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1.
In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO3) 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+ (86Rb) 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 asymmetry with respect to transported K+ ions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K+ effluxes in isotonic (∼0.014 hr−1) and hypotonic (∼0.022 hr−1) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999  相似文献   

2.
The current-voltage (I/V) profiles of Ventricaria (formerly Valonia) membranes were measured at a range of external potassium concentrations, [K+] o , from 0.1 to 100 mm. The conductance-voltage (G/V) characteristics were computed to facilitate better resolution of the profile change with time after exposure to different [K+] o . The resistance-voltage (R/V) characteristics were computed to attempt resolution of plasmalemma and tonoplast. Four basic electrophysiological stages emerged: (1) Uniform low resistance between −60 and +60 mV after the cell impalement. (2) High resistance between +50 and +150 for [K+] o from 0.1 to 1.0 mm and hypotonic media. (3) High resistance between −150 and −20 mV for [K+] o of 10 mm (close to natural seawater) and hypertonic media. (4) High resistance between −150 and +170 mV at [K+] o of 100 mm. The changes between these states were slow, requiring minutes to hours and sometimes exhibiting spontaneous oscillations of the membrane p.d. (potential difference). Our analysis of the I/V data supports a previous hypothesis, that Ventricaria tonoplast is the more resistive membrane containing a pump, which transports K+ into the vacuole to regulate turgor. We associate state (1) with the plasmalemma conductance being dominant and the K+ pump at the tonoplast short-circuited probably by a K+ channel, state (2) with the K+ pump ``off' or short-circuited at p.d.s more negative than +50 mV, state (3) with the K+ pump ``on,' and state (4) with the pump dominant, but affected by high K+. A model for the Ventricaria membrane system is proposed. Received: 5 November 1998/Revised: 11 May 1999  相似文献   

3.
Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I sc ) conditions. Addition of DCCD to a concentration of 10−4 m to the tear solution gave a marked decrease in I sc ; a marked depolarization of the intracellular potential, V o ; and a significant decrease in the apical membrane fractional resistance, fR o . There were small and variable although significant changes in the transepithelial conductance, g t . The effects may be explained by a decrease in the basolateral membrane K+ conductance, in combination with a partial inhibition of the Na+/K+-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl conductance. Received: 12 August 1999/Revised: 16 November 1999  相似文献   

4.
To examine the involvement of Na+,K+,2Cl cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on 86Rb, 36Cl and 22Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward 86Rb fluxes were not different. Bumetanide decreased basal 86Rb uptake by 70–75% with a K i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect 36Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to 86Rb and 36Cl influx, bumetanide did not inhibit 22Na uptake by VSMC. BS 86Rb uptake was completely abolished in Na+- or Cl-free media. In contrast to 86Rb, basal BS 36Cl influx was not affected by Na+ o and K+ o . Hyperosmotic and isosmotic shrinkage of VSMC increased 86Rb and 36Cl influx to the same extent. Shrinkage-induced increments of 86Rb and 36Cl uptake were completely abolished by bumetanide with a K i or ∼0.3 μm. Shrinkage did not induce BS 86Rb and 36Cl influx in (Na+ or Cl)- and (Na+ or K+)-depleted media, respectively. In the presence of an inhibitor of Na+/H+ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated 22Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na+,K+,2Cl cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K+ influx is mediated by (Na+ o + Cl o )-dependent K+/K+ exchange and Na+ o -dependent K+,Cl cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996  相似文献   

5.
Hyperthermia induces transient changes in [Na+] i and [K+] i in mammalian cells. Since Cl flux is coupled with Na+ and K+ in several processes, including cell volume control, we have measured the effects of heat on [Cl] i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl] i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl] i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na+] i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl] i depended upon time and temperature. The [Cl] i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl] i increase was completely blocked. Since furosemide inhibits the Na+/K+/2Cl cotransporter, Cl channels, and even ClHCO3 exchange, these ion transporters may be involved in the heat-induced increase in [Cl] i . Received: 15 June 1995/Revised: 9 April 1996  相似文献   

6.
The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl absorption linked to Na+ were investigated in isolated perfused tubules using Cl-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl (a i Cl ) is 11.3 ± 0.5 mm, the basolateral (V 1 ), apical (V 2 ), and transepithelial (V 3 ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, a i Cl falls by 1.3 ± 0.3 mm and V 2 hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na+ from the lumen causes a i Cl to fall further by 2.3 ± 0.4 mm and V 2 to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a i Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a i Cl . The effects of luminal HCO 3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO 3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO3 exchange. Several lines of evidence favor conductive Cl transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a i Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO 3-free Ringer, the equilibrium potential for Cl does not differ from the membrane potential V 2 . Finally, the logarithmic changes in a i Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998  相似文献   

7.
Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+] i . The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (K d = 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996  相似文献   

8.
In tilapia (Oreochromis mossambicus) intestine, Mg2+ transport across the epithelium involves a transcellular, Na+- and Na+/K+-ATPase dependent pathway. In our search for the Mg2+ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na+/Mg2+ antiport or ATP-driven transport. Evidence is provided, however, that Mg2+ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl gradient stimulated Mg2+ uptake (as followed with the radionuclide 27Mg) twofold. As Cl-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg2+ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg2+ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl-stimulated Mg2+ uptake yielded a K m (Mg2+) of 6.08 ± 1.29 mmol · l−1 and a K m (Cl) of 26.5 ± 6.5 mmol · l−1, compatible with transport activity at intracellular Mg2+- and Cl-levels. We propose that Mg2+ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996  相似文献   

9.
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K+ and HCO 3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO 3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO 3 influx (J HCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO 3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO 3. With no QA+ in the DF, J HCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, J HCO3 was virtually zero. The apparent K i for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced J HCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent K i value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent K i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter. Received: 21 November 2000/Revised: 14 May 2001  相似文献   

10.
Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver   总被引:1,自引:0,他引:1  
Although both amiloride- and phloretin-sensitive Na+/Li+ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li+ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li+ by hepatocytes, preloaded with Na+ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K m 1.2 mm Li+, V max 40 μmol · (kg dry wt · min)−1, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H+ was curvilinear suggesting a modifier role of [H+] i . This system persisted in Na+-depleted cells, although with apparent K m 3.8 mm. (b) A phloretin-sensitive component, with K m 1.2 mm, V max 21 μmol · (kg · min)−1, was unaffected by pH but was inactive in Na+-depleted cells. Phloretin inhibited Li+ uptake and Na+ efflux in parallel. (c) A residual uptake increased linearly with the external Li+ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li+ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H+]. Received: 8 April 1999/Revised: 19 July 1999  相似文献   

11.
P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+] i , activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+ i , BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+,Cl cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+,Cl cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October  相似文献   

12.
Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E50, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K+, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na+-rich solution with 2.5 mm extracellular K+γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K+ over Na+. γ depended on extracellular K+ concentration (K D = 19.6 mm) and temperature (Q 10= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC50) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC50= 6.8 nm) and mast cell degranulating peptide (MCDP, IC50= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995  相似文献   

13.
MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+–K+ ATPase and Cl/HCO 3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+ 4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10−8 m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H+–K+-ATPase. This result was confirmed by the use of 5 × 10−5 m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10−5 m (a specific blocker of Cl channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na+ and Cl free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+–K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl–free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998  相似文献   

14.
The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K Ca ) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7 m (open probability (P o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P o of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl and N-methyl-d-glucamine. P o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999  相似文献   

15.
The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10−7 m bafilomycin or 4.6 × 10−8 m concanamycin were added. No significant increase in lumen-negative V t upon perfusion with these inhibitors as compared to control, was observed as well as when 10−3 m amiloride, 10−5 m benzamil or 3 mm Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative V t by 2–4 mV, was observed only when Cl channels were blocked by 10−5 m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm−2.sec−1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule V t is minor in physiological conditions, but can be demonstrated after blocking Cl channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl channel blocker, supporting coupling of H+-ATPase with Cl transport. Received: 6 July 1996/Revised: 27 December 1996  相似文献   

16.
The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion by the glands was studied by measuring unidirectional fluxes of 24Na+, 42K+, and carrier-free 134Cs+ in paired frog skins bathed on both sides with Ringer's solution, and with 10−5 m noradrenaline on the inside and 10−4 m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the 134Cs+ flux ratio, J out Cs/J in Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free 134Cs+ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux of 134Cs+ indicates that a paracellular flow of water is dragging 134Cs+ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving the secretory flux of Cs+ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na+ fluxes were measured as well, and it was found that also Na+ was subjected to secretion. The ratio of unidirectional Na+ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular route dragged by the flow of water. This result indicates that Na+ and Cs+ do not take the same pathway through the glands. The flux ratio of unidirectional K+ fluxes indicated active secretion of K+. The time it takes for steady-state K+ fluxes to be established was significantly longer than that of the simultaneously measured Cs+ fluxes. These results allow the conclusion that — in addition to being transported between cells — K+ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which accounts for stationary Na+, K+, Cl and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na+ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction of Na+ entering the lateral space from returning to the serosal bath. Thus, Na+ is secreted into the outside bath. It has to be assumed then that the Na+ permeability of the basement membrane barrier (P BM Na) is smaller than the Na+ permeability of the junctional membrane (P JM Na), i.e., P JM Na/P BM Na > 1. The secretory paracellular flow of water further requires that the Na+ reflection coefficients (σNa) of the two barriers are governed by the conditions, σBM Na > 0, and σBM Na > σJM Na. (iii) Na+ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na+ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would be hydrolyzed for every Na+ secreted to the outside bath. Received: 30 January 1996/Revised: 12 March 1996  相似文献   

17.
We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na+,K+,Cl cotransport is inhibited up to 50% by ATP via its interaction with P2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na+,K+,Cl cotransport to purinergic regulation. We did not observe any effect of ATP on Na+,K+,Cl cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na+,K+,Cl cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP3 and cAMP production and transient elevation of [Ca2+] i . In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na+,K+,Cl cotransport by P2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity. Received: 10 June 1999/Revised: 23 August 1999  相似文献   

18.
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na+/K+/2Cl cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels. Received: 2 September 1998/Revised: 30 November 1998  相似文献   

19.
We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl, acting, respectively, via G o and G i proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+] o ), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na+] o . Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of G o in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor. Received: 1 October 1997/Revised: 24 December 1997  相似文献   

20.
Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl into the cell and is negligible when Cl flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (I amil ) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced I amil by up to 80%, demonstrating that ENaC is inhibited when Cl is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I amil downregulation and the CFTR-mediated plasma membrane Cl conductance, suggesting a direct correlation. However, I amil downregulation was not affected when Cl flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I amil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in I amil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit I amil . These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl transport across the plasma membrane. Received: 15 December 1998/Revised: 5 March 1999  相似文献   

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