the absence of external cations. This yeast shows a cation uptake activity of both (86)Rb(+) and (22)Na(+) with saturation kinetics, and much higher affinity for (86)Rb(+) than for (22)Na(+). The pH dependence of the kinetics constants was similar for both cations, and although K(m) values were higher at pH 8.0, there was also an increase in the V(max) values. The accumulation of (22)Na(+) was found to be increased in cells grown in the presence of 0.6 M NaCl. (86)Rb(+) was also accumulated more in these cells, but to a slightly greater extent. The inhibition kinetics of the uptake of (22)Na(+) by K(+), and that of (86)Rb(+) by Na(+) was found to be non-competitive. It can be concluded that Na(+) in D. hansenii is not excluded but instead, its metabolic systems must be resistant to high salt concentrations.

  11. 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.

  12. Anisotonic media and glutamate-induced ion transport and volume responses in primary astrocyte cultures.

    PubMed

    Kimelberg, H K

    1987-01-01

    1. The responses of primary monolayer astrocyte cultures prepared from neonatal rat brains to hyper- and hypotonic media and to the addition of L-glutamic acid were examined as part of a systematic approach to use these cultures to obtain information on the mechanisms of the volume changes seen in astroglial cells in situ. 2. Addition of 200 mM mannitol to the medium to make it hypertonic caused cell shrinkage as measured with [14C]3-O-methyl-D-glucose, and also activated K+ and Cl- uptake measured with 86Rb+ and 36Cl- respectively. The increased ion uptake was completely inhibited by 0.1 mM bumetanide, showing that the Na+ + K+ + 2 Cl- co-transport system was being activated by cell shrinkage. 3. Studies of 86Rb+ uptake as a function of external K+ and hypertonic media showed a complex pattern. Increased bumetanide-sensitive, hypertonic-stimulated uptake of 86Rb+ was seen up to 20 mM K+0, with maximum stimulation being first reached at around 2 to 5 mM K+. At concentrations greater than 20 mM K+0 there was a further increase in bumetanide-sensitive 86Rb+ uptake, but there was no stimulation of this uptake by hypertonicity. There were also increases in bumetanide-insensitive 86Rb+ fluxes at [K+]0 higher than 20 mM that may have been due to opening of voltage-dependent K+ channels; this increased 86Rb+ flux was decreased in hypertonic medium. 4. When primary astrocyte cultures were swollen in hypotonic medium there was a rapid increase in volume as measured with [14C] 3-O-methyl-D-glucose, which then decreased in the continued presence of hypotonic medium. Thus, these cells exhibit volume regulatory decrease or RVD, as described for other cells. The possible ionic bases of this phenomenon have not yet been fully examined but the initial RVD did not appear to stimulate a furosemide-sensitive cotransport system. 5. Glutamate has been implicated as a possible endogenous effector of volume change in astrocytes. In the presence of ouabain, L-glutamate led to swelling of

  13. Calcium-activated potassium channels in isolated presynaptic nerve terminals from rat brain.

    PubMed Central

    Bartschat, D K; Blaustein, M P

    1985-01-01

    86Rb efflux was examined in isolated presynaptic nerve terminals (synaptosomes) from rat brain in a study designed to assess K permeability (PK) changes sensitive to alterations in internal Ca activity. Rb efflux from 86Rb-loaded synaptosomes into nominally Ca-free physiological saline (PSS) containing 5 mM-K was about 0.3-0.4%/s. Raising extracellular K concentration [( K]o), to depolarize the synaptosomes, stimulated the 86Rb efflux. Addition of Ca to the 5 mM-K PSS had no effect, but Ca did further stimulate 86Rb efflux into K-rich solutions. The effect of Ca was graded, with apparent half-maximal activation, KA approximately equal to 0.5 mM-Ca. These data fit the view that, during depolarization, Ca enters the terminals through voltage-regulated Ca channels, and that the rise in intracellular Ca concentration opens certain (Ca-activated) K channels. The Ca-dependent stimulation of 86Rb efflux was greatest during the initial seconds of incubation (component CT), and then declined to a much lower rate (component CS). Much of this change in rate could be attributed to inactivation of voltage-regulated Ca channels and reduced entry of Ca. The Ca-dependent increase in 86Rb efflux was completely inhibited by 100 microM-La. In the presence of Ca, but not in its absence, the Ca ionophore A23187 stimulated 86Rb efflux both in 5 and 100 mM-K PSS. The effect in 100 mM-K was quantitatively greater, perhaps because of the increased outward driving force on Rb in depolarized synaptosomes. When synaptosomes were suspended in media containing the voltage-sensitive fluorescent dye, DiS-C3-(5) (1,1'-dipentyl-2,2'-thiocarbocyanine), the addition of Ca+ A23187 decreased the fluorescence intensity (= synaptosome hyperpolarization) when the media contained 5 mM-K but not 100 mM-K. This implies that in the presence of Ca + A23187, PK was increased, and the membrane potential moved closer to the K equilibrium potential, EK. Quinine sulphate, a blocker of Ca-activated K channels

  14. Volume regulation by human lymphocytes. Role of calcium

    SciTech Connect

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

    1982-05-01

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

  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.

  16. 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.

  17. 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.

  18. Permeability of human erythrocyte membrane vesicles to alkali cations.

    PubMed

    Sze, H; Solomon, A K

    1979-02-02

    The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using 86Rb as a K analog, we have measured the rate constant of 86Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.

  19. 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.

  20. Regional and species differences in glyburide-sensitive K+ channels in airway smooth muscles as estimated from actions of KC 128 and levcromakalim.

    PubMed Central

    Kamei, K; Yoshida, S; Imagawa, J; Nabata, H; Kuriyama, H

    1994-01-01

    1. The purpose of the present experiments was to elucidate the differences in actions of two K+ channel openers, KC 128 and levcromakalim, on the carbachol-induced contraction, membrane potential and 86Rb+ efflux of the dog tracheal and bronchial smooth muscles. Furthermore, we compared the effects of these agents on guinea-pig and human airway smooth muscles. 2. In the dog tracheal and bronchial smooth muscle tissues, levcromakalim induced a concentration-dependent relaxation of the carbachol-induced contraction. The IC50 values were 0.35 microM (pIC50: 6.46 +/- 0.10, n = 9) and 0.55 microM (pIC50: 6.26 +/- 0.07, n = 5), respectively. KC 128 relaxed bronchial smooth muscles precontracted by carbachol with an IC50 value of 0.19 microM (pIC50: 6.73 +/- 0.10, n = 7). However, KC 128 had almost no effect on the contraction evoked by carbachol in the trachea (IC50 > 10 microM). The relaxations induced by levcromakalim and KC 128 were antagonized by glyburide (0.03-1 microM) but not by charybdotoxin (100 nM). 3. Levcromakalim (1 microM) hyperpolarized the membrane of both dog tracheal and bronchial smooth muscle cells, whereas KC 128 (1 microM) hyperpolarized the membrane of bronchial but not of tracheal smooth muscle cells. 4. Levcromakalim (10 microM) increased 86Rb+ efflux rate from both tracheal and bronchial smooth muscle tissues but KC 128 (10 microM) increased 86Rb+ efflux rate only from bronchial and not tracheal smooth muscle tissues. Glyburide (1 microM) prevented the hyperpolarization and the 86Rb+ efflux induced by these agents at the same concentration as observed for mechanical responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7858882

  1. 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.

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

    SciTech Connect

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

    1990-07-01

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

  3. 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.

  4. 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.

  5. Thyroid-induced alterations in myocardial sodium-potassium-activated adenosine triphosphatase, monovalent cation active transport, and cardiac glycoside binding.

    PubMed Central

    Curfman, G D; Crowley, T J; Smith, T W

    1977-01-01

    The effects of thyroid hormone on guinea pig myocardial NaK-ATPase activity, transmembrane monovalent cation active transport, and cardiac glycoside binding were were examined. NaK-ATPase activities of left atrial and left ventricular homogenates of control and triiodothyronine (T3)-treated animals were determined, and compared to activities of skeletal muscle and liver. T3 administration was associated with a significant increase of 18% in left atrial and left ventricular NaK-ATPase specific activities. This increment was less than that noted in skeletal muscle (+42%) and liver (+30%). To determine if enhanced NaK-ATPase activity was accompanied by increased monovalent cation active transport, in vitro 86Rb+ uptake by left atrial strips and hemidiaphragms was measured. Transition from the euthyroid to the hyperthyroid state resulted in a 68% increase in active 86Rb+ uptake by left atrium, and a 62% increase in active uptake by diaphragm. Passive 86Rb+ uptake was not affected in either tissue. Ouabain binding by atrial and ventricular homogenates of T3-treated animals was increased by 19 and 17%, respectively, compared to controls, in close agreement with thyroid-induced increments in NaK-ATPase activiey. Taken together, these results are consistent with enhanced myocardial NaK-ATPase activity and monovalent cation activt transport due to an increase in the number of functional enzyme complexes. PMID:138689

  6. 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.

  7. Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase

    SciTech Connect

    Reeves, W.B.; McDonald, G.A.; Mehta, P.; Andreoli, T.E. )

    1989-07-01

    ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide- and chloride-sensitive {sup 22}Na+ uptake and barium-sensitive, voltage-dependent {sup 86}Rb+ influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1 mM ATP, and 50 mM KCl, the barium-sensitive {sup 86}Rb+ influx increased from 361 {plus minus} 138 to 528 {plus minus} 120 pM/mg prot.30 sec (P less than 0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 microgram/ml) was also present in the vesicle solutions. The stimulation of {sup 86}Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.

  8. 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).

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

    SciTech Connect

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

    1991-07-01

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

  10. Tobacco smoke chemicals attenuate brain-to-blood potassium transport mediated by the Na,K,2Cl-cotransporter during hypoxia-reoxygenation.

    PubMed

    Paulson, Jennifer R; Roder, Karen E; McAfee, Ghia; Allen, David D; Van der Schyf, Cornelis J; Abbruscato, Thomas J

    2006-01-01

    Smoking tobacco, including cigarettes, has been associated with an increased incidence and relative risk for cerebral infarction in both men and women. Recently, we have shown that nicotine and cotinine attenuate abluminal (brain facing) K(+) uptake mediated by the Na,K,2Cl-cotransporter (NKCC) in bovine brain microvessel endothelial cells (BBMECs) after hypoxic/aglycemic exposure (stroke conditions). The purpose of the current study was to explore the effects of nicotine and tobacco smoke chemicals on K(+) movement through the blood-brain barrier during both hypoxia/aglycemia and reoxygenation. BBMECs were exposed to nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) in quantities designed to mimic plasma concentrations of smokers. Stroke conditions were mimicked in vitro in BBMECs through 6 h of hypoxia/aglycemia with or without 12 h of reoxygenation, after which NKCC-mediated K(+) uptake and paracellular integrity were measured with (86)Rb and [(14)C]sucrose, respectively. In addition, K(+) concentrations in brain extracellular fluid were estimated in (86)Rb-injected rats that were administered nicotine, N-CSE, or NF-CSE and on whom global ischemia/reperfusion by in vivo four-vessel occlusion was performed. Both in vitro and in vivo paradigms showed nicotine, the major alkaloid present in tobacco smoke, to be the determining factor of an inhibited response of abluminal NKCC in BBMECs during and after stroke conditions. This was measured as a decrease in abluminal brain endothelial cell NKCC activity and as an increase in brain extracellular K(+) concentration measured as the brain extracellular fluid (86)Rb/plasma ratio after in vivo four-vessel occlusion with reperfusion.

  11. The heliothis virescens 170 kDa aminopeptidase functions as "receptor A" by mediating specific Bacillus thuringiensis Cry1A delta-endotoxin binding and pore formation.

    PubMed

    Luo, K; Sangadala, S; Masson, L; Mazza, A; Brousseau, R; Adang, M J

    1997-01-01

    The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with KD ranging from 41 to 95 nM and a lower affinity site with KD in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb+ release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb+ release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb+ release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.

  12. 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%).

  13. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    SciTech Connect

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R. )

    1989-12-01

    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake.

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

    SciTech Connect

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

    1991-06-01

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

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

    PubMed

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

    2013-02-15

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

  16. 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.

  17. Wound healing: the effect of macrophage and tumour derived angiogenesis factors on skin graft vascularization.

    PubMed Central

    Arnold, F.; West, D.; Kumar, S.

    1987-01-01

    Angiogenic factors prepared from rat Walker 256 mammary carcinoma, (TAF) and activated mouse peritoneal macrophages (MAF), were tested for their ability to stimulate vascularization during healing. They were applied to one of a pair of bilaterally symmetrical, autologous, isotopic, full thickness skin grafts in mice. Blood flow to treated and untreated graft pairs was compared by their uptake of injected 86Rb Cl, at 3 and 7 days after grafting. No difference was detected after treatment with either agent. We conclude that while angiogenic factors are important in vascularization during healing, this normally occurs at a near maximal rate and cannot be further enhanced. PMID:2443156

  18. Cooling dissociates glucose-induced insulin release from electrical activity and cation fluxes in rodent pancreatic islets.

    PubMed Central

    Atwater, I; Goncalves, A; Herchuelz, A; Lebrun, P; Malaisse, W J; Rojas, E; Scott, A

    1984-01-01

    Insulin release and beta-cell membrane potentials in response to glucose at 37 and 27 degrees C have been measured simultaneously in single, micro-dissected, perifused islets of Langerhans from normal mice. Insulin release and 45Ca outflow in response to glucose at 37 and 27 degrees C have been measured simultaneously from perfused islets isolated by collagenase digestion from normal rats. The effect of cooling on beta-cell membrane potassium permeability was assessed by changes in measured membrane potential and input resistance (in the mouse) and by changes in 86Rb outflow (in the rat). Resting and active beta-cell membrane parameters (i.e. membrane potential, spike frequency, input resistance, 45Ca outflow and 86Rb outflow), in both mouse and rat islets, were affected only slightly by cooling to 27 degrees C, with temperature coefficients of 2 or lower. At 27 degrees C glucose-stimulated insulin release was inhibited completely in mouse islets and almost completely in rat islets. The temperature coefficients in both preparations were greater than 5. It is concluded that beta-cell electrical activity and changes in membrane permeability induced by glucose are not consequences of insulin release. PMID:6371219

  19. 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

  20. Extrarenal potassium adaptation: role of skeletal muscle

    SciTech Connect

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

    1986-08-01

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

  1. Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels.

    PubMed Central

    Chen, G.; Suzuki, H.; Weston, A. H.

    1988-01-01

    1. The effects of haemoglobin and methylene blue on the acetylcholine (ACh)-induced electrical and mechanical responses of smooth muscle cells were investigated in rat aorta and rat main pulmonary artery. 2. When the endothelium was intact, ACh induced a transient hyperpolarization and sustained relaxation of tissues precontracted with noradrenaline. Both hyperpolarization and relaxation were absent in preparations without endothelium. 3. Haemoglobin and methylene blue inhibited the ACh-induced relaxation, but not the transient hyperpolarization. 4. In aorta with an intact endothelium, ACh produced an increase in both the rate of 86Rb efflux and tissue cyclic GMP levels. The changes in ion flux were unaffected by either haemoglobin or methylene blue in concentrations which almost abolished the increase in cyclic GMP concentrations. 5. In arteries with an intact endothelium, indomethacin had no effect on the ACh-induced electrical and mechanical responses or on the increase in 86Rb efflux and tissue cyclic GMP levels. 6. It is concluded that in the rat aorta and rat main pulmonary artery, ACh releases two different substances, an endothelium-derived relaxing factor (EDRF) and a hyperpolarizing factor (EDHF), from the endothelial cells. Neither substance appears to be derived from a pathway dependent on cyclo-oxygenase. EDHF seems to play a minor role in the relaxation of noradrenaline-induced contractions. PMID:2851359

  2. 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.

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

    PubMed Central

    Knudsen, T.; Ferjan, I.; Johansen, T.

    1993-01-01

    1. The activity of the Na+/K(+)-pump in rat peritoneal mast cells was measured at various time intervals after induction of cellular histamine release by compound 48/80 or by the antigen-antibody reaction. The Na+/K(+)-pump activity was assessed as the ouabain-sensitive potassium uptake of the cells using 86Rb+ as a tracer for potassium (K+(86Rb+)-uptake). 2. Stimulation of the cells with compound 48/80 induced a time and concentration dependent increase of the Na+/K(+)-pump activity. The pump activity was maximal 2 min after stimulation of the cells. Then, the activity gradually decreased and reached a level not significantly different from the controls after 2 h of incubation. 3. When the cells were stimulated by the antigen-antibody reaction, there was also a rapid (within 5 min) stimulation of the Na+/K(+)-pump. In contrast to the stimulation with compound 48/80, the pump activity returned to the control level after 60 min of incubation with antigen. 4. The ouabain-resistant potassium uptake of the cells was increased after stimulation of the cells, regardless of the secretagogue used. This probably reflects the increased surface area of the cells present after the histamine release. 5. On the basis of the present results, we suggest a role for the Na+/K(+)-pump in the recovery process of the mast cell following histamine release. PMID:7679025

  4. The effects of dietary treatment with essential fatty acids on sciatic nerve conduction and activity of the Na+/K+ pump in streptozotocin-diabetic rats.

    PubMed Central

    Lockett, M. J.; Tomlinson, D. R.

    1992-01-01

    1. This study examined the effects of dietary essential fatty acid supplementation (5% (w/w) evening primrose oil) upon sciatic motor nerve conduction velocity and 86Rb+ pumping in sciatic nerve endoneurial preparations in rats with 4 to 5 weeks of streptozotocin-induced diabetes. 2. Control diabetic rats (dietary supplementation with 5% (w/w) hydrogenated coconut oil) exhibited a reduction in motor nerve conduction velocity (16%; P less than 0.05) compared to similarly-fed non-diabetic controls, but there was no significant alteration in ouabain-sensitive 86Rb+ pumping, a parameter reflecting activity of the Na+/K+ pump. 3. Treatment of diabetic rats with evening primrose oil prevented completely the development of the motor nerve conduction velocity deficit without affecting the severity of diabetes. Evening primrose oil treatment did not significantly affect motor nerve conduction velocity of non-diabetic animals. 4. Evening primrose oil treatment caused a significant reduction in activity of the Na+/K+ pump in sciatic nerves of diabetic animals (45%; P less than 0.05). 5. These results suggest that the acute conduction velocity defect arising in streptozotocin-diabetic rats, and the actions of evening primrose oil upon this, are independent of any effect on activity of the Na+/K+ pump. Other putative mechanisms are discussed. PMID:1313726

  5. 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

  6. Dopamine actions in vitro on enzyme and electrolyte secretion from normal and sympathectomized rat parotid glands.

    PubMed Central

    Danielsson, A; Henriksson, R; Sundström, S; Wester, P

    1988-01-01

    1. Adult rats were denervated unilaterally by removal of the left superior cervical ganglion or chemically denervated with 6-hydroxydopamine or reserpine. Two weeks later the parotid glands were used for in vitro secretory studies and their catecholamines and major metabolites were measured. 2. Noradrenaline concentrations were reduced 2 weeks after surgical sympathectomy and reserpine pre-treatment 18 h previously, whereas 6-hydroxydopamine pre-treatment for 3 days reduced both noradrenaline and dopamine concentrations. 3. Dopamine caused a prominent amylase release from incubated control glands. However, a subsensitivity for dopamine-induced amylase release was recorded on the denervated side. 4. Dopamine caused a prominent potassium efflux measured as 86Rb+ efflux from control glands, but was without effect in denervated glands. This is in contrast to noradrenaline-induced 86Rb+ efflux which was equally effective in both denervated and control glands. 5. Dopamine caused [3H]noradrenaline efflux in control glands, but was without effect in surgically denervated glands and in glands pre-treated with reserpine or 6-hydroxydopamine. 6. It is concluded that dopamine-induced potassium release is caused by a presynaptic action on noradrenergic nerves, whereas dopamine-induced amylase release has a presynaptic and a postsynaptic component. The results suggest a specific action of dopamine in salivary glands, with different effects on enzyme release and ionic fluxes. PMID:2473198

  7. Landscape context alters cost of living in honeybee metabolism and feeding.

    PubMed

    Tomlinson, Sean; Dixon, Kingsley W; Didham, Raphael K; Bradshaw, S Donald

    2017-02-08

    Field metabolic rate (FMR) links the energy budget of an animal with the constraints of its ecosystem, but is particularly difficult to measure for small organisms. Landscape degradation exacerbates environmental adversity and reduces resource availability, imposing higher costs of living for many organisms. Here, we report a significant effect of landscape degradation on the FMR of free-flying Apis mellifera, estimated using (86)Rb radio-isotopic turnover. We validated the relationship between (86)Rb kb and metabolic rate for worker bees in the laboratory using flow-through respirometry. We then released radioisotopically enriched individuals into a natural woodland and a heavily degraded and deforested plantation. FMRs of worker bees in natural woodland vegetation were significantly higher than in a deforested landscape. Nectar consumption, estimated using (22)Na radio-isotopic turnover, also differed significantly between natural and degraded landscapes. In the deforested landscape, we infer that the costs of foraging exceeded energetic availability, and honeybees instead foraged less and depended more on stored resources in the hive. If this is generally the case with increasing landscape degradation, this will have important implications for the provision of pollination services and the effectiveness and resilience of ecological restoration practice.

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

    PubMed Central

    Wrona, Anne F.; Epstein, Emanuel

    1985-01-01

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

  9. Effects of dietary magnesium on sodium-potassium pump action in the heart of rats

    SciTech Connect

    Fischer, P.W.; Giroux, A.

    1987-12-01

    Sprague-Dawley rats were fed a basal AIN-76 diet containing 80, 200, 350, 500 or 650 mg of magnesium per kilogram of diet for 6 wk. Ventricular slices, as well as microsomal fractions, were prepared from the hearts and were used to determine sodium-potassium pump activity. Sodium-potassium pump activity was assessed in the microsomal membranes by determining the ouabain-inhibitable Na+, K+-ATPase activity and (/sup 3/H)ouabain binding, and in the ventricular slices, by determining ouabain-sensitive /sup 86/Rb uptake under K+-free conditions. The ATPase activity increased with increasing dietary magnesium, so that in the hearts of those animals that were fed 500 and 650 mg of magnesium/kg diet, it was significantly greater than the activity in the hearts of the animals fed 80 and 200 mg/kg diet. Similarly, /sup 86/Rb uptake by heart slices from rats fed 500 and 650 mg of magnesium/kg diet was significantly greater than the uptake by heart slices from animals fed 80 and 200 mg/kg diet. (/sup 3/H)Ouabain binding did not change with increasing dietary magnesium. Thus, magnesium deficiency appears to have no effect on the number of sodium-potassium pump sites, but does decrease the activity of the pump. It is suggested that this leads to an increase in intracellular Na+, resulting in a change in the membrane potential, and may contribute to the arrhythmias associated with magnesium deficiency.

  10. Stimulation of Growth and Ion Uptake in Bean Leaves by Red and Blue Light 1

    PubMed Central

    Blum, Dale E.; Elzenga, J. Theo M.; Linnemeyer, Paul A.; Van Volkenburgh, Elizabeth

    1992-01-01

    Red and blue light both stimulate growth and ion accumulation in bean (Phaseolus vulgaris L.) leaves, and previous studies showed that the growth response is mediated by phytochrome and a blue-light receptor. Results of this study confirm that there is an additional photosynthetic contribution from the growing cells that supports ion uptake and growth. Disc expansion in the light was enhanced by exogenous K+ and Rb+, but was not specific for anions. Light increased K+ accumulation and the rate of 86Rb+ uptake by discs, over darkness, with no effect of light quality. The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, inhibited light-driven 86Rb+ uptake by 75%. Light quality caused differences in short-term kinetics of growth and acidification of the leaf surface. At comparable fluence rates (50 μmol m−2 s−1), continuous exposure to blue light increased the growth rate 3-fold after a 2-min lag, whereas red light caused a smaller growth response after a lag of 12 min. In contrast, the acidification of the leaf surface normally associated with growth was stimulated 3-fold by red light but only slightly (1.3-fold) by blue light. This result shows that, in addition to acidification caused by red light, a second mechanism specifically stimulated by blue light is normally functioning in light-driven leaf growth. PMID:16653225

  11. Stimulus-secretion coupling of arginine-induced insulin release. Uptake of metabolized and nonmetabolized cationic amino acids by pancreatic islets

    SciTech Connect

    Blachier, F.; Mourtada, A.; Sener, A.; Malaisse, W.J.

    1989-01-01

    In order to assess the possible role of L-arginine accumulation in islet cells as a determinant of its insulinotropic action, the uptake of L-arginine and other cationic amino acids (L-ornithine, L-homoarginine, D,L-alpha-methylornithine, D,L-alpha-difluoromethylornithine) by rat pancreatic islets was compared to the ionic and secretory responses of the islets to the same amino acids. A tight correlation was found between the net uptake of these amino acids and their capacity to stimulate 86Rb efflux, 45Ca uptake and efflux, and insulin release. In the latter respect, there was little difference between metabolized and nonmetabolized amino acids. Thus, although L-homoarginine and 4-amino-1-guanylpiperidine-4-carboxylic acid failed to act as a substrate for either arginase or amino acid aminotransferase in islet homogenates, they both stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin secretion in intact islets. These findings are compatible with the view that the accumulation of these positively charged amino acids in islet cells represents an essential determinant of their secretory action. Hence, the release of insulin evoked by these amino acids could be due to depolarization of the plasma membrane with subsequent gating of voltage-sensitive Ca2+ channels and/or to some other biophysical effect, as suggested by the persistence of a sizeable secretory response to L-arginine or L-ornithine in islets perifused at a high concentrations of extracellular K+ (50 mM).

  12. Bradykinin-stimulated calcium influx in cultured bovine aortic endothelial cells

    SciTech Connect

    Schilling, W.P.; Ritchie, A.K.; Navarro, L.T.; Eskin, S.G. Univ. of Texas Medical Branch, Galveston )

    1988-08-01

    Bradykinin (BK)-stimulated release of endothelium-derived relaxing factor has been linked to a rise in cytosolic Ca{sup 2+} concentration and a change of K{sup +} permeability of the endothelial cell. In the present study, measurement of BK-induced changes in fura-2 fluorescence and {sup 86}Rb{sup +} efflux were used to monitor changes in cytosolic Ca{sup 2+} and K{sup +} permeability in cultured bovine aortic endothelial cells. In the presence of normal extracellular Ca{sup 2+}, BK induced a fourfold increase in cytosolic Ca{sup 2+}, which peaked at 20 s and declined within 1 min to a value that was 50% of the peak level. Subsequently, cytosolic Ca{sup 2+} decreased and approached basal levels within 8 min. In the absence of Ca{sup 2+}, BK produced a 1.5- to 2-fold increase in cytosolic Ca{sup 2+} that peaked within 20 s and declined to basal levels within 2 min. Addition of Ca{sup 2+} to the Ca-free reaction buffer 3-5 min after addition of BK resulted in a two-to three-fold increase in cytosolic Ca{sup 2+} that declined slowly back to basal levels. Thus Ca{sup 2+} influx can occur in response to BK at a time when there is minimal elevation of cytosolic Ca{sup 2+} above the resting level. Under all conditions tested, {sup 86}Rb{sup +} efflux paralleled changes in the cytosolic Ca{sup 2+}, suggesting that efflux occurred through Ca{sup 2+}-activated K{sup +} channels. Isosmotic substitution of Na{sup +} with N-methyl-D-glucamine did not affect the BK-stimulated changes in cytosolic Ca{sup 2+} or {sup 86}Rb{sup +} efflux, suggesting that Na{sup +}-Ca{sup 2+} exchange plays little role in the BK response. These results suggest that BK stimulates Ca{sup 2+} influx via a BK receptor-operated channel or a channel activated by some internal messenger other than Ca{sup 2+}.

  13. Locally formed dopamine inhibits Na sup + -K sup + -ATPase activity in rat renal cortical tubule cells

    SciTech Connect

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J. Karolinska Institute, Stockholm )

    1988-10-01

    Dopamine, generated locally from L-dopa, inhibits Na{sup +}-K{sup +}-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na{sup +}-K{sup +}-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate ({dot Q}o{sub 2}) and {sup 86}Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive {dot Q}o{sub 2} or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive {dot Q}o{sub 2} in a concentration-dependent manner, with half-maximal inhibition (K{sub 0.5}) of 5 {times} 10{sup {minus}7} M and a maximal inhibition of 14.1 {plus minus} 1.5% at 10{sup {minus}4}M. L-Dopa also blunted the nystatin-stimulated {dot Q}o{sub 2} in a concentration-dependent manner, indicating the L-dopa directly inhibits Na{sup +}-K{sup +}-ATPase activity and not sodium entry. Ouabain-sensitive {sup 86}Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} and {sup 86}Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na{sup +}-K{sup +}-ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner.

  14. The alpha1-Na/K pump does not mediate the involvement of ouabain in the development of hypertension in rats.

    PubMed

    Orlov, Sergei N; Taurin, Sebastien; Hamet, Pavel

    2002-01-01

    The Na/K pump of vascular smooth muscle cells (VSMC) and renal epithelial cells (REC) is viewed as a target of digitalis and endogenous ouabain (EO), leading to the development of hypertension. In this study, we compared the effect of ouabain on Na/K pump activity and the intracellular content of monovalent cations in VSMC and REC obtained from rats, humans and dogs. In VSMC from the rat aorta, ouabain inhibited maximal Na/K pump activity measured as the rate of 86Rb influx in Na+-loaded cells, with an ID50 of approximately 20-30 microM without any differences between two strains of normotensive rats (WKY and BN.1x) and three substrains of spontaneously hypertensive rats (SHR). Half-maximal inhibition of the Na/K pump in REC from the rat inner medullary collecting duct was observed at approximately 20 microM of ouabain. In contrast to rat cells, half-maximal inhibition of 86Rb influx in VSMC from human coronary arteries and in REC from the Madin-Darby canine kidney was seen at approximately 0.03 and 0.1 microM ouabain, respectively. At concentrations lower than 100 microM, ouabain did not affect the intracellular content of exchangeable Na+ and K+ in rat VSMC, measured as the steady-state distribution of 22Na and 86Rb, whereas in human VSMC, it increased the intracellular Na+/K+ ratio with an ID50 of approximately 0.5 microM. Keeping in mind that the circulating level of administered digitalis and EO does not exceed 10(-9) M, our results strongly suggest that the involvement of these compounds in the pathogenesis of hypertension in rats is not mediated by inhibition of the alpha1-isoform of the Na/K pump in VSMC and REC. Alternative mechanisms of the involvement of EO and ouabain-like factors in the development of hypertension are considered.

  15. Introduction of unsaturation into the N-n-alkyl chain of the nicotinic receptor antagonists, NONI and NDNI: effect on affinity and selectivity.

    PubMed

    Sumithran, Sangeetha P; Crooks, Peter A; Xu, Rui; Zhu, Jun; Deaciuc, Agripina G; Wilkins, Lincoln H; Dwoskin, Linda P

    2005-08-29

    N-n-octylnicotinium iodide (NONI) and N-n-decylnicotinium iodide (NDNI) are selective nicotinic receptor (nAChR) antagonists mediating nicotine-evoked striatal dopamine (DA) release, and inhibiting [3H]nicotine binding, respectively. This study evaluated effects of introducing unsaturation into the N-n-alkyl chains of NONI and NDNI on inhibition of [3H]nicotine and [3H]methyllycaconitine binding (alpha4beta2* and alpha7* nAChRs, respectively), (86)Rb+ efflux and [3H]DA release (agonist or antagonist effects at alpha4beta2* and alpha6beta2*-containing nAChRs, respectively). In the NONI series, introduction of a C3-cis- (NONB3c), C3-trans- (NONB3t), C7-double-bond (NONB7e), or C3-triple-bond (NONB3y) afforded a 4-fold to 250-fold increased affinity for [3H]nicotine binding sites compared with NONI. NONB7e and NONB3y inhibited nicotine-evoked 86Rb+ efflux, indicating alpha4beta2* antagonism. NONI analogs exhibited a 3-fold to 8-fold greater potency inhibiting nicotine-evoked [3H]DA overflow compared with NONI (IC50 = 0.62 microM; Imax = 89%), with no change in Imax, except for NONB3y (Imax = 50%). In the NDNI series, introduction of a C4-cis- (NDNB4c), C4-trans-double-bond (NDNB4t), or C3-triple-bond (NDNB3y) afforded a 4-fold to 80-fold decreased affinity for [3H]nicotine binding sites compared with NDNI, whereas introduction of a C9 double-bond (NDNB9e) did not alter affinity. NDNB3y and NDNB4t inhibited nicotine-evoked 86Rb+ efflux, indicating antagonism at alpha4beta2* nAChRs. Although NDNI had no effect, NDNB4t and NDNB9e potently inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.02-0.14 microM, Imax = 90%), as did NDNB4c (IC50 = 0.08 microM; Imax = 50%), whereas NDNB3y showed no inhibition. None of the analogs had significant affinity for alpha7* nAChRs. Thus, unsaturated NONI analogs had enhanced affinity at alpha4beta2*- and alpha6beta2*-containing nAChRs, however a general reduction of affinity at alpha4beta2* and an uncovering of antagonist effects at

  16. Low efficiency of functional translation of ouabain-resistant α2-Na(+),K(+)-ATPase mRNA in C7-MDCK epithelial cells.

    PubMed

    Akimova, Olga A; Van Huysse, James; Tremblay, Johanne; Orlov, Sergei N

    2012-01-01

    Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.

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

    PubMed

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

    2015-04-01

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

  18. 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.

  19. 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.

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

    SciTech Connect

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

    1989-08-01

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

  1. Some metabolic effects of overeating in man.

    PubMed

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

    1986-12-01

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

  2. 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.

  3. Actions of vanadate on vascular tension and sodium pump activity in cat isolated cerebral and femoral arteries.

    PubMed Central

    Sánchez-Ferrer, C. F.; Marín, J.; Lluch, M.; Valverde, A.; Salaices, M.

    1988-01-01

    1. The mechanisms involved in the responses induced by sodium vanadate (Va3 VO4) on cat cerebral and femoral arteries were studied. The possibility that these responses were due to Na+, K+-ATPase inhibition was investigated by measuring the effect of vanadate on [3H]-ouabain binding to arterial membrane fractions, K+-induced vasodilatation and ouabain-sensitive 86Rb+ uptake. 2. The vanadium compounds (Na3VO4, VOSO4, VCl3 and O5V3) induced similar, concentration-dependent contractions in each kind of artery, the cerebral vessels being the most sensitive to these compounds. 3. Exposure of the arteries to a low-Na+ (25 mM) solution suppressed the contraction caused by vanadate in femoral but not in cerebral arteries. 4. Vanadate-induced contractions were reduced in Ca2+-free medium but remained unaffected by 3 x 10(-6) M phentolamine, reserpine pretreatment or 3 x 10(-6) M verapamil in both kinds of artery. 5. The addition of 7.5 mM K+ to the arteries immersed in a K+-free solution induced vasodilatation, which was not modified by 10(-3) M vanadate. 6. The consecutive administration of ouabain (10(-4) M) and vanadate (10(-3) M) (or vice versa), or the simultaneous administration of both agents (10(-8) to 10(-3) M) appeared to produce an additive contraction in both types of artery. 7. Vanadate (10(-7) to 10(-3) M) did not displace the [3H]-ouabain binding to arterial membrane fractions of these arteries, whereas 10(-4) M ouabain did. 8. In both kinds of artery, total 86Rb+ uptake was reduced by ouabain (10(-8) to 10(-3) M), in a concentration-dependent manner, whereas it was not modified by vanadate (10(-8)-10(-3) M).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3349233

  4. 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.

  5. 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.

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

    SciTech Connect

    Renz, C.D.

    1986-01-01

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

  7. Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics.

    PubMed Central

    Koski, C L; Ramm, L E; Hammer, C H; Mayer, M M; Shin, M L

    1983-01-01

    Lysis of nucleated cells by complement was studied to determine whether the lytic process by C5b-9 conforms to a one-hit mechanism as in the case of erythrocytes. Two nucleated cell lines, Molt 4 and U937, derived from human T lymphocytes and histiocytes, respectively, were employed as targets. The antibody-sensitized cells were used to develop the titration curves, measuring cell death as a function of limiting quantities of human C6 or C5,6 complex in the presence of an excess of other complement components. The cytolysis curves generated in both experiments were sigmoidal, in sharp contrast to the monotonic curves observed in lysis of erythrocytes treated similarly. The sigmoidal curves of cytolysis indicate a cooperative action of several molecules of C6 or acid-activated C5,6 complex, C(56)a. In contrast to the multi-hit characteristics of cytolysis, dose-response measurements of the release of 86Rb indicated that only one effective molecule of C6 per cell is required for assembly of a 86Rb-releasing channel. This divergence indicates that lysis requires formation of several channels or, alternatively, assembly of large channels that are formed by several molecules of C6. Because prior studies with erythrocyte ghosts have shown that only a single effective molecule of C6 is required for assembly of a transmembrane channel, regardless of size, we prefer to interpret the multi-hit characteristics of nucleated cell lysis as an indication of a multi-channel requirement, rather than channel enlargement. PMID:6602341

  8. 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.

  9. In rat hepatocytes, the hypertonic activation of Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is mediated by protein kinase C.

    PubMed

    Heinzinger, H; van den Boom, F; Tinel, H; Wehner, F

    2001-11-01

    1. The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and bis-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures. 3. Stimulation of Na(+) conductance was monitored: (i) by use of a differential approach based on Na(+) fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na(+) pulses. All three experimental protocols in concert demonstrated a block of the activation of Na(+) conductance by staurosporine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na(+)-H(+) antiport (determined fluorometrically). 6. Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na(+)/K(+)-ATPase (determined as ouabain-sensitive (86)Rb(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is probably mediated by PKC.

  10. 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.

  11. Carbohydrate analyses of Manduca sexta aminopeptidase N, co-purifying neutral lipids and their functional interactions with Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Sangadala, S; Azadi, P; Carlson, R; Adang, M J

    2001-12-01

    Bacillus thuringiensis Cry1Ac insecticidal toxin binds specifically to 120kDa aminopeptidase N (APN) (EC 3.4.11.2) in the epithelial brush border membrane of Manduca sexta midguts. The isolated 120-kDa APN is a member of a functional Cry1 toxin receptor complex (FEBS Lett. 412 (1997) 270). The 120-kDa form is glycosyl-phosphatidylinositol (GPI) anchored and converted to a 115-kDa form upon membrane solubilization. The 115-kDa APN also binds Cry1A toxins and Cry1Ac binding is inhibited by N-acetylgalactosamine (GalNAc). Here we determined the monosaccharide composition of APN. APN is 4.2mol% carbohydrate and contains GalNAc, a residue involved in Cry1Ac interaction. APN remained associated with non-covalently bound lipids through anion-exchange column purification. Most associated lipids were separated from APN by hydrophobic interaction chromatography yielding a lipid aggregate. Chemical analyses of the lipid aggregate separated from APN revealed neutral lipids consisting mostly of diacylglycerol and free fatty acids. The fatty acids were long, unsaturated chains ranging from C:14 to C:22. To test the effect of APN-associated lipids on Cry1Ac function, the lipid aggregate and 115-kDa APN were reconstituted into phosphatidylcholine (PC) vesicles. The lipid aggregate increased the amount of Cry1Ac binding, but binding due to the lipid aggregate was not saturable. In contrast the lipid aggregate promoted Cry1Ac-induced release of 86Rb(+) at the lowest Cry1Ac concentration (50nM) tested. The predominant neutral lipid component extracted from the lipid aggregate promoted Cry1Ac-induced 86Rb(+) release from membrane vesicles in the presence of APN.

  12. Membrane-permeabilizing activities of cyclic lipodepsipeptides, syringopeptin 22A and syringomycin E from Pseudomonas syringae pv. syringae in human red blood cells and in bilayer lipid membranes.

    PubMed

    Agner, G; Kaulin, Y A; Gurnev, P A; Szabo, Z; Schagina, L V; Takemoto, J Y; Blasko, K

    2000-12-01

    The pore-forming activities of cyclic lipodepsipeptides (CLPs), syringopeptin 22A (SP22A) and syringomycin E (SRE) were compared on the human red blood cell (RBC) membrane and on bilayer lipid membranes (BLMs). SP22A above a concentration of 4 x 10(5) molecules/cell significantly increased the RBC membrane permeability for 86Rb. With electric current measurements on BLM, it was proved that like SRE, the SP22A formed two types of ion channels in the membrane, small and large, the latter having six times larger conductance and longer dwell time. Both CLPs formed clusters consisting of six small channels, and the channel-forming activity of SP22A is about one order of magnitude higher than that of SRE. A Hill coefficient of 2-3 estimated from the concentration dependence of these CLPs-induced lysis gave a proof of the pore oligomerization on RBCs. Transport kinetic data also confirmed that SP22A pores were oligomers of at least three monomers. While SRE pores were inactivated in time, no pore inactivation was observed with SP22A. The 86Rb efflux through SP22A-treated RBCs approached the tracer equilibrium distribution with a constant rate; a constant integral current was measured on the BLM for as long as 2.5 h as well. The partition coefficient (Kp = 2 x 10(4) l/mol) between the RBC membrane and the extracellular space was estimated for SRE to be at least six times higher than that for SP22A. This finding suggested that the higher ion permeability of the SP22A-treated cells compared to that of SRE was the result of the higher pore-forming activity of SP22A.

  13. Isovolumetric regulation mechanisms in cultured cerebellar granule neurons.

    PubMed

    Tuz, K; Ordaz, B; Vaca, L; Quesada, O; Pasantes-Morales, H

    2001-10-01

    Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).

  14. Quaternary Benzyltriethylammonium Ion Binding to the Na,K-ATPase: a Tool to Investigate Extracellular K+ Binding Reactions†

    PubMed Central

    Peluffo, R. Daniel; González-Lebrero, Rodolfo M.; Kaufman, Sergio B.; Kortagere, Sandhya; Orban, Branly; Rossi, Rolando C.; Berlin, Joshua R.

    2009-01-01

    This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (VM)-dependent inhibition and tested the prediction that such a VM-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K+ activation of Na,K-ATPase activity and steady-state 86Rb+ occlusion. The initial rate of 86Rb+ occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb+, a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a VM-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K+-K+ exchange by the Na,K-ATPase, step changes in VM elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K+ congener, Tl+. pNBTEA- and Tl+-dependent transient currents both displayed saturation of charge moved at extreme negative and positive VM, equivalence of charge moved during and after step changes in VM, and similar apparent valence. The rate constant (ktot) for Tl+-dependent transient current asymptotically approached a minimum value at positive VM. In contrast, ktot for pNBTEA-dependent transient current was a “U”-shaped function of VM with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly-accessible sites, one of them located at K+ binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme. PMID:19621894

  15. 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.

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

    SciTech Connect

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

    1985-10-01

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

  17. Role of Na+-K+ ATPase in cyclic GMP-mediated relaxation of canine pulmonary artery smooth muscle cells

    PubMed Central

    Tamaoki, J; Tagaya, E; Nishimura, K; Isono, K; Nagai, A

    1997-01-01

    Sodium-potassium adenosine triphosphatase (Na+-K+ ATPase) plays a role in the regulation of vascular tone, but contribution of this enzyme to nitrovasodilator-induced pulmonary vasodilatation remains uncertain. We thus studied the interaction between guanosine 3′:5′-cyclic monophosphate (cyclic GMP) and Na+-K+ ATPase in smooth muscle cells isolated from canine pulmonary artery. To assess the contractile properties, changes in smooth muscle cell length were determined microscopically. Application of potassium chloride (KCl) shortened the cell length, an effect which was reduced by sodium nitroprusside and 8-bromo-cyclic GMP in a concentration-dependent manner. Pretreatment of cells with the cyclic GMP-dependent kinase inhibitor KT 5823 (2 μM) abolished the effects of sodium nitroprusside and 8-bromo-cyclic GMP. Ouabain (0.3 μM) did not alter the KCl-induced muscle shortening, but inhibited the relaxant responses to sodium nitroprusside and 8-bromo-cyclic GMP. Incubation of smooth muscle cells with sodium nitroprusside concentration-dependently increased intracellular cyclic GMP levels and ouabain-sensitive 86Rb uptake, and these values were significantly correlated. In the presence of KT 5823, sodium nitroprusside increased cyclic GMP levels but did not alter ouabain-sensitive 86Rb uptake. These results suggest that there is a link between accumulation of intracellular cyclic GMP and activation of sarcolemmal Na+-K+ ATPase in pulmonary artery smooth muscle cells and that this link may be involved in the sodium nitroprusside-induced pulmonary vasodilatation. PMID:9298536

  18. Potassium Transport in Corn Roots 1

    PubMed Central

    Kochian, Leon V.; Lucas, William J.

    1985-01-01

    It has recently been reported that plasmalemma electron transport may be involved in the generation of H+ gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K+ (86Rb+) influx, K+ (86Rb+) efflux, net apparent H+ efflux, and O2 consumption in 2-centimeter corn (Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O2 consumption and K+ uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O2 consumption but caused a dramatic inhibition of K+ influx. Moreover, net apparent H+ efflux was significantly inhibited following NADH exposure in 4-hour washed root segments. Exogenous ferricyanide inhibited K+ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H+ efflux. Additionally, both reagents substantially altered K+ efflux at both the plasmalemma and tonoplast. These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots. PMID:16664070

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

    PubMed Central

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

    1979-01-01

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

  20. Studies on the localization of the cardiac glycoside receptor

    PubMed Central

    Smith, Thomas W.; Wagner, Henry; Markis, John E.; Young, Michael

    1972-01-01

    The purpose of this study was to see whether the receptor for cardiac glycosides might be localized upon or within the plasma membrane of digitalis-sensitive cells. Ouabain and digoxin were joined covalently to several large protein molecules. These macromolecular conjugates are too large to enter intact cells; consequently, any pharmacologic or biochemical effects which they display should arise from interaction with a cell surface receptor. Conjugates were tested in several cardiac glycoside-sensitive systems: (a), contractility response of isolated cardiac muscle; (b), active 86Rb+ uptake by red cells; (c), enzymatic activity of isolated myocardial microsomal (Na+ + K+)-activated adenosine triphosphatase (ATPase); and (d), enzymatic activity of solubilized red cell (Na+ + K+)-activated ATPase. Results demonstrated that in all of these systems, the macromolecular-glycoside conjugates were 100- to 1000-fold less active than the free glycosides. Careful chromatographic examination of the various conjugates revealed that they contained a small but persistent free cardiac glycoside contaminant. The amount of this species ranged from 0.1 to 1.0% of the total macromolecule-bound glycoside, and its presence fully explains the levels of biologic activity observed with the conjugates. To try to minimize steric factors which could interfere with glycoside-receptor interaction, digoxin and ouabain were also coupled to macromolecule via long, flexible polyamide side-chains. These extended chain conjugates, in which the cardiac glycoside potentially lay some 30 A removed from the surface of the macromolecule, also exhibited negligible digitalis-like effects when tested upon isolated cardiac muscle, red cell 86Rb+ uptake, and enzymatic activity of cardiac microsomal (Na+ + K+)-ATPase. However, the extended chain conjugates were fully active when examined with the solubilized red cell (Na+ + K+)-ATPase system. To further ensure that the chemical reactions used to couple

  1. Mild Heating of Amphotericin B-Desoxycholate: Effects on Ultrastructure, In Vitro Activity and Toxicity, and Therapeutic Efficacy in Severe Candidiasis in Leukopenic Mice

    PubMed Central

    van Etten, Els W. M.; van Vianen, Wim; Roovers, Patty; Frederik, Peter

    2000-01-01

    Heated (20 min at 70°C) amphotericin B-desoxycholate (hAMB-DOC) was further characterized, as was another formulation obtained after centrifugation (60 min, 3000 × g), hcAMB-DOC. Conventional AMB-DOC consisted of individual micelles (approximately 4 nm in diameter) and threadlike aggregated micelles, as revealed by cryo-transmission electron microscopy. For both hAMB-DOC and hcAMB-DOC, pleiomorphic cobweb structures were observed with a mean particle size of approximately 300 nm as determined by laser diffraction. The potent antifungal activity of AMB-DOC against Candida albicans is not reduced by heating. Effective killing of C. albicans (>99.9% within 6 h) was obtained at 0.1 mg/liter with each of the AMB formulations. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release (86Rb+) from C. albicans of ≥50% was observed at 0.8, 0.4, and 0.4 mg/liter, respectively. After heating of AMB-DOC, toxicity was reduced 16-fold as determined by red blood cell (RBC) lysis. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, hemolysis of ≥50% was observed at 6.4, 102.4, and 102.4 mg/liter, respectively. In contrast, AMB-DOC and its derivates showed similar toxicities in terms of cation release from RBC. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release (86Rb+) of ≥50% was observed at 1.6, 0.8, and 0.8 mg/liter, respectively. In persistently leukopenic mice with severe invasive candidiasis, higher dosages of both hAMB-DOC and hcAMB-DOC were tolerated than those of conventional AMB-DOC (3 versus 0.8 mg/kg of body weight, respectively), resulting in significantly improved therapeutic efficacy. In conclusion, this new approach of heating AMB-DOC may be of great value for further optimizing the treatment of severe fungal infections. PMID:10817715

  2. Palytoxin acts on Na+,K+-ATPase but not non-gastric H+,K+-ATPase

    PubMed Central

    Guennoun-Lehmann, Saida; Fonseca, James E.; Horisberger, Jean-Daniel; Rakowski, Robert F.

    2007-01-01

    Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on non-gastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α2- and Na,K-ATPase β2-subunits; Bufo Na,K-ATPase α1- and Na,K-ATPase β2-subunits; and Bufo Na,K-ATPase β2-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μM ouabain. Functional expression was confirmed by measuring 86Rb uptake. PTX (5 nM) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β2-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α1-subunit and Na,K-ATPase β1-subunit; rat Na,K-ATPase α2-subunit and Na,K-ATPase β2-subunit; and rat Na,K-ATPase β1 or Na,K-ATPase β2 subunit alone. Measurement of increases in 86Rb uptake confirmed that both rat Na,K- and H,K-pumps were functional in HeLa cells expressing rat colonic HKα1/NKβ1 and NKα2/NKβ2. Whole-cell patch clamp measurements in HeLa cells expressing rat colonic HKα1/NKβ1 exposed to 100 nM PTX showed no significant increase of membrane current and there was no membrane conductance increase in HeLa cells transfected with rat NKβ1 or rat NKβ2 subunits alone. However, in HeLa Cells expressing rat NKα2 NKβ2, outward current was observed after pump activation by 20 mM K+ and a large membrane conductance increase occurred after 100 nM PTX. We conclude that non-gastric H,K-ATPases are not sensitive to palytoxin when expressed in these cells whereas palytoxin does act on Na,K-ATPase. PMID:17639367

  3. Housing and Husbandry of Xenopus laevis Affect the Quality of Oocytes for Heterologous Expression Studies

    PubMed Central

    Delpire, Eric; Gagnon, Kenneth B; Ledford, Jonathan J; Wallace, Jeanne M

    2011-01-01

    To assess the effect of Xenopus husbandry on oocyte quality for membrane transport physiology experiments, we compared a recirculating-water housing system with a static-water system in a 23-mo study. Two groups of frogs (n = 8) were maintained separately for the entire study: one group was housed in a multiinvestigator centrally managed Xenopus facility, which consists of 33 tanks placed on a shared and recirculating water system; the other group was housed in a satellite facility used by a single investigator and consisting of static tanks placed in a dedicated cold-room. The activity of a heterologously expressed membrane transporter was assessed every 4 to 5 wk for a total of 23 mo. Activity of the mouse cotransporter NKCC1 was assessed through isotopic 86Rb influx measurements under 2 experimental conditions: stimulation of cotransporter by coinjection of regulatory kinases and by exposure to a hypertonic solution. The results showed a significant difference in the level of ion fluxes under these 2 experimental conditions between the 2 groups of oocytes. During the entire period, oocytes isolated from frogs maintained in the static facility demonstrated consistently robust NKCC1 function, whereas oocytes isolated from frogs maintained in the recirculating facility showed inconsistent and weaker cotransporter function. Furthermore, the oocytes isolated from frogs maintained in the recirculating facility showed significant deterioration during the summer months (April to August), a seasonal variation that was muted in frog oocytes maintained in the static facility. PMID:21333163

  4. 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.

  5. 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.

  6. UV-B-Induced Damage to the Lens In Vitro: Prevention by Caffeine

    PubMed Central

    Hegde, Kavita R.; Kovtun, Svitlana

    2008-01-01

    Abstract Ultraviolet (UV) irradiation is one of the significant risk factors in the genesis of cataracts. Pathogenetically, the process can be triggered by the intraocular generation of various reactive species of oxygen that are well known to be initiated by the penetration of light, especially of the UV frequencies. The contribution of UV exposure in the etiology of this disease is likely to increase further due to ozone depletion in the upper atmosphere. The present studies were undertaken to examine if the UV effects can be attenuated with the xanthine-based alkaloids primarily present in tea and coffee. We have examined this possibility by in vitro lens culture studies with caffeine. As expected, mice lenses incubated in Tyrode solution exposed to UV at 302 nm are physiologically damaged, as evidenced by the inhibition of the active transport of 86Rb+, an ion acting as a surrogate of the K+. There was a simultaneous decrease in the levels of adenosine triphosphate and glutathione. The addition of caffeine to the medium prevented such deleterious effects. That caffeine and perhaps other xanthinoids have a protective effect against cataract formation induced by UV has hence been demonstrated for the first time. PMID:18788993

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

    PubMed

    Freed, B R; Gelbard, A S

    1982-01-01

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

  8. Identification of Yeast Genes Involved in K+ Homeostasis: Loss of Membrane Traffic Genes Affects K+ Uptake

    PubMed Central

    Fell, Gillian L.; Munson, Amanda M.; Croston, Merriah A.; Rosenwald, Anne G.

    2011-01-01

    Using the homozygous diploid Saccharomyces deletion collection, we searched for strains with defects in K+ homeostasis. We identified 156 (of 4653 total) strains unable to grow in the presence of hygromycin B, a phenotype previously shown to be indicative of ion defects. The most abundant group was that with deletions of genes known to encode membrane traffic regulators. Nearly 80% of these membrane traffic defective strains showed defects in uptake of the K+ homolog, 86Rb+. Since Trk1, a plasma membrane protein localized to lipid microdomains, is the major K+ influx transporter, we examined the subcellular localization and Triton-X 100 insolubility of Trk1 in 29 of the traffic mutants. However, few of these showed defects in the steady state levels of Trk1, the localization of Trk1 to the plasma membrane, or the localization of Trk1 to lipid microdomains, and most defects were mild compared to wild-type. Three inositol kinase mutants were also identified, and in contrast, loss of these genes negatively affected Trk1 protein levels. In summary, this work reveals a nexus between K+ homeostasis and membrane traffic, which does not involve traffic of the major influx transporter, Trk1. PMID:22384317

  9. 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.

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

    PubMed

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

    2011-03-01

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

  11. Effect of pinacidil on norepinephrine- and potassium-induced contractions and membrane potential in rat and human resistance vessels and in rat aorta

    SciTech Connect

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

    1988-01-01

    The effect of pinacidil on contractile responses to norepinephrine, potassium, and membrane potential was examined in rat and human resistance vessels. In some experiments rat aorta was also used. Pinacidil (0.1-30 microM) caused a concentration-dependent relaxation of norepinephrine-induced contractions in all vessels studied. In the same concentration range, pinacidil had only little effect on potassium (125 mM) activated rat mesenteric and femoral resistance vessels. In denervated rat mesenteric resistance vessels, a depolarization with potassium (125 mM) before superimposing a norepinephrine tone markedly diminished the effect of pinacidil. In resting rat mesenteric resistance vessels, pinacidil (1-10 microM) caused a hyperpolarization of 10-15 mV. In rat aorta, pinacidil (10 microM) caused a significant (p less than 0.001) increase in /sup 86/Rb+ efflux rate constant whereas 1 microM had no effect. The results of these experiments indicate that the vasodilating effect may be caused by a hyperpolarization of the vascular smooth muscle cell membrane.

  12. gamma-Aminobutyric acid agonists and antagonists alter chloride flux across brain membranes.

    PubMed

    Allan, A M; Harris, R A

    1986-05-01

    gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, increases membrane chloride conductance. Previously, we reported that GABA increases 36Cl- uptake by membrane vesicles (microsacs) prepared from mouse brain. Employing this technique, we found that the GABAA agonists, muscimol, isoguvacine, 4,5,6,7-tetrahydroisoxazolo(5,4-C)pyridine-3-ol, and 3-amino-1-propane sulfonate, all produced a concentration-dependent increase in 36Cl- influx, but baclofen, a GABAB agonist, failed to alter 36Cl- flux. Inhibition of GABA-dependent 36Cl- influx was produced by the convulsant drugs, bicuculline, picrotoxin, and pentylenetetrazole. Ion specificity was demonstrated by a failure of GABA agonists to stimulate influx of 45Ca2+, 86Rb+, 22Na+, or 35SO4(2). GABA-stimulated uptake of 36Cl- was largest in cortex and cerebellum and smaller in hippocampus and striatum. There was little difference in sensitivity to GABA among the areas. Analysis of subcellular fractions prepared from mouse brain demonstrated that the GABA-dependent 36Cl- influx was enriched in the synaptosomal fraction. The nonspecific (GABA-independent) uptake of 36Cl- was enriched in the myelin fraction. These experiments provide evidence for a functional coupling among GABA receptors and the chloride ionophore and suggest that the GABA-activated chloride channel is a site of action for several convulsant compounds.

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

    SciTech Connect

    Kim, M.S.; Akera, T.

    1987-02-01

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

  14. Cell volume regulation in hemoglobin CC and AA erythrocytes

    SciTech Connect

    Berkowitz, L.R.; Orringer, E.P.

    1987-03-01

    Swelling hemoglobin CC erythrocytes stimulates a ouabain-insensitive K flux that restores original cell volume. Studies were performed with the K analog, /sup 86/Rb. This volume regulatory pathway was characterized for its anion dependence, sensitivity to loop diuretics, and requirement for Na. The swelling-induced K flux was eliminated if intracellular chloride was replaced by nitrate and both swelling-activated K influx and efflux were partially inhibited by 1 mM furosemide or bumetanide. K influx in swollen hemoglobin CC cells was not diminished when Na in the incubation medium was replaced with choline, indicating Na independence of the swelling-induced flux. Identical experiments with hemoglobin AA cells also demonstrated a swelling-induced increase in K flux, but the magnitude and duration of this increase were considerably less than that seen with hemoglobin CC cells. The increased K flux in hemoglobin AA cells was likewise sensitive to anion replacement and to loop diuretics and did not require the presence of Na. These data indicate that a volume-activated K pathway with similar transport characteristics exists in both hemoglobin CC and AA red cells.

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

    SciTech Connect

    Lin, J.D.

    1988-01-01

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

  16. Early Copper-Induced Leakage of K+ from Arabidopsis Seedlings Is Mediated by Ion Channels and Coupled to Citrate Efflux1

    PubMed Central

    Murphy, Angus S.; Eisinger, William R.; Shaff, Jon E.; Kochian, Leon V.; Taiz, Lincoln

    1999-01-01

    Copper tolerance among Arabidopsis ecotypes is inversely correlated with long-term K+ leakage and positively correlated with short-term K+ leakage (A. Murphy, L. Taiz [1997] New Phytol 136: 211–222). To probe the mechanism of the early phase of K+ efflux, we tested various channel blockers on copper and peroxide-induced K+ efflux from seedling roots. The K+ channel blockers tetraethyl ammonium chloride and 4-aminopyridine (4-AP) both inhibited short-term copper-induced K+ efflux. In contrast, peroxide-induced K+ efflux was insensitive to both tetraethyl ammonium chloride and 4-AP. Copper-induced lipid peroxidation exhibited a lag time of 4 h, while peroxide-induced lipid peroxidation began immediately. These results suggest that short-term copper-induced K+ efflux is mediated by channels, while peroxide-induced K+ efflux represents leakage through nonspecific lesions in the lipid bilayer. Tracer studies with 86Rb+ confirmed that copper promotes K+ efflux rather than inhibiting K+ uptake. Short-term K+ release is electroneutral, since electrophysiological measurements indicated that copper does not cause membrane depolarization. Short-term K+ efflux was accompanied by citrate release, and copper increased total citrate levels. Since citrate efflux was blocked by 4-AP, K+ appears to serve as a counterion during copper-induced citrate efflux. As copper but not aluminum selectively induces citrate production and release, it is proposed that copper may inhibit a cytosolic form of aconitase. PMID:10594125

  17. Role of cell volume variations in Na(+)-K(+)-ATPase recruitment and/or activation in cortical collecting duct.

    PubMed

    Coutry, N; Farman, N; Bonvalet, J P; Blot-Chabaud, M

    1994-05-01

    The aim of this study was to examine whether cell volume variations could play a role in the previously reported Na(+)-K(+)-ATPase pump recruitment and/or activation induced by an increase in intracellular Na concentration (Nai) in cortical collecting ducts (CCD). Isolated CCD from kidneys of aldosterone-repleted mice were incubated in hyper-, hypo-, or isosmotic solutions with and without Na to modify Nai and cell volume independently. Nai, cell volume, and the number of basolateral pumps were measured using 22Na, image analysis, and specific [3H]ouabain binding, respectively. Ouabain-sensitive 86Rb uptake was also measured. In CCD with high Nai, pump recruitment and/or activation was observed only when an increase in tubular volume was associated with Na load. Pump recruitment and/or activation was also induced by cell swelling in the absence of Na load. Recruited and/or activated pumps display an affinity for ouabain and a specific activity (ouabain-sensitive Rb uptake per pump unit) similar to basal pumps. We conclude that 1) cell swelling is implied in the process of Nai-dependent pump recruitment and/or activation, 2) cell swelling can promote pump recruitment and/or activation independently of Na load, 3) basal and recruited and/or activated pumps probably correspond to the same Na(+)-K(+)-ATPase isoform.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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).

  3. 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.

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

    PubMed

    Ciapa, B; Allemand, D; Payan, P

    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 86Rb uptake and amiloride-sensitive 24Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na+/H+ and Na+/K+ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na+/H+ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na+/H+ 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 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na+ pump, Na+/H+ exchange) without eliciting corresponding regulatory mechanisms (Na+ stat, pH stat).

  5. 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.

  6. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells.

    PubMed

    Venglarik, C J; Bridges, R J; Frizzell, R A

    1990-08-01

    We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-[cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl)oxy]acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

  7. Mechanisms for optimising photodynamic therapy: second-generation photosensitisers in combination with mitomycin C.

    PubMed Central

    van Geel, I. P.; Oppelaar, H.; Oussoren, Y. G.; Schuitmaker, J. J.; Stewart, F. A.

    1995-01-01

    Mechanisms for improving photodynamic therapy (PDT) were investigated in the murine RIF1 tumour using meso-tetrahydroxyphenylchlorin (m-THPC) or bacteriochlorin a (BCA) as photosensitisers and comparing these results with Photofrin-mediated PDT. The 86Rb extraction technique was used to measure changes in perfusion at various times after interstitial PDT. Non-curative combinations of light doses with m-THPC and BCA PDT markedly decreased vascular perfusion. This decrease was more pronounced for both new photosensitisers than for Photofrin. Comparison of tumour perfusion after PDT with tumour response revealed an inverse correlation for all three photosensitisers, but the relationship was less clear for m-THPC and BCA. In vivo/in vitro experiments were performed after Photofrin or m-THPC PDT in order to assess direct tumour kill (immediate plating) vs indirect vascular effects (delayed plating). For both photosensitisers, there was little direct cell killing but clonogenic survival decreased as the interval between treatment and excision increased. When m-THPC PDT was combined with mitomycin C (MMC), light doses could be decreased by a factor of 2 for equal tumour effects. Lower light and m-THPC doses could be used compared with Photofrin PDT in combination with MMC. BCA PDT with MMC did not result in a greater tumour response compared with BCA PDT alone. Reduction in both light and photosensitiser does for effective PDT regimes in combination with MMC offers substantial clinical advantages, since both treatment time and skin photosensitisation will be reduced. PMID:7640216

  8. Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle.

    PubMed

    Wong, J A; Gosmanov, A R; Schneider, E G; Thomason, D B

    2001-08-01

    Na(+)-K(+)-Cl(-) cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive (86)Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na(+)-K(+)-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na(+)-K(+)-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

  9. Nicotinic activation of mesolimbic neurons assessed by rubidium efflux in rat accumbens and ventral tegmentum.

    PubMed

    Rowell, Peter P; Volk, Kelly A

    2004-01-01

    Dopaminergic mesolimbic neurons, with cell bodies in the ventral tegmental area (VTA) projecting to the nucleus accumbens (NAc), have been shown to be involved in the development of drug dependence. The application of nicotine to either the VTA or NAc produces an increase in dopamine release; however, the positive reinforcement produced by the systemic injection of nicotine is primarily due to stimulation of nicotinic acetylcholine receptors (nAChRs) in the VTA. Because the brain levels of nicotine would likely be the same in both brain areas, the nAChRs in the NAc may be less sensitive than those in the VTA. This study was undertaken to make a direct comparison of the native nAChRs in intact slices of NAc and VTA by measuring nicotine-stimulated efflux of (86)Rb(+) in a superfusion assay. The potency of nicotine and several other agonists was similar in both brain areas, but nicotine was somewhat more efficacious in the NAc. The effects of treatment duration, calcium and nicotinic antagonists were also determined. The results suggest that the predominant effect of nicotine in the VTA following systemic administration is due to differences in neuronal circuitry or firing patterns rather than inherent differences in the two nAChR populations.

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

    PubMed

    Terstappen, Georg C

    2004-10-01

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

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

    PubMed Central

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

    2012-01-01

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

  12. Discovery of highly potent and selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonists containing an isoxazolylpyridine ether scaffold that demonstrate antidepressant-like activity. Part II.

    PubMed

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

    2012-11-26

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

  13. Synthesis and biological evaluation of novel hybrids of highly potent and selective α4β2-Nicotinic acetylcholine receptor (nAChR) partial agonists.

    PubMed

    Zhang, Han-Kun; Eaton, J Brek; Fedolak, Allison; Gunosewoyo, Hendra; Onajole, Oluseye K; Brunner, Dani; Lukas, Ronald J; Yu, Li-Fang; Kozikowski, Alan P

    2016-11-29

    We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4β2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3β4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [(3)H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4β2* nAChR partial agonists with Ki values of 0.5-51.4 nM for α4β2 and negligible affinities for α3β4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC50 and IC50 values of 15-50 nM) of the parent azetidine-containing compounds 3 and 4 in the (86)Rb(+) ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCube(®) platform and classical forced swim tests, supporting the potential use of α4β2 partial agonists for treatment of depression.

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

    SciTech Connect

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

    1991-07-01

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

  15. 2-(2-Piperidyl)- and 2-(2-pyrrolidyl)chromans as nicotine agonists: synthesis and preliminary pharmacological characterization.

    PubMed

    Efange, S M; Tu, Z; von Hohenberg, K; Francesconi, L; Howell, R C; Rampersad, M V; Todaro, L J; Papke, R L; Kung, M P

    2001-12-20

    As part of an effort to develop a new class of subtype selective nicotine agonists, we have synthesized and tested a group of 12 hydroxylated 2-(2-piperidyl)- and 2-(2-pyrrolidyl)chromans. In rat brain membranes, all 12 compounds displayed poor affinity for [(125)I]-alpha-bunagarotoxin binding sites. In contrast, three compounds, 17c, 24, and 26, displayed moderate to high affinity for [(3)H]cytisine binding sites, while three (17b, 18b,c) and six (17a,d,e and 18a,d,e) compounds showed weak and poor affinity, respectively, for these same sites. In subsequent studies, compounds 17a and 17c were found to stimulate the efflux of (86)Rb(+) from rat cortical synaptosomes, an indication of agonist activity. Further, both 17c and 26 displayed high intrinsic activity in stimulating the release of [(3)H]dopamine from striatal synaptosomes; however, only 17c was effective at stimulating the release of [(3)H]acetylcholine from cortical synaptosomes, suggesting differential selectivity. In cloned human nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes, both 17c and 26 activated alpha7 and alpha3beta2 receptor subtypes in a dose-dependent manner, but 26 was clearly the more potent agonist. Last, neither compound displayed dose-dependent activation of alpha4beta2 nAChRs. We conclude that 2-(2-azacyclic)chromans appear to be a promising new class of nicotine agonists.

  16. Effects of peptides cleaved from human fibrinogen by plasmin on rabbit kidney cells in culture

    SciTech Connect

    Stachurska, J.; Janik, M.; Kobus, M.; Luczak, M.; Szmigielski, S.; Roszkowski, M.; Gerdin, B.; Saldeen, T.; Kopec, M.

    1983-02-15

    Low molecular weight fibrinogen degradation products (LMW-FDP) containing a mixture of dialysable peptides cleaved from human fibrinogen by plasmin are cytotoxic to an established line of rabbit kidney cells and to primary cultures of rabbit kidney cells. The presence of LMW-FDP in a concentration of 50 micrograms/ml during the cell cultivation caused a considerable release of /sup 51/Cr from prelabelled cells and inhibited /sup 3/H-thymidine and /sup 86/Rb uptake. Among three isolated peptides of established primary structure only one, 6D: Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys, induced a significant effect, i.e. it enhanced /sup 3/H-thymidine incorporation. Two others, 6A: Ala-Arg-Pro-Ala-Lys and 6E: Thr-Ser-Glu-Val-Lys, did not influence the examined parameters. Hence other components of LMW-FDP must be assumed to be responsible for the cytotoxic effect on kidney cell cultures.

  17. Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis[C][W

    PubMed Central

    Osakabe, Yuriko; Arinaga, Naoko; Umezawa, Taishi; Katsura, Shogo; Nagamachi, Keita; Tanaka, Hidenori; Ohiraki, Haruka; Yamada, Kohji; Seo, So-Uk; Abo, Mitsuru; Yoshimura, Etsuro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2013-01-01

    Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K+ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K+ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using 86radioactive rubidium ion (86Rb+) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses. PMID:23396830

  18. Role of phospholipid metabolism in bromobenzene- and carbon tetrachloride-dependent hepatocyte injury

    SciTech Connect

    Lamb, R.G.; McCue, S.B.; Taylor, D.R.; McGuffin, M.A.

    1984-09-30

    Sprague-Dawley rats and cultured rat hepatocytes exposed to bromobenzene (BB) and carbon tetrachloride (CCl4) display rapid and significant increases and decreases in hepatic phospholipase C (PLC) and sn-glycerol-3-phosphate acyltransferase (GPAT) activities, respectively. Primary cultures of adult rat hepatocytes were used to determine if the BB- and CCl4-dependent alterations in phospholipid metabolism were related to the hepatotoxicity of these agents. Cultured hepatocytes exposed to BB and CCl4 exhibited a rapid (1 to 5 min). PLC-mediated reduction (20 to 80%) in (32P)phosphatidylserine content. Other phospholipids were also reduced; however, phosphatidylserine was preferentially degraded by hepatotoxin-activated PLC. A time course of CCl4-and BB-induced cellular events showed that these agents (1) rapidly activate liver cell PLC activity; (2) accelerate 86Rb release; (3) decrease GPAT acyltransferase activity; and (4) cause a release of intracellular enzymes (GOT and GPT). All of these BB- and CCl4-mediated effects on the functional integrity of liver cells were blocked or reduced by agents (EDTA and chlorpromazine) that reduce the BB- and CCl4-dependent rise in PLC activity. Therefore, BB- and CCl4-dependent alterations in the functional and structural integrity of liver cells may be a result of accelerated phospholipid degradation and a corresponding inability of the cell to repair injured membranes by generating new phospholipids.

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

    SciTech Connect

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

    1986-03-01

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

  20. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells

    SciTech Connect

    Venglarik, C.J.; Bridges, R.J.; Frizzell, R.A. )

    1990-08-01

    We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-(cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl-oxy)acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

  1. High-resolution structure of the open NaK channel

    SciTech Connect

    Alam, Amer; Jiang, Youxing

    2009-04-08

    We report the crystal structure of the nonselective cation channel NaK from Bacillus cereus at a resolution of 1.6 {angstrom}. The structure reveals the intracellular gate in an open state, as opposed to the closed form reported previously, making NaK the only channel for which the three-dimensional structures of both conformations are known. Channel opening follows a conserved mechanism of inner helix bending using a flexible glycine residue, the gating hinge, seen in MthK and most other tetrameric cation channels. Additionally, distinct inter and intrasubunit rearrangements involved in channel gating are seen and characterized for the first time along with inner helix twisting motions. Furthermore, we identify a residue deeper within the cavity of the channel pore, Phe92, which is likely to form a constriction point within the open pore, restricting ion flux through the channel. Mutating this residue to alanine causes a subsequent increase in ion-conduction rates as measured by {sup 86}Rb flux assays. The structures of both the open and closed conformations of the NaK channel correlate well with those of equivalent K{sup +} channel conformations, namely MthK and KcsA, respectively.

  2. Identification of yeast genes involved in k homeostasis: loss of membrane traffic genes affects k uptake.

    PubMed

    Fell, Gillian L; Munson, Amanda M; Croston, Merriah A; Rosenwald, Anne G

    2011-06-01

    Using the homozygous diploid Saccharomyces deletion collection, we searched for strains with defects in K(+) homeostasis. We identified 156 (of 4653 total) strains unable to grow in the presence of hygromycin B, a phenotype previously shown to be indicative of ion defects. The most abundant group was that with deletions of genes known to encode membrane traffic regulators. Nearly 80% of these membrane traffic defective strains showed defects in uptake of the K(+) homolog, (86)Rb(+). Since Trk1, a plasma membrane protein localized to lipid microdomains, is the major K(+) influx transporter, we examined the subcellular localization and Triton-X 100 insolubility of Trk1 in 29 of the traffic mutants. However, few of these showed defects in the steady state levels of Trk1, the localization of Trk1 to the plasma membrane, or the localization of Trk1 to lipid microdomains, and most defects were mild compared to wild-type. Three inositol kinase mutants were also identified, and in contrast, loss of these genes negatively affected Trk1 protein levels. In summary, this work reveals a nexus between K(+) homeostasis and membrane traffic, which does not involve traffic of the major influx transporter, Trk1.

  3. Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis.

    PubMed

    Osakabe, Yuriko; Arinaga, Naoko; Umezawa, Taishi; Katsura, Shogo; Nagamachi, Keita; Tanaka, Hidenori; Ohiraki, Haruka; Yamada, Kohji; Seo, So-Uk; Abo, Mitsuru; Yoshimura, Etsuro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2013-02-01

    Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K(+) uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K(+) channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using (86)radioactive rubidium ion ((86)Rb(+)) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.

  4. 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

  5. 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

  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. Triggering of monoclonal human lymphoma B cells with antibodies to IgM heavy chains: differences of response obtained with monoclonal as compared to polyclonal antibodies.

    PubMed Central

    Godal, T; Ruud, E; Heikkilä, R; Funderud, S; Michaelsen, T; Jefferis, R; Ling, N R; Hildrum, K

    1983-01-01

    A comparative study of human B lymphoma cells activation by monoclonal (murine hybridoma) antibodies to mu heavy chains (Ma-mu) as compared to polyclonal (rabbit) antibodies to mu heavy chains (Ra-mu) has been carried out. Early events related to calmodulin activation such as 86Rb influx and changes in cell volume at 4 h could be induced by Ma-mu. One antibody (AF6) approached Ra-mu with regard to the strength of response obtained. However, Ma-mus including AF6 were deficient in inducing DNA synthesis under conditions where this was achieved with Ra-mu. Studies in one lymphoma, where stimulation of re-expressed surface IgM could be studied, revealed that Ma-mu was deficient in stimulating re-expressed sIgM. These findings raise questions with regard to polyclonal antibody to surface Ig as a model for B cell triggering by antigen and suggest that antigen-induced B cell triggering may be more complex than indicated by previous studies with polyclonal antibody. PMID:6418424

  8. 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.

  9. 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.

  10. Measurement of the hyperfine splitting of 133Cs atoms in superfluid helium

    NASA Astrophysics Data System (ADS)

    Imamura, K.; Furukawa, T.; Yang, X. F.; Mitsuya, Y.; Fujita, T.; Hayasaka, M.; Kobayashi, T.; Hatakeyama, A.; Ueno, H.; Odashima, H.; Matsuo, Y.

    2015-04-01

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

  11. Cl- secretion in a model intestinal epithelium induced by a neutrophil-derived secretagogue.

    PubMed Central

    Madara, J L; Parkos, C; Colgan, S; MacLeod, R J; Nash, S; Matthews, J; Delp, C; Lencer, W

    1992-01-01

    A secreted product of activated neutrophils, NDS (neutrophil-derived secretagogue), elicits a short circuit current (Isc) in epithelial monolayers derived from the human intestinal cell line T84 (J. Clin. Invest. 1991. 87:1474-1477). Here, we identify and characterize the source of this Isc and examine associated signaling pathways. 125I efflux studies suggested that NDS activates an anion conductive channel. Bidirectional 22Na 36Cl flux studies showed that electrogenic Cl- secretion fully accounts for the NDS-induced Isc response. NDS behaved in many respects as a cAMP-mediated secretagogue: NDS did not further increase maximal cAMP-induced Cl- secretion; NDS potentiated Ca(2+)-mediated Cl secretion; and NDS elicited measurable 125I but not 86Rb effluxes. However, NDS did not elicit a detectable rise in intracellular cAMP. Such data suggest that NDS may elicit Cl- secretion by effecting distal events in the cAMP-mediated pathway. Data derived from cell volume assays of isolated guinea pig intestinal crypt cells indicated that NDS also directly elicits Cl- secretion from natural intestinal epithelia. Additionally, since NDS activity is released from PMN by stimuli normally present in the colonic lumen, since NDS is active when applied apically to this model intestinal epithelium, and since the NDS-elicited Isc response is indicative of electrogenic chloride secretion, we speculate NDS may contribute to the secretory diarrhea encountered in many patients with inflammatory intestinal disease. Images PMID:1602001

  12. Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis.

    PubMed Central

    Kurtz, M B; Douglas, C; Marrinan, J; Nollstadt, K; Onishi, J; Dreikorn, S; Milligan, J; Mandala, S; Thompson, J; Balkovec, J M

    1994-01-01

    The pneumocandins are natural lipopeptide products of the echinocandin class which inhibit the synthesis of 1,3-beta-D-glucan in susceptible fungi. The lack of a corresponding pathway in mammalian hosts makes this mode of action an attractive one for treating systemic infections. Substitution by an aminoethyl ether at the hemiaminal and dehydration and reduction of the glutamine of pneumocandin B0 produced a semisynthetic compound (L-733,560) with intrinsic water solubility, significantly increased potency, and a broader antifungal spectrum. To evaluate the mechanism for the improved antifungal efficacy, we determined that L-733,560 was a more potent inhibitor of glucan synthase activity in vitro, did not affect the other membrane-bound enzymes tested, conferred susceptibility to lysis in the absence of osmotic support, and did not disrupt currents in liposomal bilayers or 86Rb+ fluxes from liposomes. In Aspergillus species L-733,560 also produced the same morphological alterations as pneumocandin B0. A stereoisomer of L-733,560 with poor antifungal activity was a weak inhibitor of glucan synthase. All of these results support the notion that the enhanced antifungal activity of L-733,560 is achieved by superior inhibition of glucan synthesis and not by nonspecific membrane effects or a second mode of action. Images PMID:7695257

  13. Amino acid sequence of TsTX-V, an alpha-toxin from Tityus serrulatus scorpion venom, and its effect on K+ permeability of beta-cells from isolated rat islets of Langerhans.

    PubMed

    Marangoni, S; Toyama, M H; Arantes, E C; Giglio, J R; da Silva, C A; Carneiro, E M; Gonçalves, A A; Oliveira, B

    1995-04-13

    Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of 86Rb+ release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 micrograms/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 microM veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

  14. Dual Mechanisms of Ion Absorption in Relation to Long Distance Transport in Plants

    PubMed Central

    Luttge, Ulrich; Laties, George G.

    1966-01-01

    The characteristics of ion transport to the shoots of young corn seedlings were studied with respect to the nature of the isotherm through a wide concentration range, the competitive influence of closely related ions upon the transport of a given ion, and the influence of the counter-ion. Both with respect to 36Cl and 86Rb transport, the characteristics of the process in every way resemble uptake by non-vacuolate root tips wherein the plasma membrane is the only membrane involved in absorption, and where system 1 — of the 2 systems which can be shown to participate in absorption by vacuolate tissue — is the only system operative. Net ion uptake by the roots per se was shown to display both the high affinity (system 1) and low affinity (system 2) mechanisms. It is concluded that the symplastic theory of ion movement to the xylem is valid, and that the contention that system 1 operates at the plasma membrane while system 2 functions at the tonoplast is strengthened. PMID:16656435

  15. Early, anti-immunoglobulin induced events prior to Na+-K+ pump activation: an analysis in a monoclonal human B-lymphoma cell population.

    PubMed

    Heikkilä, R; Iversen, J G; Godal, T

    1983-10-01

    Events following F(ab)2 anti-delta immunoglobulin stimulation of monoclonal (leukemic) human B cells prior to Na+-K+ pump activation were investigated in vitro. This pump activation, measured by ouabain-sensitive 86Rb+ uptake, appeared susceptible to the phospholipid-interacting drugs tetracaine and quinacrine, to the antioxydant nordihydroguaiaretic acid (NDGA), and to the calmodulin antagonist trifluoperazine, while much less susceptible to the methylation inhibitor-3-deazaadenosine. The Ca++ ionophore A 23187 appeared to induce pump activation in a way similar to anti-delta, as it was susceptible to the same drugs and as anti-delta had no additional stimulating effect on A 23187-stimulated cells. However, whereas the anti-delta-induced activations appeared independent of the extracellular Ca++ activity, [Ca++]e, the activation by A 23187 was potentiated by addition of the Ca++ chelator ethyleneglycol-bis (beta-aminoethyl ether) N, N'-tetracetic acid (EGTA). Estimations by fluorescent chelator method (quin 2) showed anti-delta to increase the intracellular Ca++ activity, [Ca++]i both in the absence and presence of EGTA. A 23187 increased [Ca++]i strongly in Ca++ medium, but was weaker, more similar to the anti-delta response, in EGTA medium. It is suggested that Na+-K+ pump activation after anti-Ig stimulation in B cells may follow Ca++ mobilization from internal stores. The trifluoperazine susceptibility suggests that calmodulin regulation is involved.

  16. Effect of dietary sodium on the Na-K ATPase inhibitor in patients with essential hypertension

    SciTech Connect

    Ashida, T.; Kuramochi, M.; Kojima, S.; Yoshimi, H.; Kawano, Y.; Kimura, G.; Abe, H.; Imanishi, M.; Yoshida, K.; Kawamura, M. )

    1989-07-01

    To study the circulating humoral factor modifying transmembrane sodium transport, plasma was obtained from 12 patients with essential hypertension (EH) fed a high sodium diet (NaCl 15 to 17 g/d) for seven days and thereafter a low sodium diet (NaCl 2 to 3 g/d) for seven days. Ouabain-sensitive {sup 86}Rb+ influx into the red blood cells (RBC) obtained from a healthy subject, and incubated with the plasma obtained during the high sodium diet was significantly lower than that incubated with the plasma obtained during the low sodium diet (3.74 +/- 0.26 v 3.97 +/- 0.30 nmol/10(8) cells, P less than .05). The changes in mean blood pressure from the high to low sodium diet showed a significant positive correlation with the changes in the ouabain-sensitive Rb influx into RBC in the plasma from the high to low sodium diet. These results suggest that a humoral factor modifying the sodium pump might be altered by sodium balance in EH, especially in salt-sensitive hypertension.

  17. Actions of arachidonic acid on erythrocyte membrane Rb permeability.

    PubMed

    Dwight, J F; Hendry, B M

    1995-07-14

    The effects of non-esterified arachidonic acid (AA) on erythrocyte membrane ion permeability have been studied using 86Rb flux measurements. [14C]AA was used to quantify membrane incorporation of AA and to show AA removal by albumin washing. The actions of vitamin E and other antioxidants on the effects of AA were examined. Reversible membrane incorporation of 700-2000 nmol AA per ml cells was achieved without significant haemolysis or morphological change. AA incorporation caused a reversible mean increase in bumetanide-sensitive Rb influx of 34% (S.E.M. 4.5, n = 23). This action could be partially prevented by co-incubation with vitamin E, but not by Trolox or dithioerythritol. AA incorporation caused an irreversible mean increase in residual Rb permeability (bumetanide and ouabain insensitive) of 130% (S.E.M. 22, n = 20), associated with a rise in intracellular Na and a fall in intracellular K concentrations. This action was also partially prevented by co-incubation with vitamin E. The effects of AA incorporation on Na,K-ATPase function were difficult to quantify because of the concomitant rises in intracellular Na but the data are consistent with approximately 20% inhibition of activity. Modulation of membrane ion permeability by AA appears to be partially mediated by lipid peroxidation and may have pathophysiological significance.

  18. Thallium and rubidium permeability of human and rat erythrocyte membrane.

    PubMed

    Skulskii, I A; Manninen, V; Glasunov, V V

    1990-02-01

    Transport of Tl+ and Rb+ in human and rat erythrocytes was investigated in the presence of ouabain. The chloride-dependent cotransport of Tl+, Rb+ and Na+ was precluded by replacement of Cl- by NO3-. The inward and outward rate constants for the residual fluxes of the cations were determined by measuring the transport of 204Tl and 86Rb in double label experiments. The rate of passive transport of Tl+ exceeded that of Rb+ by one-two orders of magnitude in human as well as rat erythrocytes. The membrane barrier which contributes to the maintenance of ion gradients was shown not to be a barrier for Tl+ which easily penetrates the membrane by an unknown mechanism. In rat erythrocytes the barrier for Rb+ was 10-15 times weaker than that in human red blood cells, while the corresponding ratio of rat/human Tl+ permeabilities was about 1.8-2.0. It follows that Tl+ permeability is only slightly affected by factors modifying the permeability to alkali cations. The increase of temperature from 20 degrees to 37 degrees C resulted in a three-fourfold stimulation of the passive transport of Tl+ both in human and rat erythrocytes. The movement of Tl+ and Rb+ through the erythrocyte membrane differed substantially from their diffusion along the excitable membrane channels characterized both by poor Tl+/K+ selectivity and weak temperature dependence.

  19. Regulation of erythrocyte Na-K-2Cl cotransport by threonine phosphorylation.

    PubMed

    Matskevich, Ioulia; Hegney, Karen L; Flatman, Peter W

    2005-08-01

    A method is described to measure threonine phosphorylation of the Na-K-2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.

  20. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

    PubMed Central

    Rufo, P A; Jiang, L; Moe, S J; Brugnara, C; Alper, S L; Lencer, W I

    1996-01-01

    Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals. PMID:8903326

  1. Selective Inhibition of K+, Na+, Cl−, and PO43− Uptake in Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Pathotoxin

    PubMed Central

    Mertz, Stuart M.; Arntzen, Charles J.

    1977-01-01

    Pathotoxin preparations were obtained from either axenic culture filtrate of race T of Bipolaris maydis (Nisikado) Shoemaker (new culture media and toxin purification procedures are described) or extracts of maize leaves infected with the fungus. The toxins (10−6 to 10−8m) caused inhibition of [86Rb]K+ uptake in leaf discs and apical root segments of Zea mays L. cv W64A Texas (Tcms) and normal (N) cytoplasms. Significant inhibition was measurable as early as 5 min after adding toxin. In Tcms per cent inhibition was increased by increasing toxin concentration and time in toxin, by using solution at pH 5 rather than pH 7, by decreasing external KCl concentration over the range 50 to 0.1 mm (in the presence of 0.5 mm CaSO4), or by exposing leaf discs to light rather than dark during the uptake period in toxin. Root uptake of 22Na+ and 36Cl− was inhibited to a lesser extent than K+. Inhibition of 32PO43− uptake occurred after 40 min when cyclosis had ceased. When combined with data in the literature, our data indicate that the plasmalemma is the probable primary site of toxin action in N and Tcms maize. Comparison of the effects of toxin on K+ uptake in N and Tcms maize suggests the existence of more than one mode of toxin action: a weak disruptive effect in N and Tcms, and in addition, specific membrane sites in Tcms involved in monovalent ion uptake. Six genotypes in N or Tcms cytoplasm which exhibited different degrees of disease susceptibility in the field showed a corresponding gradation of susceptibility to the toxin when a K+ uptake bioassay was used. This correlation is strong evidence that the sites of toxin action affecting K+ transport have characteristics closely related to cellular factors regulating susceptibility to fungal attack. PMID:16660094

  2. Effects of extracellular HCO3(-) on fatigue, pHi, and K+ efflux in rat skeletal muscles.

    PubMed

    Broch-Lips, Martin; Overgaard, Kristian; Praetorius, Helle A; Nielsen, Ole Baekgaard

    2007-08-01

    Elevated plasma HCO(3)(-) can improve exercise endurance in humans. This effect has been related to attenuation of the work-induced reduction in muscle pH, which is suggested to improve performance via at least two mechanisms: 1) less inhibition of muscle enzymes and 2) reduced opening of muscle K(ATP) channels with less ensuing reduction in excitability. Aiming at determining whether the ergogenic effect of HCO(3)(-) is related to effects on muscles, we examined the effect of elevating extracellular HCO(3)(-) from 25 to 40 mM (pH from 7.4 to 7.6) on fatigue, intracellular pH (pH(i)), and K(+) efflux in isolated rat skeletal muscles contracting isometrically. Fatigue induced by 30-Hz stimulation at 30 and 37 degrees C was similar between soleus muscles incubated in high and normal HCO(3)(-) concentrations. In extensor digitorum longus muscles stimulated at 60 Hz, elevated HCO(3)(-) did not affect fatigue at 30 degrees C. In soleus muscles, 30-Hz stimulation induced a approximately 0.2 unit reduction in pH(i), as determined by using the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. This reduction in pH(i) was not affected by elevated HCO(3)(-). Estimation of K(+) efflux using (86)Rb(+) showed that elevated HCO(3)(-) did not affect K(+) efflux at rest or during contractions. Similarly, other modifications of the intra- and extracellular pH had little effect on K(+) efflux during contraction. In conclusion, elevated extracellular HCO(3)(-) had no significant effect on muscle fatigue, pH(i), and K(+) efflux. These findings indicate that alternative mechanisms must be considered for the ergogenic effect of HCO(3)(-) observed in integral exercise studies.

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

    PubMed

    Keep, R F; Xiang, J

    1995-06-01

    The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide-sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 +/- 0.02, 0.44 +/- 0.04, and 0.84 +/- 0.06 min-1 in 240, 300, and 424 mOsm/kg solutions, respectively (p < 0.001). This increase in efflux with osmolality, an opposite effect to that found in many cells, was solely due to enhanced K cotransport. The increased cotransport may be involved in limiting brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide-sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 +/- 1, 19 +/- 3, 13 +/- 2, and 6 +/- 1 nmol/mg/min after 0, 10, 30, and 60-min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide-sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.

  4. 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

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

    PubMed Central

    Taylor, P W; Kroll, H P

    1983-01-01

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

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

    PubMed

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

    2004-10-01

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

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

    SciTech Connect

    Hunter, D.D.

    1986-01-01

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

  8. Control of membrane permeability in animal cells by divalent cations

    SciTech Connect

    Otero, M.J.; Carrasco, L.

    1987-04-01

    The permeability of several cell lines, including HeLa, L929, 3T6 and T3, to various compounds is affected by the concentration of divalent cations in the culture medium. In the absence of Mg/sup 2 +/ ions but with 4-8 mM CaCl/sub 2/ in the medium, HeLa and L929 cells become permeabilized, as measured by the entry of the aminoglycoside antibiotic hygromycin B. However, 33 and 3T6 cells become much more permeable when calcium and magnesium are both absent from the medium. Addition of Mg/sup 2 +/ above 2 mM abolishes the permeabilization induced by Ca/sup 2 +/. Basic pH favors permeabilization, whereas acidic pH inhibits the entry of hygromycin B. Increased entry of macromolecules, such as the toxin alpha-sarcin, horseradish peroxidase (HRP) and luciferase, is also observed under permeabilization conditions, suggesting that this method could be of general use, since it is not harmful to cells and is fully reversible. Exit of /sup 86/Rb/sup +/ ions and (/sup 3/H)uridine-labelled nucleotides was also assayed. The authors did not observe increased release of these compounds from preloaded cells under various calcium concentrations. Finally, the effects of several inhibitors of endocytosis and other membrane functions on the permeabilization process were also analyzed. The entry of alpha-sarcin was not affected by nifedipine, dibucaine or mepacrine, but was partially inhibited by NH/sub 4/Cl, amantadine and chloroquine.

  9. Action Spectra for Guard Cell Rb+ Uptake and Stomatal Opening in Vivia faba

    PubMed Central

    Hsiao, Theodore C.; Allaway, W. G.; Evans, L. T.

    1973-01-01

    Abaxial epidermal strips, containing guard cells as the only viable cells, were prepared from leaves of Vicia faba following a period in darkness, and floated, under CO2-free air, on 2 mm RbCl + 0.1 mm CaCl2 labeled with 86Rb+. Under white light (high pressure mercury vapor lamp), stomatal opening in these strips approached its maximum at less than 0.02 calorie per square centimeter per minute. Under light of different wavelengths, 20 nanometers apart, and at a low quantum flux density of 7 × 1014 quanta per square centimeter per second, Rb+ uptake and stomatal opening were activated only in the blue and long ultraviolet regions, with a peak at 420 to 460 nanometers. The action spectrum suggests that the underlying process is not photosynthesis. At higher quantum flux density (38 × 1014 quanta per square centimeter per second), uptake and opening also responded to red (600-680 nanometers) and somewhat to green light, with a minimum at 540 to 560 nanometers, indicating a possible involvement of the photosynthetic process. This light-induced opening appeared not to be mediated by a lowering of CO2 concentration, since CO2-free air was used in all treatments and controls. Stomatal opening paralleled Rb+ uptake in all cases. This constitutes further evidence for the potassium transport hypothesis of stomatal movement. In the abaxial surface of leaf discs under air of normal CO2 concentration, stomatal opening in white light approached its maximum at an intensity similar to that for epidermal strips. At both quantum flux densities, the action spectra for opening in leaf discs were very similar to those for epidermal strips. Thus, these light-linked processes for stomatal opening are likely to be the same in leaves as in epidermal strips. PMID:16658302

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

    SciTech Connect

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

    1987-11-01

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

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

    PubMed

    Monette, Michelle Y; Forbush, Biff

    2012-01-13

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

  12. 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.

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

    PubMed Central

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

    2011-01-01

    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-A1; its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl−-dependent uptake of 86Rb+ 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

  14. 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.

  15. Developmental changes in metabolism and transport properties of capillaries isolated from rat brain.

    PubMed Central

    Betz, A L; Goldstein, G W

    1981-01-01

    1. Capillaries were isolated from the brains of 1- to 45-day-old rats in order to study the development of metabolic and transport aspects of the blood-brain barrier. 2. The hydroxyproline content of capillary hydrolysates increased nearly threefold between 5 and 45 days of age. This finding is consistent with histological studies showing thickening of capillary basement membrane during development. 3. The activities of L-DOPA decarboxylase and monoamine oxidase were greatest in capillaries from 10-day-old rat brain. Thus, the metabolic blood-brain barrier for amine precursors is present during early development. 4. Capillaries from all ages were able to metabolize glucose, beta-hydroxybutyrate and palmitate. The rate of glucose oxidation more than doubled between 21 and 30 days of age but subsequently decreased. In contrast, beta-hydroxybutyrate and palmitate oxidation increased throughout development. These data suggest a sparing effect by alternate fuels on glucose metabolism. 5. Capillary glucose uptake was similar at 10 and 30 days of age and activity of the ouabain-sensitive K+ pump (measured using 86Rb+) was relatively constant at all ages. In contrast, Na+-dependent neutral amino acid transport was not present until after 21 days of age. Since this transport system may be responsible for the active efflux of neutral amino acids from brain to blood, it is likely that this process does not occur at the immature blood-brain barrier. 6. We conclude that various aspects of brain capillary functions show distinct developmental patterns which may be related to changes in blood-brain barrier permeability during development. PMID:7264999

  16. 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.

  17. Embryonic Expression of the Putative γ Subunit of the Sodium Pump Is Required for Acquisition of Fluid Transport Capacity during Mouse Blastocyst Development

    PubMed Central

    Jones, D. Holstead; Davies, Tyler C.; Kidder, Gerald M.

    1997-01-01

    The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic α subunit and a noncatalytic, glycosylated β subunit. Recently, a putative third subunit, the γ subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the α and β subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the γ subunit in preimplantation mouse development, where Na+,K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the γ subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the γ subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the α and β subunits, the γ subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the γ subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+,K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay (“back door” phosphorylation) applied to plasma membrane preparations. These results indicate that the γ subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse. PMID:9396759

  18. Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells

    PubMed Central

    Mettler, Irvin J.; Leonard, Robert T.

    1979-01-01

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

  19. Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2.

    PubMed

    Wehner, Frank; Lawonn, Peter; Tinel, Hanna

    2002-03-01

    We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.

  20. Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon

    PubMed Central

    Barmeyer, Christian; Rahner, Christoph; Yang, Youshan; Sigworth, Frederick J.; Binder, Henry J.

    2010-01-01

    KCNN4 channels that provide the driving force for cAMP- and Ca2+-induced anion secretion are present in both apical and basolateral membranes of the mammalian colon. However, only a single KCNN4 has been cloned. This study was initiated to identify whether both apical and basolateral KCNN4 channels are encoded by the same or different isoforms. Reverse transcriptase-PCR (RT-PCR), real-time quantitative-PCR (RT-QPCR), and immunofluorescence studies were used to clone and identify tissue-specific expression of KCNN4 isoforms. Three distinct KCNN4 cDNAs that are designated as KCNN4a, KCNN4b, and KCNN4c encoding 425, 424, and 395 amino acid proteins, respectively, were isolated from the rat colon. KCNN4a differs from KCNN4b at both the nucleotide and the amino acid level with distinct 628 bp at the 3′-untranslated region and an additional glutamine at position 415, respectively. KCNN4c differs from KCNN4b by lacking the second exon that encodes a 29 amino acid motif. KCNN4a and KCNN4b/c are identified as smooth muscle- and epithelial cell-specific transcripts, respectively. KCNN4b and KCNN4c transcripts likely encode basolateral (40 kDa) and apical (37 kDa) membrane proteins in the distal colon, respectively. KCNN4c, which lacks the S2 transmembrane segment, requires coexpression of a large conductance K+ channel β-subunit for plasma membrane expression. The KCNN4 channel blocker TRAM-34 inhibits KCNN4b- and KCNN4c-mediated 86Rb (K+ surrogate) efflux with an apparent inhibitory constant of 0.6 ± 0.1 and 7.8 ± 0.4 μM, respectively. We conclude that apical and basolateral KCNN4 K+ channels that regulate K+ and anion secretion are encoded by distinct isoforms in colonic epithelial cells. PMID:20445171

  1. 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.

  2. Inhibition by glibenclamide of the vasorelaxant action of cromakalim in the rat.

    PubMed Central

    Buckingham, R. E.; Hamilton, T. C.; Howlett, D. R.; Mootoo, S.; Wilson, C.

    1989-01-01

    1. In rat isolated thoracic aortic rings pre-contracted with noradrenaline (10(-6) M), cromakalim (3 x 10(-7)-3 x 10(-5) M) produced concentration-related relaxation. This effect was progressively inhibited by increasing concentrations of the anti-diabetic sulphonylurea drug, glibenclamide (10(-6)-10(-5) M). 2. In rat isolated portal veins, cromakalim (3 x 10(-8)-10(-6) M) produced concentration-related inhibition of the spontaneous contractive activity and glibenclamide (3 x 10(-7)-3 x 10(-6) M) prevented this inhibitory action in a concentration-dependent manner. 3. In both rat aortic rings and portal veins, cromakalim (10(-5) M) stimulated 86Rb efflux. Prior exposure to glibenclamide (10(-7)-10(-6) M) produced a concentration-related inhibition of this response. 4. In conscious rats, cromakalim, 0.075 mg kg-1 i.v., produced a rapid and sustained fall in arterial blood pressure which was not influenced by pretreatment (2 h) with a large oral dose of glibenclamide (100 mg kg-1). 5. In conscious rats, the hypotensive action of cromakalim, 0.075 mg kg-1 i.v., was abolished by pretreatment (30 min) with glibenclamide, 20 mg kg-1, given by the intravenous route. 6. The results suggest that the vasorelaxant and hypotensive actions of cromakalim involve a K+ channel which can be inhibited by glibenclamide, but which may be distinct from the ATP-sensitive K+ channel of the pancreatic beta-cell. PMID:2497925

  3. The effect of cromakalim on the smooth muscle of the guinea-pig urinary bladder.

    PubMed Central

    Foster, C. D.; Fujii, K.; Kingdon, J.; Brading, A. F.

    1989-01-01

    1. The actions of cromakalim were studied on the detrusor muscle from guinea-pig urinary bladder. Cromakalim reduced the frequency and amplitude of spontaneous contractile activity of the smooth muscle of the guinea-pig urinary bladder at 5 x 10(-8)M and abolished the activity at concentrations above 5 x 10(-7)M. 2. Electrophysiological experiments demonstrated that cromakalim increased membrane conductance, caused a dose-dependent hyperpolarization of the cell membrane and loss of spike activity. These events are consistent with the opening of K+ channels. 3. The effects of 10(-6)M and 10(-5)M cromakalim on the contractile responses to carbachol, potassium and transmural nerve stimulation were studied. Cromakalim did not prevent the detrusor from responding to these agents, although it significantly reduced the contractile response to K+ at concentrations below 70 mM. 4. Uptake and efflux experiments using 86Rb+ were unable to demonstrate any significant effect on transmembrane movement produced by cromakalim (10(-5)M). 5. 43K+ efflux showed a dose-dependent increase in the rate constant on addition of cromakalim. The difference in the selectivity for K+ over Rb+ was confirmed in dual label uptake experiments. 6. Substitution experiments in which the K+ ions in the tissue were gradually replaced by Rb+ demonstrated that cromakalim had a progressively decreasing effect on spontaneous activity as internal K+ was lowered. When all the K+ was replaced by Rb+, cromakalim no longer inhibited spontaneous activity, confirming that the channel opened by cromakalim appears relatively impermeant to Rb+. PMID:2720312

  4. Rubidium chloride tolerant callus cultures of rice (Oryza sativa L.) accumulate more potassium and cross tolerate to other salts.

    PubMed

    Naga Amrutha, R; Jogeswar, G; Srilaxmi, P; Kavi Kishor, P B

    2007-09-01

    Callus cultures from salt tolerant (CSR-10) and susceptible (Swarnadhan) varieties of Oryza sativa L. were established in Murashige and Skoog's (MS) medium containing lethal concentrations (50 mM) of rubidium chloride (RbCl) as a selective agent. While 95-100% cells were viable in callus cultures grown without RbCl, viability was 75% in 50 mM RbCl selected cultures. Growth of RbCl selected calli in presence of salt was comparable to that of callus grown without it. Cells tolerant to RbCl showed more vacuoles and accumulated more K(+) in comparison with their corresponding controls. Suspension cultures were established and uptake of (86)Rb(+) was measured at 10 and 20 min intervals, which revealed a linear relationship between the absorption of K(+) and time. Callus cultures (560-day-old) tolerant to 50 mM RbCl regenerated shoots with 35-40% frequencies in both the varieties, but the same age-old callus grown in the medium devoid of RbCl did not show any organogenesis. Callus cultures that are tolerant to 50 mM RbCl when exposed to 25 mM LiCl, 50 mM NaCl, 50 mM KCl and 25 mM CsCl also exhibited cross tolerance in both the varieties. This is the first time that a callus line of rice resistant to RbCl was raised and shown to accumulate a major cation K(+ )and also an increased influx of it.

  5. Nicotine-induced dystonic arousal complex in a mouse line harboring a human autosomal-dominant nocturnal frontal lobe epilepsy mutation.

    PubMed

    Teper, Yaroslav; Whyte, Douglas; Cahir, Elizabeth; Lester, Henry A; Grady, Sharon R; Marks, Michael J; Cohen, Bruce N; Fonck, Carlos; McClure-Begley, Tristan; McIntosh, J Michael; Labarca, Cesar; Lawrence, Andrew; Chen, Feng; Gantois, Ilse; Davies, Philip J; Petrou, Steven; Murphy, Mark; Waddington, John; Horne, Malcolm K; Berkovic, Samuel F; Drago, John

    2007-09-19

    We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the alpha4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1-2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on alpha4S248Fbeta2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.

  6. Ouabain-like compound changes rapidly on physical exercise in humans and dogs: effects of beta-blockade and angiotensin-converting enzyme inhibition.

    PubMed

    Bauer, Natali; Müller-Ehmsen, Jochen; Krämer, Ulrike; Hambarchian, Njde; Zobel, Carsten; Schwinger, Robert H G; Neu, Horst; Kirch, Ulrike; Grünbaum, Ernst-Günther; Schoner, Wilhelm

    2005-05-01

    Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by (86)Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5+/-0.5 to 86.0+/-27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3+/-1.7 to 129.8+/-51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1+/-1.2 nmol/L and at 30+/-1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7+/-0.8 to 166.9+/-91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the beta1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

  7. 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.

  8. WNK2 Kinase Is a Novel Regulator of Essential Neuronal Cation-Chloride Cotransporters*

    PubMed Central

    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.

    2011-01-01

    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 86Rb+ 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. TiO2 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

  9. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    SciTech Connect

    Delamere, N.A.; Socci, R.R.; King, K.L. )

    1990-10-01

    The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin.

  10. 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

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

    SciTech Connect

    Ailenberg, M.; Fritz, I.B.

    1989-03-01

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

  12. 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.

  13. Down-regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy.

    PubMed

    Obara-Michlewska, Marta; Pannicke, Thomas; Karl, Anett; Bringmann, Andreas; Reichenbach, Andreas; Szeliga, Monika; Hilgier, Wojciech; Wrzosek, Antoni; Szewczyk, Adam; Albrecht, Jan

    2011-12-01

    Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

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

    SciTech Connect

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

    1988-02-01

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

  15. Original 2-alkylamino-6-halogenoquinazolin-4(3H)-ones and K(ATP) channel activity.

    PubMed

    Somers, F; Ouedraogo, R; Antoine, M H; de Tullio, P; Becker, B; Fontaine, J; Damas, J; Dupont, L; Rigo, B; Delarge, J; Lebrun, P; Pirotte, B

    2001-08-02

    A series of 6-substituted 2-alkylaminoquinazolin-4(3H)-ones structurally related to 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides were synthesized and tested as putative K(ATP) channel openers on isolated pancreatic endocrine tissue as well as on isolated vascular, intestinal, and uterine smooth muscle. Most of the 6-halogeno-2-alkylaminoquinazolin-4(3H)-ones were found to inhibit insulin release from pancreatic B-cells and to exhibit vasorelaxant properties. In contrast to their pyridothiadiazine dioxide isosteres previously described as more active on the endocrine than on the smooth muscle tissue, quinazolinones cannot be considered as tissue selective compounds. Biological investigations, including measurements of (86)Rb, (45)Ca efflux from pancreatic islet cells and measurements of vasodilator potency in rat aortic rings exposed to 30 or 80 mM KCl in the presence or the absence of glibenclamide, were carried out with 6-chloro- and 6-iodo-3-isopropylaminoquinazolin-4(3H)-ones. Such experiments showed that, depending on the tissue, these new compounds did not always express the pharmacological profile of pure K(ATP) channel openers. Analyzed by X-ray crystallography, one example of quinazolinones appeared to adopt a double conformation. This only suggests a partial analogy between the 2-alkylaminoquinazolin-4(3H)-ones and the 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides. In conclusion, the newly synthesized quinazolinones interfere with insulin secretion and smooth muscle contractile activity. Most of the compounds lack tissue selectivity, and further investigations are required to fully elucidate their mechanism(s) of action.

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

    SciTech Connect

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

    1987-03-01

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

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

    PubMed

    Loiseau, A; Leroy, C; Castaing, M

    1997-11-13

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

  18. 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.

  19. 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

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

    PubMed

    Van Dyke, R W; Scharschmidt, B F

    1987-11-01

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

  1. (Na,K)-ATPase-mediated cation pumping in cultured rat hepatocytes. Rapid modulation by alanine and taurocholate transport and characterization of its relationship to intracellular sodium concentration.

    PubMed

    Van Dyke, R W; Scharschmidt, B F

    1983-11-10

    (Na,K)-ATPase is thought to maintain the transmembrane electrochemical sodium gradient which powers secondary active sodium-coupled transport of a variety of solutes including amino acids and bile acids. However, little is known regarding the effect of sodium-coupled solute transport on intracellular sodium concentration ( [Na]ic) and on (Na,K)-ATPase-mediated cation pumping in the intact cell. In order to address this question, we have measured 22Na uptake rate, steady state 22Na content, and ouabain-suppressible 86Rb uptake rate in primary cultures of adult rat hepatocytes under a variety of conditions. Compared with control conditions (sodium uptake rate = 6.00 +/- 0.40 nmol X min-1 X mg-1; [Na]ic = 11.96 +/- 0.54 mM; cation pumping = 2.53 +/- 0.18 nmol X min-1 X mg-1), cation pumping was increased by taurocholate (less than or equal to 158%), alanine (less than or equal to 246%), monensin (less than or equal to 400%), and cold exposure (less than or equal to 525%), and this increase was accompanied by increases in Na uptake and [Na]ic. In contrast, preincubation in low sodium medium decreased all three variables. These changes in cation pumping were blocked in the absence of extracellular sodium and were not accompanied by changes in ouabain-suppressible ATP hydrolysis measured in cell homogenate. An overall plot of cation pumping versus [Na]ic yielded a sigmoid-shaped curve. Values for KNa (17.8 +/- 1.4 mM) and Vmax (8.98 +/- 0.62 nmol X min-1 X mg-1) for cation pumping were estimated assuming three sodium sites per pump unit. These findings indicate that: 1) uptake of alanine and taurocholate is associated with a rapid increase in (Na,K)-ATPase cation pumping; 2) this increase probably results from an increase in pumping per pump unit rather than an increase in the total number of pump units, and it appears to be mediated via an increase in sodium influx and [Na]ic; 3) [Na]ic under control conditions is close to the apparent KNa of cation pumping, implying

  2. Stimulation of insulin release from the MIN6 cell line by a new imidazoline compound, S-21663: evidence for the existence of a novel imidazoline site in β cells

    PubMed Central

    Le Brigand, Laurence; Virsolvy, Anne; Peyrollier, Karine; Manechez, Dominique; Godfroid, Jean-Jacques; Guardiola-Lemaître, Béatrice; Bataille, Dominique

    1997-01-01

    The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic β cells was used to study the insulin-releasing capacity and the cellular mode of action of S-21663, a newly synthesized imadizoline compound known for its antidiabetic effect in vivo and its ability to release insulin from perfused pancreas. S-21663, at concentrations ranging from 10−5 M to 10−3 M was able to release insulin from MIN6 cells; its activity peaked at 10−4 M, a drop in the stimulant factor being noted between 10−4 and 10−3 M. Its efficacy, which did not differ whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP-1. In contrast to tGLP-1, S-21663 did not change the cyclic AMP content, whereas it increased Ca2+ influx via verapamil- and nifedipine-sensitive voltage-dependent calcium channels, the insulin release being a direct consequence of this Ca2+ entry. The S-21663-induced Ca2+ influx appears to be essentially the consequence of closure of K+ channels which differ from the ATP-dependent K+ (K-ATP) channels as determined by measurement of 86Rb efflux and use of a K-ATP channel opener. Comparison of the effects of S-21663 to that of efaroxan, another imidazoline compound shown to act on insulin release in a glucose-dependent way via binding sites distinct from the imidazoline I1 and I2 sites, suggested that S-21663 acts through a novel site which displays a remarkably stable expression along the cell culture. It is concluded that S-21663 is a very efficient, glucose-independent insulin secretagogue acting through a novel imidazoline site, linked to K+ channels, distinct from the I1, I2 and ‘efaroxan' binding sites. In vitro and in vivo features of S-21663 indicate that this compound, or new drugs drived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin-dependent) diabetes. PMID:9375978

  3. ‘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

  4. Osmolyte and Na+ transport balances of rat hepatocytes as a function of hypertonic stress.

    PubMed

    Wehner, F; Tinel, H

    2000-11-01

    The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an influx of Na+ that is then exchanged for K+ via stimulation of Na+/K+-adenosine triphosphatase (ATPase). In this study, we analysed the activation pattern of the Na+ transporters underlying RVI as a function of the degree of hypertonic stress. In confluent primary cultures, four hypertonic conditions were tested (changes from 300 to 327, 360, 400 or 450 mosmol/l) and the activities of Na+ conductance, Na+/H+ antiport, Na+-K+-2Cl- symport and Na+/K+-ATPase were quantified using intracellular microelectrodes, microfluorometry and time-dependent, furosemide- or ouabain-sensitive 86Rb+ uptake, respectively. Neither Na+ conductance nor Na+-K+-2Cl- symport responded to 327 mosmol/A. At 360, 400 and 450 mosmol/l, uptake via these transporters would lead to increases of cell Na+ by 33.0, 49.0 and 49.0 and by 4.5, 10.4 and 9.2 mmol/l per 10 min, respectively. In contrast, Na+/H+ antiport exhibited 65% of its maximal activation already at 327 mosmol/l. At the four osmolarities tested, this transporter would augment cell Na+ by 6.9, 8.9, 9.8 and 10.6 mmol/l per 10 min. The sums of Na+ import were consistent with the amounts of Na+ exported via Na+/K+-ATPase plus the actual increases of cell Na+ (21.2, 58.5, 63.6 and 68.3 mmol/l per 10 min and 2.2, 4.0, 6.3 and 8.2 mmol/l, respectively). In addition, these elevations of cell Na+ plus the increases of cell K+ (via Na+/K+-ATPase) that amounted to 5.0, 6.5, 17.5 and 18.4 mmol/l were consistent with the increases of intracellular osmotic (cationic) activity of 2.5, 11.5, 21.0 and 28.5 mmol/l, respectively, computed from RVI data. It is concluded that the principle of rat hepatocyte RVI, i.e. an initial uptake of Na+ that is then exchanged for K+ via Na+/K+-ATPase, is realized over the entire range of 9-50% hypertonicity tested. The set-point for the activation of RVI clearly lies below 327 mosmol/l. Na+/H+ antiport is the most sensitive Na+ importer

  5. 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

    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

    2014-04-01

    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 - (K i > 10(4) nmol/L) and α7-nAChRs (K i > 10(4) 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 (86)Rb(+) 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.

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

    PubMed Central

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

    2014-01-01

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

  7. The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype

    PubMed Central

    Eaton, J. Brek; Lucero, Linda M.; Stratton, Harrison; Chang, Yongchang; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.

    2014-01-01

    Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb+ efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(−)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit–specific [125I]mAb295 labeling. However, maximum function is approximately five times greater on a “per-receptor” basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(−)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(−)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely

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

    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.

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

    PubMed

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

    2005-05-01

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

  10. Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis

    PubMed Central

    Stewart, Andrew K.; Kedar, Prabhakar S.; Shmukler, Boris E.; Vandorpe, David H.; Hsu, Ann; Glader, Bertil; Rivera, Alicia; Brugnara, Carlo

    2011-01-01

    We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na+ efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na+ efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl− transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl− transport activity, but R730 substitution with I, E, or H inactivated Cl− transport. AE1 R730C expression substantially increased endogenous oocyte Na+-K+-ATPase-mediated 86Rb+ influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pHo) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pHo-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pHo insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl− influx by acid pHo. Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb+ and Li+ influxes. Selective rescue of acid pHo-stimulated sulfate uptake and conferral of acid pHo-stimulated Cl− influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and

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

    PubMed

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

    1994-09-01

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

  12. The effects of tert-butyl hydroperoxide on human erythrocyte membrane ion transport and the protective actions of antioxidants.

    PubMed

    Dwight, J F; Hendry, B M

    1996-05-30

    The oxidising actions of tert-butyl hydroperoxide (tBH) (0-3 mmol/l) on human erythrocyte membrane ion transport have been studied using measurements of 86Rb+ influx. Ouabain and bumetanide were used to distinguish Rb+ flux via the sodium pump (Na,K-ATPase), Na,K,2Cl cotransporter and through residual membrane permeability. The protective actions of antioxidants and related molecules (vitamin E, vitamin E acetate, Trolox, butylated hydroxytoluene (BHT) and dithioerythritol (DTE) were studied. The effects of tBH were concentration dependent and the mean residual (ouabain and bumetanide insensitive) Rb+ permeability was increased by a factor of 8.5 (S.E.M. 2.2, n = 15) by a 5-min exposure to 2 mmol/l tBH. This action was almost completely prevented by co-incubation with Trolox or BHT, and partially prevented by the presence of vitamin E or DTE. Incubation with 2 mmol/l tBH for 5 min increased intracellular Na+ by a factor of 1.8 (S.E.M. 0.1, n = 8) and reduced intracellular K+ by a factor of 0.93 (S.E.M. 0.03, n = 8). These effects were prevented by Trolox and partially prevented by vitamin E, whereas DTE and vitamin E acetate were ineffective. Incubation with 2 mmol/l tBH for 5 min reduced the mean apparent sodium pump Vmax by 43% (S.E.M. 4, n = 8). This effect was completely prevented by Trolox and partially prevented by vitamin E. Vitamin E acetate had no effect. The mean bumetanide-sensitive Rb+ influx via the Na,K,2Cl cotransporter was reduced by 30% (S.E.M. 8.7, n = 25) by a 5-min exposure to 2 mmol/l tBH. This action was variable and no significant actions of the antioxidants studied could be demonstrated. This study suggests that tBH-mediated oxidative damage occurs from a hydrophilic site and involves increased non-selective membrane cation permeability and inhibition of specific transport systems.

  13. Kinetics of K-Cl cotransport in frog erythrocyte membrane: effect of external sodium.

    PubMed

    Gusev, G P; Agalakova, N I; Lapin, A V

    1999-12-01

    In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K(m)) of 8.2 +/- 1.3 mm and maximal velocity (V(max)) of 10.4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl(-)-dependent K(+) influx increased both K(m) (12.8 +/- 1.7 mm, P < 0.05) and V(max) (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) by NMDG(+) markedly decreased V(max) (3.2 +/- 0.7 mmol/l/hr, P < 0.001) and increased K(m) (15.7 +/- 2.1 mm, P < 0.03) of Cl(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong

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

    PubMed

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

    1996-12-13

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