Prostaglandin E2 increases 7-pS Cl- channel density in the apical membrane of A6 distal nephron cells

1997 ◽  
Vol 273 (2) ◽  
pp. C548-C557 ◽  
Author(s):  
K. E. Kokko ◽  
P. S. Matsumoto ◽  
Z. R. Zhang ◽  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Prostaglandin E2 ◽  
Channel Blocker ◽  
Short Circuit ◽  
Apical Cell ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Distal Nephron ◽  
Open Probability ◽  
Channel Density ◽  
Cl Channel

In A6 distal nephron cells, short-circuit current (Isc) was increased by basolateral exposure to prostaglandin E2 (PGE2; peak response at 1 microM). The effect was only partially abolished by either apical amiloride, an Na+ channel blocker, or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl- channel blocker. In apical cell-attached patches, we observed a 7-pS Cl- channel with a linear current-voltage relationship, a reversal potential near resting membrane potential, and open probability > 0.5. The channel was blocked by diphenylamine-2-carboxylate, glibenclamide, and NPPB but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The frequency of observed Cl- channel activity increased 7-fold with 10-min exposure to PGE2 and 3.7-fold with longer (10-50 min) exposure to PGE2. The PGE2-induced increase in Cl- channel activity was due primarily to an increase in the number of functional channels. The following conclusions were made: 1) activation of apical, 7-pS Cl- channels in A6 cells accounts for the PGE2-induced increase in the amiloride-insensitive Isc, and 2) 7-pS Cl- channel activation was mediated via an increase in channel density without substantial effects on channel kinetics.

Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6)

1994 ◽  
Vol 267 (5) ◽  
pp. C1414-C1425 ◽  
Author(s):  
K. E. Kokko ◽  
P. S. Matsumoto ◽  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Prostaglandin E2 ◽  
Apical Cell ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
Channel Inhibition ◽  
Camp Levels

We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.

A novel cGMP-activated Cl- channel in renal proximal tubules

1995 ◽  
Vol 268 (2) ◽  
pp. F323-F329 ◽  
Author(s):  
N. Darvish ◽  
J. Winaver ◽  
D. Dagan
Keyword(s):  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Open Probability ◽  
Cl Channel ◽  
Bath Application ◽  
Cl Channels ◽  
Dose Dependent

Cl- channels activated by natriuretic peptides were detected in cultured rat proximal convoluted tubule (PCT) cells with the use of patch-clamp methodology. Bath application of atrial natriuretic peptide (ANP) activates a 150-pS Cl- channel with the open probability (Po) of the channel increasing from 0.0008 +/- 0.0003 to 0.021 +/- 0.008. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a membrane-permeable analogue of cGMP, increased channel activity in the on-cell mode. In inside-out patches the channel was activated by cGMP in a dose-dependent manner. Channel activity decreased after washing out and increased on reapplication of cGMP. A similar activation was observed also in presence of either of two protein kinase inhibitors, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride or KT5823, or a phosphatase inhibitor. Bath application of urodilatin mimicked the action of ANP. Po of the channel was found to be independent of both voltage and Ca2+, and gating activity could be blocked by the stilbene, 4,4-dinitrostilbene-2,2-disulfonic acid. These results demonstrate a Cl- conductance in PCT cells modulated by ANP and urodilatin via their second messenger, cGMP.

Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers

1989 ◽  
Vol 256 (4) ◽  
pp. C902-C912 ◽  
Author(s):  
R. J. Bridges ◽  
R. T. Worrell ◽  
R. A. Frizzell ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Kinetic Scheme ◽  
Disulfonic Acid ◽  
Lipid Bilayer Membranes ◽  
Plasma Membrane Vesicles ◽  
Open Probability ◽  
Cl Channel ◽  
Cl Channels

We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.

Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells

1997 ◽  
Vol 273 (3) ◽  
pp. C999-C1007 ◽  
Author(s):  
A. Stutzin ◽  
A. L. Eguiguren ◽  
L. P. Cid ◽  
F. V. Sepulveda
Keyword(s):  
Hela Cells ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Common Channel ◽  
Inward Current ◽  
Cl Channel ◽  
Organic Osmolyte ◽  
Efflux Pathway ◽  
Taurine Efflux

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.

Cl- channels of the gastric parietal cell that are active at low pH

1993 ◽  
Vol 264 (6) ◽  
pp. C1609-C1618 ◽  
Author(s):  
J. Cuppoletti ◽  
A. M. Baker ◽  
D. H. Malinowska
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Parietal Cell ◽  
Asymmetric Reduction ◽  
Ion Selectivity ◽  
Channel Activity ◽  
Open Probability ◽  
Gastric Parietal Cell ◽  
Cl Channel ◽  
Cl Channels

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Chloride channels in primary cultures of seawater fish (Dicentrarchus labrax) gill

1997 ◽  
Vol 273 (3) ◽  
pp. C874-C882 ◽  
Author(s):  
C. Duranton ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Chloride Channels ◽  
Primary Cultures ◽  
Ion Homeostasis ◽  
Dicentrarchus Labrax ◽  
Disulfonic Acid ◽  
Inward Rectification ◽  
Open Probability ◽  
Pipette Solution ◽  
Cl Channel ◽  
Respiratory Cells

Patch-clamp experiments were undertaken on primary cultures of respiratory cells originating from sea bass (Dicentrarchus labrax) gills. A small-conductance Cl- channel of 8 pS was characterized in cell-attached configuration with 140 mM N-methyl-D-glucamine-Cl in the pipette and bath solutions. No activity was recorded below a membrane holding potential of +20 mV (-Vp, referenced to the pipette solution), and the channel showed an inward rectification. In the inside-out configuration the Cl- channel was active at all membrane holding potentials. Its open probability strongly increased with membrane depolarization. The channel activity could be increased by the application of protein kinase A+ATP. This channel was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid, diphenylamino-2-carboxylic acid, and I- and was insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of Cl- channels in the apical membrane of respiratory cells provides additional evidence for an important role of this cell type in the control of ion homeostasis of seawater fish.

scholarly journals Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

1999 ◽  
Vol 277 (3) ◽  
pp. C469-C479 ◽  
Author(s):  
Wolfgang Zeiske ◽  
Ilse Smets ◽  
Marcel Ameloot ◽  
Paul Steels ◽  
Willy van Driessche
Keyword(s):  
Kinetic Parameters ◽  
Channel Blocker ◽  
Na Transport ◽  
Open Probability ◽  
Experimental Conditions ◽  
Channel Density ◽  
A6 Cells ◽  
The Individual ◽  
The Relationship ◽  
Volume Controlled

We report, for the epithelial Na+ channel (ENaC) in A6 cells, the modulation by cell pH (pHc) of the transepithelial Na+ current ( I Na), the current through the individual Na+channel ( i), the open Na+ channel density ( N o), and the kinetic parameters of the relationship between I Na and the apical Na+ concentration. The i and N o were evaluated from the Lorentzian I Na noise induced by the apical Na+ channel blocker 6-chloro-3,5-diaminopyrazine-2-carboxamide. pHc shifts were induced, under strict and volume-controlled experimental conditions, by apical/basolateral NH4Cl pulses or basolateral arrest of the Na+/H+exchanger (Na+ removal; block by ethylisopropylamiloride) and were measured with the pH-sensitive probe 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The changes in pHc were positively correlated to changes in I Na and the apically dominated transepithelial conductance. The sole pHc-sensitive parameter underlying I Na was N o. Only the saturation value of the I Na kinetics was subject to changes in pHc. pHc-dependent changes in N o may be caused by influencing P o, the ENaC open probability, or/and the total channel number, N T = N o/ P o.

Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon

2001 ◽  
Vol 280 (3) ◽  
pp. G400-G405 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
John Geibel ◽  
Henry J. Binder
Keyword(s):  
Channel Blocker ◽  
Apical Membrane ◽  
Distal Colon ◽  
Proton Gradient ◽  
Disulfonic Acid ◽  
High Dose ◽  
Cl Channel ◽  
Rat Distal Colon ◽  
Na Uptake ◽  
Crypt Cells

A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven 22Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant ( K m) for Na of ∼24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K m for H of ∼1.5 μM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant ( K i) of ∼480.2, 1.1, and 9.5 μM, respectively; 4) is inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K i of ∼280.6 and 18.3 μM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.

G protein-regulated large-conductance chloride channels in freshly isolated fetal type II alveolar epithelial cells

1993 ◽  
Vol 265 (4) ◽  
pp. L323-L329 ◽  
Author(s):  
P. J. Kemp ◽  
G. G. MacGregor ◽  
R. E. Olver
Keyword(s):  
Epithelial Cells ◽  
G Protein ◽  
Alveolar Epithelial Cells ◽  
Disulfonic Acid ◽  
Single Channels ◽  
Type Ii ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Alveolar Epithelial ◽  
Cl Channel

Using the patch-clamp technique, we have recorded single channels in cell-attached and inside-out excised patches from the plasma membrane of type II alveolar epithelial cells freshly isolated from fetal guinea pig lung by elastase digestion and differential filtration. In cell-free patches the channels were highly selective for Cl- (PCl:Pcat = 9:1), had a large unitary conductance (375 pS +/- 23 pS), and current reversal of 0 mV in either symmetrical Na(+)-rich solutions or when the inner membrane leaflet was bathed in a K(+)-rich solution. The large-conductance Cl- channel exhibited little or no voltage inactivation at positive potentials, remained open for a significant amount of time at potentials negative to -40 mV, and was blocked at all potentials by 0.1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid. Channel activity was independent of intracellular calcium concentration. Bath addition of the nonmetabolizable analogue of GTP, GTP gamma S (0.1 mM), caused a voltage-dependent inhibition of channel activity [open probability (Po) plot was shifted by at least +25 mV]. Smaller channels (25 +/- 3 pS) were recorded in the cell-attached configuration with a current-voltage (I-V) relationship which was compatible with a Cl- conductance. On excision, the patches previously containing small-conductance channels exhibited only large-conductance Cl- channel behavior. These large-conductance, G protein-regulatable Cl- channels may provide a route for alveolar cell Cl- exit and as such may be an integral part of the mechanism responsible for secretion of fetal lung fluid.

Role of chloride ions in lower esophageal sphincter tone and relaxation

1992 ◽  
Vol 263 (1) ◽  
pp. G115-G126 ◽  
Author(s):  
J. K. Saha ◽  
J. N. Sengupta ◽  
R. K. Goyal
Keyword(s):  
Lower Esophageal Sphincter ◽  
Channel Blocker ◽  
Chloride Ions ◽  
Disulfonic Acid ◽  
Gamma Aminobutyric Acid ◽  
Inhibitory Neurotransmitter ◽  
Cl Channel ◽  
Esophageal Sphincter ◽  
Resting Tone

Studies were performed in strips of opossum lower esophageal sphincter (LES) muscle in vitro. External Cl(-)-free Krebs solution (0[Cl-]o) inhibited resting tone. Treatment with the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 0.3-100 microM) caused a concentration-dependent relaxation of LES muscle, as did treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1 microM-3 mM), a Cl(-)-HCO3- exchange blocker, and bumetanide (0.3-100 microM), a blocker of the Na(+)-K(+)-2Cl- cotransport. DIDS and bumetanide are also known to cause Cl- channel block. The calculated pD2 and Emax values for DPC, DIDS, and bumetanide were 5.24 +/- 0.28 (n = 5), 3.38 +/- 0.16 (n = 5), and 4.49 +/- 0.23 M (n = 5), and 78.80 +/- 5.38, 74.80 +/- 6.54, and 83.70 +/- 10.20%, respectively. The neuronal Cl- channel activators gamma-aminobutyric acid and glycine had no effect on the resting tone. DPC, DIDS, and bumetanide appear to have acted directly on smooth muscle rather than indirectly through the release of inhibitory neurotransmitters because LES relaxation by these agents was not influenced by tetrodotoxin (10 microM), which blocks action potentials in nerves, or by omega-conotoxin (1 microM), which inhibits the release of neurotransmitters from nerve terminals. LES muscle relaxed by exposure to 0[Cl-]o, DPC, DIDS, and bumetanide contracted with the addition of carbachol (30 microM); muscle so treated was resistant to the inhibitory neurotransmitter-mediated relaxation ordinarily induced by electrical field stimulation (EFS, 0.12-32 Hz). This effect was not nonselective, as the EFS-resistant muscle relaxed fully with isoproterenol (0.1-100 microM). HCO(3-)-free Krebs in the nominal absence of CO2 did not affect the resting tone and its relaxation. The Ca2+ channel blocker nifedipine decreased resting tone but did not antagonize the relaxation of carbachol-contracted muscle induced by either EFS or isoproterenol. These studies suggest that Cl- may play an important role in LES tone and relaxation due to inhibitory neurotransmitter released from intramural nerves.

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