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1.
Yamada T Konno N Matsuda K Uchiyama M 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2007,177(5):503-508
In our previous study, it was suggested that ANP and cGMP may increase Na+ absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na+ transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly
isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na+ channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 ± 0.2 pS, long open and closed
times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including
10−6 M amiloride. These characteristics were similar to those of amiloride-sensitive Na+ channels (ENaC). Addition of 10−9 M ANP activated and significantly increased the ENaC activity from 0.58 ± 0.09 to 1.47 ± 0.34. On the other hand, mean amplitudes
and conductance of single channel did not change significantly after the addition of ANP. Addition of 10−5 M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 ± 0.10 to 2.00 ± 0.33. The
addition of ANP failed to activate the ENaC in the presence of 10−6 M amiloride. These results suggested that ANP and cGMP activate Na+ transport via ENaC in the epithelial cells of frog urinary bladder. 相似文献
2.
《生物化学与生物物理学报:生物膜》1986,858(1):145-152
The transport characteristics of the skin of neotenic Ambystoma tigrinum were investigated using ion substitution and circuit analysis. When bathed with sodium Ringer solution on both sides, a transepithelial potential of up to 50 mV (inside positive) and a short-circuit current (Isc) of up to 10 μA/cm2 were observed. When amiloride was added or Na+ was replaced by tetramethylammonium in the apical solution, Isc was decreased from 3.7 ± 0.4 to 1.5 ± 0.2 μA/cm2 (n = 10). When K+ replaced Na+, there was a smaller change in Isc from 5.8 ± 0.6 to 3.7 ± 0.5 μA/cm2 (n = 10). Although barium had no effect when added to 100 K Ringer on normal skin, it inhibited Isc on skins taken from K+-loaded animals. Nystatin caused substantial increases in Isc with either Na+ or K+ as the dominant cation in the apical solution. Current voltage analysis using amiloride was used to estimate the resistances and electromotive forces (EMF) associated with ion transport. The EMF for ion transport was partially dependent on K+ in the basolateral solution and it was similar to that observed in other epithelia. The resistance of the transport pathway was high, consistent with the low Isc. These results suggest that there is an amiloride-sensitive Na+ channel in parallel with a small K+ conductance in the apical membrane of this preparation. 相似文献
3.
I.I. Ismailov B.K. Berdiev A.L. Bradford M.S. Awayda C.M. Fuller D.J. Benos 《The Journal of membrane biology》1996,149(2):123-132
The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component,
an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of
renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and
uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements,
and apparent amiloride equilibrium dissociation constant (K
amil
i
) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed
a substantial decrease in K
amil
i
following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even
allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC recognized a 70 kDa polypeptide component
of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins.
Received: 5 June 1995/Revised: 29 September 1995 相似文献
4.
Toru Ishikawa Yoshinori Marunaka Daniela Rotin 《The Journal of general physiology》1998,111(6):825-846
The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P
o), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP
o) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+. 相似文献
5.
Mitzy Canessa José R. Romero Nelson Ruiz-Opazo Victoria L. M. Herrera 《The Journal of membrane biology》1993,134(2):107-122
The properties of the α1 Na+-K+ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( i ) and external ( o ) Na+ and K+ concentrations. Kinetic parameters of several modes of operation, i.e., Na+/ K+, K+/K+, Na+/Na+ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na+ + 5 mm K+, ouabain-sensitive K+ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na+ efflux and Na i were similar in both strains. Thus, the coupling ratio between unidirectional Na+∶K+ fluxes was significantly higher in DS than in DR cells at similar RBC Na+ content. In the presence of 140 mm Na o , activation of ouabain-sensitive K+ influx by K o had a lower K m and V max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K m 0.74 ± 0.09 mm; V max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K m 2.31 ± 0.16 mm; v max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na o removal. The activation of ouabain-sensitive K+ influx by Na i had significantly lower V max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K m. These data suggest that the low K+ influx in DS cells is caused by a defect in modulation by Na o and Na i . Na+ efflux showed no differences in Na i activation or trans effects by Na o and K o , thus accounting for the different Na+∶K+ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na+ and K+ fluxes, where the net ouabain-sensitive Na+ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K+ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na+ influx and K+ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na+ influx by K o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K o could activate higher K+ influx in DR pumps without a complete inhibition of ouabain-sensitive Na+ influx. This behavior is consistent with K o interaction with distinct Na+ and K+ transport sites. In addition, the inhibition of K+ efflux by Na, was different between Dahl strains. Ouabain-sensitive K+ efflux at Na i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K50 for Na i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na+ modulation of K+ transport is altered at both sides of the membrane. The dissociation of Na+ modulatory sites of K+ transport from Na+ transport sites observed in RBCs of Dahl strains suggests that K+ transport by the Na+-K+ pump is controlled by Na+ allosteric sites different from the Na+ transport sites. The alterations in K+ transport may be related to the amino acid substitution (Leu/Gln276) reported for the cDNA of the α1 subunit of the Na+-K+ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na+-K+ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990. 相似文献
6.
Marie-Christine Broillet Jean-Daniel Horisberger 《The Journal of membrane biology》1993,134(3):181-188
K+ channels sensitive to intracellular ATP (KATP channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K+ channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 ± 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of ?83 ± 1 mV and was inward rectifying in spite of the outwardly directed K+ gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 ± 3.0 μm and 0.114 ± 0.018 μm for tolbutamide and glibenclamide respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 mm) and insulin (250 μU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K+ conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP. 相似文献
7.
M. M. Reddy J. R. Riordan P. M. Quinton 《In vitro cellular & developmental biology. Plant》1988,24(9):905-910
Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and
related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with
electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V
a
) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na+−K+ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized
both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na+ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (Va) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na+ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture.
Cl− substitution at the apical side by the impermeant anion gluconate depolarized the V
a
of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized
(ΔV
a
=−8.3±1.2mV,n=5) or depolarized (ΔV
a
=8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest
that CD cells, like MPD cells, retain significant Na+ and Cl− conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared
to MPD cells.
This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette
Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation. 相似文献
8.
In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na+ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na+ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I
sc) and the amiloride-sensitive Na+ current (I
ami) across Xenopus alveolar epithelium was downregulated by high apical Na+ concentrations. Apical application of gadolinium (Gd3+) increased I
sc in a dose-dependent manner (EC
50 = 23.5 µM). The effect of Gd3+ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na+ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na+ channels from Na+-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na+ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd3+ on transepithelial Na+ transport. PHMB increased I
sc and this stimulation was similar to the effect of Gd3+. Co-application of PHMB and Gd3+ had no additive effects on I
sc. In cell-attached patches on Xenopus oocytes extracellular Gd3+ increased the open probability (NP
o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na+-dependent non-hormonal control of transepithelial Na+ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd3+ directly modulates the activity of ENaCs. 相似文献
9.
Hideo Takanashi Tohru Sawanobori Kazuaki Kamisaka Hidenori Maezawa Masayasu Hiraoka 《Journal of cellular physiology》1994,161(3):537-543
The patch-clamp technique of cell-attached and inside-out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K+ concentrations. A linear single-channel current-voltage relationship was obtained at the voltage range of -80 to -20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K+ concentration of 148 mM in the cell-attached patch membrane, the I-V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K+]0) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K+]0, indicating that this channel was highly selective for K+. Open probabilities (P0) of the channel were 73-93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K+ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34-36°C. P0 in inside-out patches was not changed by application of 1 μM Ca2+ nor 1 mM Mg2+ to the internal side of patch membranes. It is concluded that a novel type of the K+ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca2+]i, from those in other species. © 1994 Wiley-Liss, Inc. 相似文献
10.
We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na+ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na+ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na+, we decided to use whole-cell patch clamp techniques to investigate whether the Na+ conductance in salivary duct cells is also regulated by extracellular Na+. Using Na+-free pipette solutions, we observed that the whole-cell Na+ conductance increased when the extracellular Na+ was increased, whereas the whole-cell Na+ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na+ conductance on extracellular Na+ could be described by the Michaelis-Menten equation with a K
m
of 47.3 mmol/1 and a maximum conductance (G
max) of 2.18 nS. To investigate whether this saturation of the Na+ conductance with increasing extracellular Na+ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of
the noise generated during the onset of blockade of the Na+ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel
conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na+ solutions. We found that Na+ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing
extracellular Na+. On the other hand, the single-channel current saturated with increasing extracellular Na+ and, consequently, whole-cell Na+ permeability declined. In other words, the decline in Na+ permeability in salivary duct cells with increasing extracellular Na+ concentration is due simply to saturation of the single-channel Na+ conductance rather than to inactivation of channel activity.
Received: 27 July 1995/Revised: 7 December 1995 相似文献
11.
The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mg i 2+ , interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mg i 2+ . This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mg i 2+ leads to a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mg i 2+ is decreased upon increasing intracellular or extracellular Na+ concentration, which suggests that Mg?' interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mg i 2+ for the sodium channel has been calculated to be 8.65 ± 1.51 mM, with its binding site located at 0.26 ± 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Na i +, which is 83.76 ± 7.60 mM with its binding site located at 0.75 ± 0.23. The low dissociation constant of Mg i 2+ reflects its high affinity for the sodium channel. 相似文献
12.
C. Korbmacher T. Volk A. S. Segal E. L. Boulpaep E. Frömter 《The Journal of membrane biology》1995,146(1):29-45
We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA
89:10262–10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na+, K+, Rb+, Cs+, and Li+. It has a linear I-V relation with a conductance of 22.7±0.5 pS (n=78) at room temperature. The Pcation/ Panion ratio is about 60 and there is no measurable conductance for NMDG, Ca2+, Ba2+, and Mn2+. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10–6
m and depolarization increases channel activity (NP
o
). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10–4
m and 10–3
m, ATP reduces NP
o
by 23% and 69%, respectively.Furthermore, since ADP (10–3
m) reduces NP
o
by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a -phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10–4
m) or by cGMP-dependent protein kinase (10–7
m) in the presence of 8-Br-cGMP (10–5
m) and ATP (10–4
m). The NSC channel is not sensitive to amiloride (10–4
m cytoplasmic and/or extracellular) but flufenamic acid (10–4
m) produces a voltage-dependent block, reducing NP
o
by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages.We conclude that the NSC channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.The expert technical assistance of U. Fink and I. Doering-Hirsch is gratefully acknowledged. We thank A. Rabe and Dr. J. Disser for programming the computer software.This work was supported by a grant from the Deutsche Forschungsge-meinschaft (DFG grant Fr 233/9-1) and a grant from the National Institutes of Health (NIH grant DK-17433). 相似文献
13.
Weifeng Song Shipeng Wei Yongjian Zhou Ahmed Lazrak Gang Liu James D. Londino Giuseppe L. Squadrito Sadis Matalon 《The Journal of biological chemistry》2010,285(13):9716-9728
We investigated the mechanisms by which chlorine (Cl2) and its reactive byproducts inhibit Na+-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na+ channels (ENaC) by measuring AFC in mice exposed to Cl2 (0–500 ppm for 30 min) and Na+ and amiloride-sensitive currents (INa and Iamil, respectively) across Xenopus oocytes expressing human α-, β-, and γ-ENaC incubated with HOCl (1–2000 μm). Both Cl2 and HOCl-derived products decreased AFC in mice and whole cell and single channel INa in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased INa in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of INa by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the γ-ENaC subunit restored the ability of HOCl to inhibit INa. Finally, INa of oocytes expressing wild type α- and γ-ENaC and a mutant form of βENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage. 相似文献
14.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
15.
Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate
that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl− channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V
te
) of −31.4 ± 1.3 mV and a transepithelial resistance (R
te
) of 1151 ± 28 Ωcm2. The amiloride-sensitive short circuit current (I
sc
) was −28.0 ± 1.1 μA/cm2. The ATP-mediated activation of Cl− channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+]i increase was paralleled by a rapid and transient R
te
decrease (297 ± 51 Ωcm2). In the presence of CPA, basolateral ATP led to an R
te
increase by 144 ± 17 Ωcm2 and decreased V
te
from −31 ± 2.6 to −26.6 ± 2.5 mV. I
sc
dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated
inhibition of Na+ absorption. Lowering [Ca2+]i by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pHi alkalinization and inhibited by pHi acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect.
In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+]i, does not involve PKC and is to a small part mediated via intracellular acidification.
Received: 9 February 2001/Revised: 17 May 2001 相似文献
16.
Summary The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of “endogenous”
CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to
overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus
Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed
two fundamental components of absorption, a cAMP activated Cl− conductance (Gcl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10−5
M) significantly increased the Cl− diffusion potential (Vt) generated by a luminally directed Cl− gradient from − 15.3 ± 0.7 mV to −23.9 ± 1.1 mV,n=39; and decreased the transepithelial resistance (Rt) from 814.8 ± 56.3 Ω.cm2 to 750.5 ± 47.5 Ω.cm2,n=39, (n=number of cultures). cAMP activation, anion selectivity (Cl−>I−>gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10−5
M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 ± 3.1% and 77.9 ± 2.6%, respectively, and an increase in Rt by 7.2 ± 0.8%,n=36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue. It provides an opportunity to study the properties and role of CFTR in the context
of absorptive function in immortalized epithelial cells. 相似文献
17.
The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na+ with Li+, K+, Rb+, or Cs+ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with PK/PNa = 0.1 and GK/GNa = 0.7 and PRb/PNa = 0.03 and GRb/GNa = 0.3. Stimulation of Cl− currents when Na+ was replaced with Ca2+, Sr2+, or Ba2+ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na+ in a hyperbolic manner, consistent with an apparent affinity (Km) for Na+ conduction of 25 mM. Nitrogen-containing cations, including NH4+, NH3OH+, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na+ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na+ (Km = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule. 相似文献
18.
With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na+ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na+ over K+ (as for Cs+, NH4
+, Ca2+, Ba2+) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li+-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722. 相似文献
19.
Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl− secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5′-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5′-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y2 receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl− channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl− transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N2,2′-O-dibutyrylguanosine 3′,5′-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl−. 相似文献