Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.     

Ca2+-activated Cl- channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion.

This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC-1) and CFTR-deficient (CFPAC-1; DeltaF508 mutation) cell lines and measured HCO3- extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC-1 cells, cAMP causes parallel activation of Cl- channels and of HCO3- extrusion by DIDS-sensitive and Na+-independent Cl-/HCO3- exchange, both effects being inhibited by Cl- channel blockers NPPB and glibenclamide. 2) In CFPAC-1 cells, cAMP fails to stimulate Cl-/HCO3- exchange and Cl- channels, except after promoting surface expression of DeltaF508-CFTR by glycerol treatment. Instead, raising intracellular Ca2+ concentration to 1 micromol/l or stimulating purinergic receptors with ATP (10 and 100 micromol/l) leads to parallel activation of Cl- channels and HCO3- extrusion. 3) K+ channel function is required for coupling cAMP- and Ca2+-dependent Cl- channel activation to effective stimulation of Cl-/HCO3- exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl-/HCO3- exchange is directly correlated to activation of apical membrane Cl- channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP-activated Cl- channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca2+-dependent Cl- channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.     

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Inhibition of heart calcium and chloride currents by sodium iodide. Specific attenuation in cAMP-dependent protein kinase-mediated regulation.

The enzymes cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate the activity of cardiac ion channel proteins. In this study the whole-cell arrangement of the patch clamp technique was used to examine the effect of NaI on PKA-stimulated Cl- and Ca2+ channels in isolated guinea pig ventricular myocytes. Cl- currents (ICl) activated either by the beta-adrenergic agonist isoproterenol or the membrane-soluble cAMP analogue, 8-chlorphenylthio (8-CPT) cAMP, were greatly reduced in amplitude after substitution of an external solution containing 140 mM NaCl with a solution containing 140 mM NaI. This reduction was accompanied by a shift of -7 mV in the reversal potential (Erev) for ICl and could be reversed upon return to the NaCl external solution. Inhibition of ICl by NaI occurred in a concentration-dependent manner and was more pronounced for inward ICl (IC50 = 19 mM at -60 mV) than for outward ICl (IC50 = 60 mM at +60 mV). In contrast to ICl activated by PKA, ICl activated by PKC was slightly augmented in the presence of NaI and the Erev was found to shift by -15 mV. Based on these data, the relative permeability of I- to Cl- (PI/PCl) for this channel was calculated to be 1.79. NaI produced no change in the amplitude of inward calcium currents (ICa) recorded under basal conditions, but strongly inhibited ICa augmented by isoproterenol and 8-CPT cAMP, and during dialysis of cells with the catalytic subunit of PKA (CS). The in vitro incorporation of [gamma-32P]ATP into histone IIA and Kemptide, measured in the presence of PKA and cAMP, was not significantly different in assay mixtures containing salts of Cl- and I-. However, the ability of isoproterenol to augment basal ICa in whole-cell experiments was attenuated when experiments were carried out entirely in NaI external solution. Thus, the reduction in ICl and ICa observed in this study may result from a direct effect of I- on the phosphorylation/dephosphorylation of cardiac ion channel proteins or associated regulatory proteins.     

Pharmacological and biophysical properties of swelling-activated chloride channel in mouse cardiac myocytes

The present study was designed to observe the properties of swelling-activated chloride channel (ICl.swell) in mouse cardiac myocytes using patch clamp techniques. In whole-cell recordings, hypotonic solution activated a chloride current that exhibited outward rectification, weak voltage-dependent inactivation, and anion selectivity with permeability sequence of I- > Br- > Cl-. The current was sensitive to Cl- channel blockers tamoxifen, NPPB and DIDS. In single-channel recordings, cell swelling activated a single channel current which showed outward rectification with open probability of 0.76 +/- 0.08 and conductance of 38.1 +/- 2.5 pS at +100 mV under [Cl-] symmetrical condition. I-V relation revealed the reversal potential as expected for a Cl(-)-selective channel. These results suggested that in mouse cardiac myocytes, swelling-activated, outward rectifying chloride channel with a single channel conductance of 38.1 +/- 2.5 pS (at +100 mV under [Cl-] symmetrical condition) underlies the volume regulatory Cl- channel.     

Potassium-chloride promiscuous channels in mitochondrial membranes

Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels.     

Cl- channel blockers inhibit transition of quiescent (G0) fibroblasts into the cell cycle

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl- channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl- channel blocker 5-N-2-(3-phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4-aminopyridine (4-AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0-to-G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10-14 h. NPPB applied only at 0-8 or 8-14 h after serum addition inhibited entry into S phase. Cl- permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki-67-negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl- channel blockers (NPPB and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid [DIDS]) but not the K+ channel blocker (4-AP) significantly decreased the rate of reduction in number of Ki-67-negative cells. These data indicate that an increase in Cl- permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint.     

Cl-]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.     

Quercetin and NPPB-induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl- secretion over 30-fold due to activation of the Na+/K+/2Cl- cotransporter and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC), and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity, and alpha-ENaC mRNA expression was diminished by 24h-pretreatment with quercetin (an activator of Na+/K+/2Cl- cotransporter participating in Cl- entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl- channel participating in Cl- release from the cytosolic space), while 24h-pretreatment with bumetanide (a blocker of Na+/K+/2Cl- cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of the Na+/K+/2Cl- cotransporter, and that modification of activity of Cl- transporter/channel participating in the transepithelial Cl- secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport).     

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.     

Chloride transport in human fibroblasts is activated by hypotonic shock

Incubation of human skin fibroblasts in hypotonic media induced the activation of 36Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of 36Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also 36Cl- influx was enhanced by hypotonic medium.     

Cellular mechanisms of carbachol-stimulated Cl- secretion in rat epididymal epithelium

Neurotransmitter-controlled Cl- secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca2+ activated Cl- channel blocker disulfonic acid stilbene (DIDS, 300 microM) only inhibited part of the CCH-induced Isc response, while nonselective Cl- channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 microM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca2+-chelating agent BAPTA/AM (50 microM) and an endoplasmic reticulum Ca2+ pump inhibitor, Thapsigagin (Tg, 1 microM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways.     

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Removal of the nitro and phenyl groups from NPPB decreases its inhibitory effect on cytoplasmic streaming in the alga Nitella hookeri.

Structural analogues of the arylaminobenzoate 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), prepared using a simple reductive amination sequence, were tested for their effects on cytoplasmic streaming rates in the alga Nitella hookeri. Cytoplasmic streaming was sensitive to NPPB, with an IC50 value of 24 micromol/L. Removal of the nitro group from the benzoate ring decreased the IC50 to 455 micromol/L. The introduction of an extra carbon or double bond into the aliphatic chain had no effect on activity. Loss of the phenyl group decreased potency, with an IC50 of 6.4 mmol/L. These data are the first documenting the relative inhibitory effects of structural changes to arylaminobenzoates in algae. Patch-clamp data and the effects of tetrapentyl ammonium chloride on streaming suggest that the nitro and phenyl groups may act by inhibiting both K+ and Cl- channels. This is likely, through changes in the membrane potential, to affect Ca2+ fluxes and action potentials, thereby slowing cytoplasmic streaming.     

Chloride channels in the small intestinal cell line IEC-18

Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion.     

Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.     

Stimulating effects of dopamine on chloride transport across the rat caudal epididymal epithelium in culture

The present study investigated the effects of dopamine on chloride transport across cultured rat caudal epididymal epithelium. The results showed that dopamine induced a biphasic short-circuit current (Isc) in a concentration-dependent manner. The dopamine-induced response consisted of an initial rapid spike followed by a sustained phase. The alpha and beta adrenoreceptor inhibitors, phentolamine and propranolol, inhibited the initial spike and the sustained phase, respectively, suggesting a contribution of adrenergic receptors. The response was almost abolished by removing the extracellular Cl-, suggesting that the dopamine-induced short-circuit current is primarily a Cl- current. The response was inhibited by the apical Cl- channel blocker, diphenylamine-dicarboxylic acid, and the Ca2+-activated Cl- channel blocker, disulfonic acid stilbene, indicating that Cl- may pass through two types of Cl- channels on the apical side. Preloading monolayers with the intracellular Ca2+ chelator BAPTA/AM abolished the initial spike and greatly reduced the second phase in the Isc response to dopamine. Pretreating the monolayers with an adenylate cyclase inhibitor, MDL12330A, inhibited all of the second Isc response and part of the initial spike. Also, characteristics of the Cl- currents induced by dopamine were observed in whole-cell patch-clamp recording. The increases of intracellular cAMP and Ca2+ induced by dopamine were also measured. The results suggest that extracellular dopamine activates Ca2+-dependent and cAMP-dependent regulatory pathways, leading to activation of both Ca2+-dependent and cAMP-dependent Cl- conductances in epididymal epithelial cells.     

Hypotonic shock activated Cl- and K+ pathways in human fibroblasts.

The exposure of human fibroblasts to hypotonic medium (200 mosmolal) evoked the activation of both 36Cl- influx and efflux, which were insensitive to inhibitors of the anion exchanger and of the anion/cation cotransport, and conversely were inhibited by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 36Cl- efflux was linked to a parallel efflux of 86Rb+; thus conductive K+ and Cl- pathways are activated during volume regulation in human fibroblasts. This conclusion is supported by evidence that, in hypotonic medium, 36Cl- influx and 86Rb+ efflux were both enhanced by depolarization of the plasma membrane. Depletion of the intracellular K+ content, obtained by preincubation with the ionophore gramicidin in Na(+)-free medium, had no effect on Cl- efflux in hypotonic medium. This result has been interpreted as evidence for independent activation of K+ and Cl- pathways. It is also concluded that the anion permeability is the rate-limiting factor in the response of human fibroblasts to hypotonic stress.