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1.
Summary Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10–8 m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10–8 m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10–8 m) resulted in a rapid and transient increase in [Ca2+] i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca2+ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/P i cotransport.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation and the Geigy-Jubiläumsstiftung. We are grateful to Denise Rossi and Christa Knellwolf for their excellent secretarial assistance.  相似文献   

2.
Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm2 from a mean value of 138±13 · cm2 before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl in the medium. Subsequent addition of bumetanide (10–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba2+ (1mm), a known inhibitor of basolateral K+ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K+ conductance.The results suggest that forskolin-induced Cl secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl conductance of the apical membrane but does not affect K+ or Cl conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl and the basal Ba2+-sensitive K+ conductance are sufficiently large to maintain the Cl efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl channel blockers NPPB and DPC on basolateral conductance rather than apical Cl conductance.  相似文献   

3.
Summary Cell K activity,a k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO3 Ringer on the apical surface.Under control conditionsa k r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm2). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina k r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa k r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.  相似文献   

4.
Summary The effect of extracellular and intracellular Na+ (Na o + , Na i + ) on ouabain-resistant, furosemide-sensitive (FS) Rb+ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na++1 K++2 Cl) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na+–K+) cotransport, and modeii) a Na o + -independent 11 K o + /K i + exchange requiring Na i + which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb+ uptake through Rb o + /K i + exchange contributes about 25% to total Rb+ uptake in 145mm NaCl media containing 5mm RbCl at normal Na i + (pH 7.4). Na+–K+ cotransport into the cells occurs largely additive to K+/K+ exchange. Inward Na+–Rb+ cotransport exhibits a substrate inhibition at high Rb o + . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K+/K+ exchange (apparent pK close to pH 7.4). The apparentK m Rb o + of Na+–K+ cotransport is low (2mm) and almost independent of pH, and high for K+/K+ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na++1 K++2 Cl) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl-dependent K+ transport pathway that is independent of both Na o + and Na i + . This pathway differs in many properties from the basal, Na o + -independent K+/K+ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na o + -independent FS K+ transport pathway which most probably is not identical to basal K+/K+ exchange. K o + o +
  • o + o 2+ reduce furosemide-resistant Rb+ inward leakage relative to choline o + .  相似文献   

  • 5.
    We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca2+ uptake was inhibited by TG up to 10 nm (apparent Ki4.2 nm, Ca2+ pool I). An additional increase of inhibition up to 85–90% of total Ca2+ uptake could be achieved at 15 to 20 nm of TG (apparent Ki12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30–40% of total Ca2+ uptake was inhibited by 100 m of vanadate (apparent Ki18 m, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki300 m) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at 25% over a wide range of Ca2+ filling. About 10–20% remained unreleasable by IP3. Reduction of IP3 releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki 3.0 nm for IP3-induced Ca2+ release as compared to 4.2 nm for Ca2+ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations >10 nm which blocked Ca2+ pool II the apparent Ki values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent Ki values were 18 m for Ca2+ uptake and 7 m for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were 300 and 200 m, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 m. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 m. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 m. These data allow the conclusion that there are at least three different Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and IP3 insensitive but highly vanadate-sensitive Ca2+ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca2+ pools with different TG-, vanadate- and IP3-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.  相似文献   

    6.
    Cyclic AMP and intracellular ionic activities innecturus gallbladder   总被引:2,自引:0,他引:2  
    Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E m ) and intracellular sodium, potassium, and chloride activities (a Na i ,a K i ,a Cl i ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E Tr ) was also measured. In the presence of cAMP,a Cl i fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa Na i decreased anda K i increased,E m was unchanged andE Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina K i was abolished when Cl-free incubation media were used. Addition of the Ca++-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE m ,E Tr , ora Cl i . When A23187 was added to the mucosal medium,E m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca++ concentration.  相似文献   

    7.
    We have used a well-differentiated A6-cell preparation (A6-C1) to study cellular location and vasopressin control of Na/H-exchange activity. After cell acidification, cell pHi (measured by BCECF-fluorescence) only recovered by the addition of Na medium to the basolateral cell surface; this pHi recovery was inhibited by dimethylamiloride (2 m) consistent with basolateral location of Na/H-exchange activity. Addition of vasopressin produced stimulation of Na/H-exchange activity and increased the affinity of the exchanger for Na+. Stimulation of Na/H exchange was mimicked by pharmacological activation of protein kinase A (forskolin, 8-Br-cAMP) and not by pharmacological activation of protein kinase C (TPA). It is concluded that basolaterally located Na/H-exchange in A6-C1 cells is activated by vasopressin.  相似文献   

    8.
    Summary Cell Na activity,a Na c , was measured in the short-circuited frog skin by simulaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditionsa Na c averaged 8±2mm (n=9,sd). Apical addition of amiloride (20 m) or Na replacement reduceda Na c to 3mm in 6–15 min. Sequential decreases in apical [Na] induced parallel reductions ina Na c and cell current,I c . On restoring Na after several minutes of exposure to apical Na-free solutionI c rose rapidly to a stable value whilea Na c increased exponentially, with a time constant of 1.8±0.7 min (n=8). Analysis of the time course ofa Na c indicates that the pump Na flux is linearly related toa Na c in the range 2–12mm. These results indicate thata Na c plays an important role in relating apical Na entry to basolateral active Na flux.  相似文献   

    9.
    Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells. In this study, we identify and characterize this basolateral, barium-sensitive K conductance. Porcine uterine tissues were mounted in Ussing chambers and bathed with KMeSO4 Ringer solution. Amphotericin B (70 μm) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of the basolateral K conductance after activation by 100 μm CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of −53 mV when standard Ringer solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5 mm barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential of −56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However, the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying K channels are present in endometrial epithelial cells from different species. Received: 6 February 1997/Revised: 10 July 1997  相似文献   

    10.
    Cl conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na+and K+-free internal and external solutions, the basal whole-cell current was predominantly a Cl current. Cl-sensitive current (I Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl] o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl] < 40 mm, and was not saturated with a physiological Cl gradient. The Cl current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl current. The cAMP- or forskolin-activated and basal Cl current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca2+] i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca2+] i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.  相似文献   

    11.
    Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm2. The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na+, and serosal Na+ is substituted by K+, Cs+, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na+ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca2+. In Cl-free SO 4 2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K+. This pathway can be blocked by serosal amiloride (K i=13.1 m) or serosal Na+ ions (K i 10 to 20mm). It also conducts Li+ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec–1 at 0 mV.  相似文献   

    12.
    Summary The specific activity of the Na+/K+/Cl cotransporter was assayed by measuring the initial rates of furosemide-inhibitable86Rb+ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable86Rb+ influx on external Na+ and K+ was hyperbolic with apparentK m values of 46 and 4mm, respectively. The dependence on Cl was sigmoidal. Assuming a stoichiometry of 112 for Na+/K+/Cl, aK m of 78mm was obtained for Cl. In quiescent smooth muscle cells cotransport activity was approximately equal to Na+ pump activity with each pathway accounting for 30% of total86Rb+ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na+ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca2+ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited86Rb+ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.  相似文献   

    13.
    Summary Na–K–Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics withK 1/2 values of 5 and 4.5mm and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship withK 1/2 of 20mm and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (I sc) were also determined. TheK 1/2 for Na was 7mm with a Hill coefficient of 0.9 and theK 1/2 for Cl was 46mm with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na1K2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 21. Therefore Na recycling from serosa to mucosa does not significantly contribute to theI sc. Addition of serosal ouabain (100 m) inhibited Rb influx, indicating that Na–K–Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na–K-ATPase on the basolateral membrane and the apical Na–K–2Cl cotransporter.  相似文献   

    14.
    Summary The effect of the K-sparing diuretic amiloride was assessed electrophysiologically in the isolated cortical collecting tubule of the rabbit, a segment which absorbs Na and secretes K. Low concentrations of amiloride in the perfusate caused a rapid, reversible, decrease in the magnitude of the lumen negative transepithelial potential difference,V te, transepithelial conductanceG te, and equivalent short-circuit current,I sc, with an apparentK 1/2 of approximately 7×10–8 m. The effects of a maximum inhibitory concentration of amiloride (10–5 m) were identical to those observed upon Na removal from lumen and bath (Na removal from the bath alone has no effect). Removal of Na in the presence of 10–5 m amiloride had no affect onV te,G te, orI sc, and is consistent with the view that amiloride blocks the Na conductive pathways of the apical cell membrane. Further, in the absence of Na, the subsequent addition of amiloride had no influence. In tubules where active Na absorption was either spontaneously low, or abolished by removal of Na from lumen and bath, the elevation of K from 5 to 155 meq/liter in the perfusate caused a marked change of theV te in the negative direction and an increase in theG te. These effects could be attributed to a high K permeability of the apical cell membrane and not of the tight junctions. Amiloride (10–5 m) had no effect on these responses to K. It is concluded that amiloride selectively blocks the apical cell membrane Na channels but has no effect on the K conductive pathways(s). This selective nature of amiloride may indicate that Na and K are transported across the apical cell membrane via separate conductive pathways.  相似文献   

    15.
    Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for ClBr>NO 3 >I. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.  相似文献   

    16.
    The respective effects of aldosterone and arginine vasopressin (AVP) were examined on the number of active Na+-K+-ATPase and their pumping activity in nonperfused microdissected mouse cortical collecting tubules (CCD) by measuring specific 3H-ouabain binding and ouabain-sensitive 86Rb uptake. In adrenalectomized (ADX) animals, incubation of CCD with AVP (10–8 m for 5 min) had no effect on the number of pumps. In contrast, in ADX animals replete with aldosterone, AVP induced a 40% increase in the number of pumps. This was accompanied by a 60–65% increase in ouabain-sensitive Rb uptake. AVP effect was dose-dependent (10–10–10–8 m) and was reproduced by dDAVP, forskolin and 8-Br cAMP, indicating a V2 pathway. It was inhibited by amiloride 10–5 m, and did not occur in CCD incubated in hyperosmotic solution, suggesting that the signal was transmitted via apical sodium entry and cell swelling. Finally, the AVP-dependent increase in the number of pumps was rapid (within 5 min) and transient (<25 min).These results demonstrate that, in the CCD, aldosterone and AVP act synergistically to increase not only the apical sodium entry but also the basolateral Na+-K+-ATPase transport capacity: AVP allows a rapid recruitment and/or activation of an aldosterone-dependent pool of latent Na+-K+-ATPase.  相似文献   

    17.
    Summary The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm–2hr–1, andI sc, 0.50±0.50(8) eq cm–2hr–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 21 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation.  相似文献   

    18.
    Summary Ion-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na] i , electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na] i : 9.2 to 12.1mm; transepithelial potential difference, ms : –1.5 mV (lumen negative); basolateral cell membrane potential, es : –62 mV (cell interior negative); sodium conductance of the luminal cell membrane,g Na: 12 mho cm–2; active transcellular sodium flux, 88 to 101 pmol cm–2 sec–1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.  相似文献   

    19.
    Summary Patch-clamp and single cell [Ca2+] i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca2+] i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward KATP current, (ii) evoked Ca2+ spike potentials, and (iii) caused a sharp rise in [Ca2+] i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca2+ spike potentials and attenuated the secretagogue-induced rise in [Ca2+] i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K+ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K+ channels, reversed the effects of cromakalim on membrane potential and KATP currents.  相似文献   

    20.
    Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent22Na transport and tracer [3H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK 0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK 0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and22Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.  相似文献   

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