Volume-activated Cl Research Articles

Downregulation of volume-activated Cl- currents during muscle differentiation.

We have used the whole cell configuration of the patch-clamp technique to investigate volume-activated Cl- currents in BC3H1 and C2C12 cells, two mouse muscle cell lines that can be switched from a proliferating to a differentiated musclelike state. Reducing the extracellular osmolality by 40% evoked large Cl- currents in proliferating BC3H1 and C2C12 cells. These currents were outwardly rectifying and had an anion permeability sequence as follows: I- > Br- > Cl- >> gluconate. They were inhibited by >50% by flufenamic acid (500 microM), niflumic acid (500 microM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) but were relatively insensitive to tamoxifen (100 microM). A reduction in the serum concentration in the culture medium induced growth arrest in both cell lines, and the cells started to differentiate into spindle-shaped nonfusing muscle cells (BC3H1) or myotubes (C2C12). This differentiation was accompanied by a drastic decrease in the magnitude of the volume-activated Cl- currents. The close correlation between volume-activated Cl- currents and cell proliferation suggests that these currents may be involved in cell proliferation.

Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i activates small-conductance Cl- channels in endothelial cells, which coexist with the volume-activated Cl- channels described previously.

Volume regulatory taurine release in human tracheal 9HTEo- and multidrug resistant 9HTEo-/Dx cells.

The intracellular taurine release evoked by hypotonic shock is accomplished by volume-activated Cl- channels whose activity has been related to the expression of the multidrug resistance protein (MDR-1). We studied taurine transport in 9HTEo- cells and in the derived cell line 9HTEo-/Dx expressing MDR-1. [3H]taurine release from preloaded cells increased upon reduction of extracellular osmolality. This process was not inhibited by preincubation with phorbol 12-myristate 13-acetate but was reduced by inhibitors of volume-sensitive Cl- channels such as 1,9-dideoxiforskolin, La3+, and arachidonate. Verapamil, a substrate of MDR-1, increased the osmotically evoked taurine efflux. Replacement of extracellular Cl- with I- or gluconate or of extracellular Na+ with Li+ significantly reduced the taurine efflux, whereas substitution of N-methyl-D-glucamine for Na+ increased it. Application of ATP and 2-chloroadenosine stimulated the efflux in isotonic medium. No differences were seen between 9HTEo- and 9HTEo-/Dx cells with respect to hypotonically induced taurine efflux and the response to phorbol ester, channel blockers, ion replacement, and purinergic agents. Our results reveal novel properties of the osmotically induced taurine release and demonstrate its independence from MDR-1 gene expression.

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene.

The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl- channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl- currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotonic-induced chloride conductance in neuroblastoma cells.

Association of ClC-3 Channel with Cl − Transport by Human Nonpigmented Ciliary Epithelial Cells

Electrophysiologic and volumetric evidence link the swelling-activated Cl- channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl(-)-channel/Cl(-)-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl- channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl- channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 microM) which blocks > 90% of the MDR1-associated Cl- currents, forskolin had no effect on the volume-activated Cl- channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mM) and niflumic acid (IC50 approximately 200 microM) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl(-)-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2(+)-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrink-age. These results support the concept that the volume-sensitive protein pICln regulates the Cl- channels, and that the same conduits subserve volume- and staurosporine-activated Cl- release. Of the cloned and sequenced Cl- channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl- channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl- channels of human nonpigmented ciliary epithelial cells.

Volume regulation in the bovine lens and cataract. The involvement of chloride channels.

The purpose of this study was to investigate volume regulation in the lens and its involvement in lens opacification (cataract) and the role of chloride channels in these processes. Single, isolated lens fiber cells from the lens were whole cell patch clamped. When exposed to hypotonic solution, and outwardly rectifying whole-cell current was activated. The current increased from 1.0 to 32.6 pA/pF, reversed at the chloride reversal potential (Ec1 = O mV), and was blocked by the chloride channel blockers 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB) and tamoxifen. Replacing all but 5mM of the external chloride with gluconate caused the reversal potential to shift +33 mV, consistent with a CL- current with a gluconate/chloride permeability ratio of 0.26. When the whole lens of the eye was exposed to hypotonic solution, there was an initial increase in anterior-posterior diameter (5-8 min), representing lens swelling of 6.5%. This was followed by a decrease in volume to a new steady state value that lasted for up to 2 h. In the longer term (> or = 2h), the lenses began to swell again. The simultaneous exposure to hypotonic solution and tamoxifen or NPPB caused swelling and prevented this volume regulation. Lenses incubated in hypotonic solution and hypotonic solution containing tamoxifen becane ipaque after a 2-h incubation period. We conclude that the lens is able to volume regulate. It possesses volume-activated Cl- channels, the inhibition of which results in inhibition of volume regulation, lens swelling and opacification. Our data suggest the long-term prophylactic use of tamoxifen may make the patient more susceptible to cataract.

Characterization of volume-activated chloride currents in endothelial cells from bovine pulmonary artery.

We have measured the kinetic and pharmacological properties of volume-activated Cl- currents (ICl, vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current ICln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP. In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery (CPAE) contain pICln. The expression of this protein has been shown to be closely associated with the ICln current. ICl, vol showed however, in contrast with ICln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current related to MDR-1. Activation of ICl, vol at potentials more negative than -80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current. The antiviral nucleoside analogue AZT (3'-azido-3'-deoxythymidine) inhibited ICl, vol by 21 +/- 2.7% (n = 10), at a concentration of 100 microM. Another antiviral drug, acyclovir (ACV, 9-[2-hydroxyethoxy) methyl]guanine) blocked ICl, vol by 27 +/- 6.2% at 100 microM (n = 11). Both blocking effects are much smaller than those reported for ICln. The phenol derivative gossypol, which blocks ICln-related currents, efficiently inhibited ICl, vol in CPAE cells (67 +/- 2.1% at 1 microM, n = 7, KI = 0.4 microns). The presence of pICln in CPAE cells and the similar qualitative pharmacological profile of ICl, vol and ICln support the hypothesis that pICln is a good molecular candidate for ICl, vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation phenomena due to the large amplitudes of these currents.

The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of ICl,vol occurred at a hypotonicity of 27.5+/-1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect ICl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 micromol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence ICl,vol. Trifluoperazine and tamoxifen fully blocked ICl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 micromol/l respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 micromol/l) and genistein (100 micromol/l) did not affect ICl,vol. Exposing CPAE cells to lysophosphatidic acid (1 micromol/l), an activator of p42 MAPkinase and the focal adhesion kinase p125(FAK) in endothelial cells, neither evoked a Cl- current nor affected ICl,vol. Neither wortmannin (10 micromol/l), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/l), which interferes with the p70S6 kinase pathway, affected ICl,vol. Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl- current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.

Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration

Combined patch clamp and Ca2+-measurements (Fura-2) were used to study the dependence of volume-activated Cl--currents (ICl,vol) of endothelial cells from bovine pulmonary artery on the intracellular Ca2+-concentration [Ca2+]i. Loading the cells with high concentrations of EGTA or BAPTA via ruptured membrane patches or by preincubating them with 50 microM BAPTA-AM caused a substantial decrease of ICl,vol. This reduction was independent of the activation state of the current: the current amplitude was not only diminished if [Ca2+]i was lowered at the peak of the volume-activated current, but this low Ca2+-concentration also prevented activation of the current by a second hypotonic challenge.ICl,vol is already maximally activated at intracellular Ca2+-concentrations between 50 and 100 nmol/l, a further increase of [Ca2+]i does not affect the size of ICl,vol.These results indicate that a sustained full activation of ICl,vol in endothelial cells requires submicromolar concentrations of Ca2+, and that changes in [Ca2+]i do not modulate the current.

Blockers of volume-activated Cl? currents inhibit endothelial cell proliferation

Volume-activated Cl- currents (ICl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (1), inhibits both ICl,vol and cell proliferation with IC50 of 3.8 and 4.8 micromol/l respectively.NPPB and quinine, which also block ICl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that ICl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.

Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye.

When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K+ and Cl-. This is primarily caused by a dramatic increase in Cl- permeability, which may reflect the opening of volume-sensitive channels (11). To test this hypothesis, we measured volume-activated Cl- currents in HL-60 cells using the patch-clamp technique. The whole cell Cl- conductance (in nS/pF at 100 mV) increased from 0.09 +/- 0.06 to 1.15 +/- 0.19 to 1.64 +/- 0.40 as the tonicity (in mosmol/kgH2O) of the external medium was decreased from 334 to 263 to 164, respectively. Cl- currents showed no significant inactivation during 800-ms pulses. Current-voltage curves exhibited outward rectification and were identical at holding potentials of 0 or -50 mV, suggesting that the gating of the channels is voltage independent. The selectivity sequence, based on permeability ratios (PX/PCl) calculated from the shifts of the reversal potentials, was SCN- > I- approximately NO3- > Br- > Cl- >> gluconate. 4-Acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with approximately 10-fold increased affinity at potentials greater than +40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of the membrane potential. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [diBA-(5)-C4] with a concentration that gives half inhibition (IC50) of 1.8 microM at room temperature. A similar apparent IC50 value (1.2 microM) was observed for net 36Cl- efflux into a Cl(-)-free hypotonic medium at 21 degrees C. It seems likely, therefore, that the volume-activated Cl- channels are responsible for the net Cl- efflux during RVD. These Cl- channels have properties similar to the "mini-Cl-" channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.

Volume-activated chloride currents in pancreatic duct cells.

We have used the patch clamp technique to study volume-activated Cl- currents in the bicarbonate-secreting pancreatic duct cell. These currents could be elicited by a hypertonic pipette solution (osmotic gradient 20 mOsm/l), developed over about 8 min to a peak value of 91 +/- 5.8 pA/pF at 60 mV (n = 123), and were inhibited by a hypertonic bath solution. The proportion of cells which developed currents increased from 15% in freshly isolated ducts to 93% if the ducts were cultured for 2 days. The currents were ATP-dependent, had an outwardly rectifying current/voltage (I-V) plot, and displayed time-dependent inactivation at depolarizing potentials. The anion selectivity sequence was: ClO4 = I = SCN > Br = NO3 > Cl > F > HCO3 > gluconate, and the currents were inhibited to a variable extent by DIDS, NPPB, dideoxyforskolin, tamoxifen, verapamil and quinine. Increasing the intracellular Ca2+ buffering capacity, or lowering the extracellular Ca2+ concentration, reduced the proportion of duct cells which developed currents. However, removal of extracellular Ca2+ once the currents had developed was without effect. Inhibiting protein kinase C (PKC) with either the pseudosubstrate PKC (19-36), calphostin C or staurosporine completely blocked development of the currents. We speculate that cell swelling causes Ca2+ influx which activates PKC which in turn either phosphorylates the Cl- channel or a regulatory protein leading to channel activation.

Inhibition of volume‐activated chloride currents in endothelial cells by chromones

1. We have studied the effects of the reported chloride channel blocker, sodium cromoglycate, on volume-activated Cl- currents in endothelial cells from bovine pulmonary artery by means of the whole-cell patch clamp technique. Cl- currents were activated by challenging the cells with a hypotonic extracellular solution of 60% of the normal osmolarity. 2. Half maximal activation of the current at +95 mV occurred after exposure of the cells for 148 +/- 10 s (n = 6) to hypotonic solution (HTS). At the same membrane potential but in the presence of 100 microM sodium cromoglycate (disodium-1,3-bis (2'-carboxylate-chromone-5'-yloxy)-2-hydroxy-propane) activation was delayed (253 +/- 25 s, n = 6) and the maximal current amplitude was reduced to 63 +/- 7% of the control (n = 13). 3. In comparison, an equimolar concentration of NPPB (5-nitro-2(3-phenyl) propylamino-benzoic acid), another Cl- channel blocker, completely blocked the volume-activated current in less than 20 s. 4. Sodium cromoglycate, applied at the time when the HTS-induced current was completely activated, dose-dependently inhibited this current with a concentration for half maximal inhibition of 310 +/- 70 microM. Data for nedocromil sodium were not significantly different from those for sodium cromoglycate. 5. Sodium cromoglycate, loaded into the endothelial cells via the patch pipette in ruptured patches, resulted in a decline of the HTS activated current with a time course that was compatible with diffusion of the compound from the pipette into the cell. Intracellulary applied sodium cromoglycate was also more effective and at 50 microM caused a decrease in the amplitude of the current to 25 +/- 6% (n = 10) of the control current.6 It is concluded that blockade of volume-activated Cl- currents by extracellular sodium cromoglycatemay be due to an intracellular action following its permeation across the cell membrane.

Volume-activated chloride currents are not correlated with P-glycoprotein expression

It has been proposed that P-glycoprotein, the product of the human MDR1 gene, may function not only as a drug transporter but, depending on the conditions, as a volume-activated Cl- channel [Valverde, Diaz, Sepúlveda, Gill, Hyde and Higgins (1992) Nature (London) 355, 830-833; Gill, Hyde, Higgins, Valverde, Mintenig and Sepúlveda (1992) Cell 71, 23-32]. To verify this hypothesis, we have compared volume-activated Cl- currents with the level of MDR1 mRNA and its protein product in the human KB3 (epitheloid lung cancer) and HeLa cell lines. The related MDR2 was also included to find out whether it could account for observed discrepancies between Cl- current and MDR1 expression. A 40% decrease in osmolarity evoked a Cl- current in both cell types (at +80 mV: 50.3 +/- 4.3 pA/pF in KB3, n = 13; 28.2 +/- 3.3 pA/pF in HeLa, n = 16). The blocking of this current in both cell types by 5-nitro-2-(3-phenylpropylamino)-benzoic acid and by 1,9-dideoxyforskolin is similar to that of the presumed P-glycoprotein associated Cl- channel. As measured by reverse-transcriptase polymerase chain reaction, KB3 cells expressed only an extremely small amount of the messengers for MDR1 and MDR2. The signal observed for MDR1 in HeLa cells was at least an order of magnitude more intense than in KB3 cells, while MDR2 mRNA was undetectable. A clear difference in MDR1 expression between KB3 and HeLa was also observed at the protein level. These data are difficult to reconcile with the hypothesis that in HeLa and KB3 cells MDR1- or MDR2- encoded P-glycoproteins are associated with volume-activated Cl- channels.

Whole cell membrane currents in cultured pig endocardial endothelial cells

The whole cell mode of the patch-clamp technique was applied to cultured endocardial endothelial cells from the porcine right ventricle to study their electrophysiological properties. With isotonic pipette and bathing solutions (300-310 mosmol/kgH2O), single endocardial endothelial cells had resting membrane potentials ranging from -20 to -90 mV (mean = -55 +/- 20 mV, n = 48). In voltage-clamp experiments, the main membrane current was an inwardly rectifying K+ current with all characteristics described for the inwardly rectifying K+ current in vascular endothelium. Outward currents at positive clamp potentials were small, but when cell swelling was induced by means of a hypertonic pipette or hypotonic bathing solution and ATP (5 mM) was present in the pipette solution, a large outwardly rectifying current developed. This volume-activated current was insensitive to extracellular K+ or Na+ concentration variations but sensitive to changes in extracellular Cl- concentrations. It was inhibited in the presence of 4,4'-diisothiocyanostilbene-2,2 disulfonic acid (100-300 microM) and flufenamic acid (50-100 microM). Volume-activated Cl- channels are different from the stretch-activated cationic channels described in vascular endothelium and might be involved in the regulation of cell volume or the response to mechanical stretch.

Molecular Cloning of the Human Volume-Sensitive Chloride Conductance Regulatory Protein, pI Cln, from Ocular Ciliary Epithelium

Chloride channels in the ocular ciliary epithelium are believed to play a key role in aqueous humor formation. We isolated a cDNA clone from a λUni-ZAP cDNA library of human nonpigmented ciliary epithelial (NPE) cells encoding the swelling-induced chloride channel/channel regulator PI Cln. The human clone contains an open reading frame of 237 amino acids (M r 26,293). The deduced human amino-acid sequence shows 90.2% and 92.7% identity with counterparts isolated from rat kidney and the canine kidney epithelial cell line MDCK. Human NPE cell lines exhibited significant levels of pI Cln transcripts. Complementary perforated-patch, whole-cell patch clamping demonstrated that swelling activates Cl − channels of the NPE cells, as suggested by ruptured-patch measurements. The results document the molecular isolation and identification of a human cDNA clone of a Cl − conductance regulator from ocular cells displaying volume-activated Cl −channels.

PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln

Swelling activates and protein kinase C (PKC) downregulates Cl- channels in cultured nonpigmented ciliary epithelial (NPE) cells. We now report that the PKC inhibitor staurosporine upregulates whole cell Cl- currents isosmotically. The kinetics and current-voltage relationship are similar to those of volume-activated Cl- channels of these cells. These properties are inconsistent with cloned ClC-0, ClC-1, ClC-2, and MDR1 channels but could reflect the cystic fibrosis transmembrane conductance regulator (CFTR) channel or the Cl- channel regulator pICln. CFTR mRNA was undetectable by Northern analysis of cultured NPE cells or ciliary body tissue. In contrast, a human pICln probe obtained by polymerase chain reaction cloning and showing 90% identity with the rat cDNA clone detected high levels of transcripts in NPE cells. The level was low in tissue, where the NPE message was diluted by RNA from other cells. We conclude that NPE cells display staurosporine-activated Cl- channels [gSt(Cl)] likely identical with the volume-activated channels. The same cells expressing gSt(Cl) transcribe mRNA for a novel homologue (pHCBICln) of pICln that may regulate Cl- transport into the aqueous humor.

Regulatory volume decrease in carp red blood cells: mechanisms and oxygenation-dependency of volume-activated potassium and amino acid transport

Hypo-osmotic swelling of carp red blood cells (RBCs) induced a regulatory volume decrease (RVD), which restored the original cell volume within 140 min in oxygenated RBCs, whereas volume recovery was incomplete in deoxygenated RBCs. The complete RVD in oxygenated RBCs resulted from a sustained volume-activated release of K+, Cl- and amino acids (AAs). In the absence of ouabain, the cells also lost Na+ as released K+ was partially regained via the Na+/K+ pump. Inorganic osmolytes contributed approximately 70 %, and organic osmolytes approximately 30 %, to the RVD of oxygenated RBCs. Oxygenation in isotonic medium per se activated a K+ efflux from the RBCs. Hypo-osmotic cell swelling stimulated an additional K+ release. The oxygenation-activated and the volume-activated K+ efflux were both inhibited by DIDS and by the replacement of Cl- with NO3-, showing that both types of K+ efflux were Cl--dependent and probably occurred via the same K+/Cl- cotransport mechanism. Once activated by oxygenation, the K+/Cl- cotransport was further stimulated by cell swelling. Deoxygenation inactivated the oxygenation-induced Cl--dependent K+ release and cell swelling was not a sufficient stimulus to reactivate it significantly. In deoxygenated RBCs, the volume-induced K+ release was transient and primarily Cl--independent and, in the absence of ouabain, the cell K+ content recovered towards control values via the Na+/K+ pump. The Cl--independent K+ efflux seemed to involve K+/H+ exchange, but other transport routes also participated. Swelling-activated AA release differed in kinetics between oxygenated and deoxygenated RBCs but was important for RVD at both oxygenation degrees. Approximately 70 % of the AA release was inhibited by DIDS and substitution of NO3- for Cl- produced a 50 % inhibition, suggesting that the AA permeation was partly Cl--dependent. In oxygenated RBCs, a reduction in pH lowered the volume-activated Cl--dependent K+ efflux but not the AA efflux. In deoxygenated RBCs, the acute volume-stimulated K+ and AA release were both increased by acidification. The data are discussed in relation to possible transducer mechanisms and physiological implications.

Lack of inhibition by dideoxy-forskolin and verapamil of DIDS-sensitive volume-activated Cl − secretion in human squamous lung carcinoma epithelial cells

The effect of osmotic stress on Cl − permeability in human squamous lung carcinoma epithelial (S1) cells was investigated using a macroscopic 125I efflux assay. Hypotonic challenge of monolayers led to a significant ( P < 0.01) dose-related increase in efflux from pre-loaded cells, returning to pre-activation rates within 10 min. A similar magnitude of response could be produced by challenge with an isotonic low chloride-containing solution. Neither 100 mM dideoxy-forskolin nor 100 mM verapamil inhibited the increase in Cl − secretion after hypotonic challenge, whereas 100 mM DIDS inhibited volume-activated Cl − secretion by 55%. Both Northern and Western blot analysis confirmed the absence of MDR1 mRNA and P-glycoprotein in the S1 cells. We conclude that these cells have a volume-regulated Cl − secretory pathway that is independent of the ABC transporter, P-glycoprotein.

Volume-activated Cl- currents in different mammalian non-excitable cell types.

The existence and properties of volume-activated Cl- currents were studied in 15 different cell types (endothelium: human umbilical vein, human aorta, bovine pulmonary artery; fibroblasts: Swiss 3T3, L, C3H 10T1/2 and COS-1; epithelium: KB3, HeLa and A6; blood cells: RBL-2H3 and Jurkat; endothelioma cells derived from both subcutaneous and thymic hemangiomas; skin: IGR1 melanoma). Volume-activated Cl- currents with common characteristics, i.e. small conductance, outward rectification, higher permeability for iodide than for chloride and sensitivity to block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) could be elicited in all cells. The block of this current by tamoxifen and dideoxyforskolin is different for the various cell types, as well as the time course and the amplitude of the responses induced by repetitive applications of hypotonicity. Volume-activated Cl- channels with similar biophysical properties are therefore wide-spread among mammalian cells. This may reflect either a single Cl- channel that is ubiquitously expressed or a family of functionally related Cl- channels with cell specific expression patterns.