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• W2046728004 abstract "We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented rat aortic smooth muscle cell proliferation, which was related to cell volume regulation. In the present study, we further characterized the regulation of intracellular Cl- concentrations ([Cl-]i) via volume-regulated ClC-3 Cl- channels in an embryo rat aortic vascular smooth muscle cell line (A10 cell) and ClC-3 cDNA-transfected A10 cells (ClC-3-A10) using multiple approaches including [Cl-]i measurement, whole cell patch clamp, and application of ClC-3 antisense and intracellular dialysis of an anti-ClC-3 antibody. We found that hypotonic solution decreased [Cl-]i and evoked a native ICl.vol in A10 cells. The responses of [Cl-]i and ICl.vol to hypotonic challenge were enhanced by expression of ClC-3, and inhibited by ClC-3 antisense. The currents in A10 (ICl.vol) and in ClC-3-A10 cells (ICl.ClC-3) were remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Reduction in [Cl-]i and activation of ICl.vol and ICl.ClC-3 in A10 and ClC-3-A10 cells, respectively, were significantly inhibited by activation of protein kinase C (PKC) by phorbol-12,13-dibutyrate (PDBu) and inhibition of tyrosine protein kinase by genistein. Sodium orthovanadate (vanadate), a protein-tyrosine phosphatase inhibitor, however, enhanced the cell swelling-induced reduction in [Cl-]i, accompanied by the activation of ICl.vol and ICl.ClC-3 in a voltage-independent manner. Our results suggest that the volume-regulated ClC-3 Cl- channels play important role in the regulation of [Cl-]i and cell proliferation of vascular smooth muscle cells. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented rat aortic smooth muscle cell proliferation, which was related to cell volume regulation. In the present study, we further characterized the regulation of intracellular Cl- concentrations ([Cl-]i) via volume-regulated ClC-3 Cl- channels in an embryo rat aortic vascular smooth muscle cell line (A10 cell) and ClC-3 cDNA-transfected A10 cells (ClC-3-A10) using multiple approaches including [Cl-]i measurement, whole cell patch clamp, and application of ClC-3 antisense and intracellular dialysis of an anti-ClC-3 antibody. We found that hypotonic solution decreased [Cl-]i and evoked a native ICl.vol in A10 cells. The responses of [Cl-]i and ICl.vol to hypotonic challenge were enhanced by expression of ClC-3, and inhibited by ClC-3 antisense. The currents in A10 (ICl.vol) and in ClC-3-A10 cells (ICl.ClC-3) were remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Reduction in [Cl-]i and activation of ICl.vol and ICl.ClC-3 in A10 and ClC-3-A10 cells, respectively, were significantly inhibited by activation of protein kinase C (PKC) by phorbol-12,13-dibutyrate (PDBu) and inhibition of tyrosine protein kinase by genistein. Sodium orthovanadate (vanadate), a protein-tyrosine phosphatase inhibitor, however, enhanced the cell swelling-induced reduction in [Cl-]i, accompanied by the activation of ICl.vol and ICl.ClC-3 in a voltage-independent manner. Our results suggest that the volume-regulated ClC-3 Cl- channels play important role in the regulation of [Cl-]i and cell proliferation of vascular smooth muscle cells. Cell swelling occurs in many physiological responses and pathological processes. In most cells, the cell swelling occurs in the early phase of cell proliferation probably caused by water influx that accompanies changes in cell metabolism (such as obligatory uptake of amino acids) in the cell cycle (1Eggermont J. Trouet D. Carton I. Nilius B. Cell Biochem. Biophys. 2001; 35: 263-274Crossref PubMed Scopus (91) Google Scholar). An increase in cell volume usually evokes regulatory volume decrease (RVD) process through activation of various transporters and ion (K+ and Cl-) channels, which induces the effluxes of K+, Cl-, and H2O, and returns the cell volume to normal size. It is generally understood that RVD is mainly mediated by Cl- efflux through a volume-regulated Cl- channel (VRC). 1The abbreviations used are: VRC, volume-regulated Cl- channel; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 4α-PMA, 4-α-phorbol 12-myristate 13-acetate; PDBu, phorbol-12,13-dibutyrate; PKC, protein kinase C; VSOAC, volume-sensitive osmolyte and anion channels; MEQ, 6-methoxy-N-ethylquinolinium iodide; F, farad; gp, glycoprotein. Therefore, VRC may play an essential role in cell proliferation through regulation of cell volume. In our previous study, we determined effects of different kinds of Cl- channel blockers on endothelin-1-induced proliferation in cultured rat aortic vascular smooth muscle cells. It was found that the aortic vascular smooth muscle cell proliferation was only inhibited by DIDS (2Xiao G.N. Guan Y.Y. He H. Life Sci. 2002; 70: 2233-2241Crossref PubMed Scopus (32) Google Scholar). Furthermore, ClC-3 antisense inhibited the functional expression of ClC-3 and endothelin-1-induced proliferation in cultured rat aortic vascular smooth muscle cells (3Wang G.L. Wang X.R. Lin M.J. He H. Lan X.J. Guan Y.Y. Cir Res. 2002; 91: e28-e32Crossref PubMed Google Scholar). These results provide evidence that ClC-3 may be the gene responsible for ICl.vol and mediate volume regulation in vascular smooth muscle cells. Although it has been suggested that the ClC-3 gene may encode VRC and mediate the volume regulation process in guinea pig ventricular myocytes (4Duan D. Winter C. Crowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar), canine pulmonary smooth muscle cells (5Duan D. Zhong J.M. Hermoso M. Satterwhite C.M. Rossow C.F. Hatton W.J. Yamboliev I. Horowitz B. Hume J.R. J. Physiol. 2001; 531: 437-444Crossref PubMed Scopus (82) Google Scholar), bovine non-pigmented ciliary epithelial cells (6Wang L. Chen L. Jacob T.J. J. Physiol. 2000; 524: 63-75Crossref PubMed Scopus (114) Google Scholar), HeLa cells, and Xenopus laevis oocytes (7Hermoso M. Satterwhite C.M. Andrade Y.N. Hidalgo J. Wilson S.M. Horowittz B. Hume J.R. J. Biol. Chem. 2002; 277: 40066-40074Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), others have presented results against the ClC-3 hypothesis (8Sardini A. Amey J.S. Weylandt K.H. Nobles M. Valverde M.A. Higgins C.F. Biochim. Biophys. Acta. 2003; 1619: 153-162Crossref Scopus (138) Google Scholar, 9Nilius B. Droogmans G. Acta Physiol. Scand. 2003; 177: 119-147Crossref PubMed Scopus (372) Google Scholar, 20Li X. Shimada K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; 275: 35994-35998Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar, 22Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Therefore, molecular identification of VRC has not been determined. In the present study, we further determined the relationship between volume-regulated Cl- and ClC-3 channels in A10 and ClC-3-A10 vascular smooth muscle cells by continuously monitoring the change in [Cl-]i, whole cell patch clamp, ClC-3 antisense, and intracellular dialysis of an anti-ClC-3 antibody techniques. Our results strongly suggest that the ClC-3 channel is responsible for swelling-induced Cl- current and Cl- movement and mediates volume regulation in A10 vascular smooth muscle cell. Cell Culture—A10 vascular smooth muscle cells from the American Type Culture Collection. A10 cells were grown in Dulbecco's modified Eagle's medium/F12 medium with 10% fetal calf serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. Cultures were maintained at 37 °C in a humidified incubator in a 95% O2 plus 5% CO2 atmosphere. For electrophysiological experiments, the cells were subcultured in coverslips for 1–2 days. gpClC-3 cDNA Transfection—Cells were plated in 24-well Corning tissue culture plates. Twenty-four hours later, cells were transfected with 1 μg/ml gpClC-3/pc DNA 3.1 plasmid, which contains a full-length gpClC-3 cDNA, and pcDNA3.1 vector (gpClC-3/pcDNA3.1 was kindly provided by Dr. J. R. Hume, University of Nevada School of Medicine, Reno, Nevada). The vector contains a geneticin-resistant marker. Transfection was performed with Lipofectamine2000 reagent according to the manufacturer (Invitrogen, Life Technologies, Inc.). Stably transfected clonal cell lines were selected using geneticin (G418) at 400 μg/ml for 2 weeks following the transfection. The surviving G418-resistant cells were further plated and passed in the presence of 200 μg/ml G418. The expression of ClC-3 protein was detected by Western blot analysis. Transfections of Antisense, Sense, or Missense Oligonucleotides—The antisense and sense oligonucleotides corresponding specifically to the initiation codon region of the human ClC-3 mRNA were synthesized (Sangon, Shanghai, China) as reported previously (6Wang L. Chen L. Jacob T.J. J. Physiol. 2000; 524: 63-75Crossref PubMed Scopus (114) Google Scholar). The antisense sequence was 5′-TCC ATT TGT CAT TGT-3′. The sense oligonucleotide had the sequence 5′-ACA ATG ACA AAT GGA-3′. We designed missense oligonucleotide 5′-TCT ATT CCT GTA TTG-3′, which consisted of the same bases employed in the antisense probe, but in a “random” order, and did not recognize any known sequence available in Gen-Bank™. The first three bases at either end in all oligonucleotides were phosphorothioated. To examine the uptake of oligonucleotide by A10 cells, the oligonucleotides were labeled with fluorescence. For transient transfection, the cells in the quiescent state were transfected with oligonucleotides by incubation for 48 h with Lipofectamine2000 (5 μl/ml). Western Blot Analysis—To examine ClC-3 protein expression, ClC-3-A10 cells were washed with phosphate-buffered saline, and lysis buffer: Tris-Cl 50 mm, NaCl 150 mm, NaN3 0.02%, Nonidet P-40 1%, SDS 0.1%, sodium deoxycholate 0.5%, 5 μg/ml leupeptin, and 1 μg/ml aprotinin. The protein content of cell lysates was quantified with Coomassie Brilliant Blue, separated by SDS-PAGE, and transferred to nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked at room temperature for 1 h in PBST containing (mm): 103 NaCl, 2.5 KCl, 10 Na2HPO4, 1.5 KH2PO4, 0.1% Tween 20, and 5% bovine serum albumin, pH 7.4), incubated initially with primary antibodies (Anti-ClC-3, Alomone Labs, 1 h at room temperature or overnight at 4 °C), and then with the appropriate secondary peroxidase-conjugated antibodies (horseradish peroxidase-linked anti-rabbit secondary antibody and horseradish peroxidase-linked anti-biotin antibody, 1 h at room temperature). Final detection was carried out with LumiGLO chemiluminescent reagent (New England Biolabs) as described by the manufacturer. The density of target bands was accurately determined by the computer-aided one-dimensional gel analysis system. Measurement of [Cl-]i—6-Methoxy-N-ethylquinolinium iodide (MEQ) was reduced to its cell-permeable derivative 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ) as described previously (10Biwersi J. Verkman A.S. Biochemistry. 1991; 30: 7879-7983Crossref PubMed Scopus (130) Google Scholar). The MEQ reduction was carried out by addition of 32 μm sodium borohydride to MEQ solution at room temperature under flowing nitrogen in the dark for 30 min. Dihydro-MEQ was freshly prepared before experiment. Cells were incubated with 100–150 μm diH-MEQ in a Ringer's buffer solution containing (mm):119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 1.3 MgSO4, 2.5 CaCl2, 26 NaHCO3, and 11 glucose, pH 7.4 at room temperature in the dark for 30 min. In cytoplasm, dihydro-MEQ is quickly oxidized to MEQ, which is sensitive to [Cl-]i. Fluorescence quenching induced by Cl- was monitored by MetaFluor Imaging software (Universal Imaging Systems, Chester, PA) with 350-nm excitation wavelength and 435-nm emission wavelength. Electrophysiological Experiments—Cells in a chamber of 500 μl in volume were continuously superfused at the rate of 2 ml per min. The Cl- currents were recorded with an Axopatch 200B Amplifier (Axon Instrument, Foster City, CA) using conventional whole cell recording technique. Patch pipettes were made from borosilicate glass using a two-stage puller (pp-83, Narishige, Tokyo, Japan) and had the resistances of 3–5 Ω when the pipettes were filled with the pipette solution. A 3 mm KCl-agar salt bridge between the bath and the Ag-AgCl reference electrode was used to minimize the changes of liquid junction potentials. To determine the whole cell current-voltage curve, the cell was held at -40 mV, and test potentials were applied from -100 mV to +120 mV for 400 ms in +20 mV increments at an interval of 5 s. To obtain time-dependent changes in current amplitude, cells were clamped from a holding potential of -40 mV to a hyperpolarizing potential of -80 mV for 250 ms, then back to -40 mV for 25 ms, and then to a depolarizing potential of +100 mV for 250 ms. This protocol was repeated every 30 s. Currents were filtered at a frequency of 2 kHz and digitized at 5 kHz using pCLAMP8.0 software (Axon Instruments). The data were directly entered into the hard drive of a PC-compatible computer. All experiments were performed at room temperature (25 °C). The hypotonic bath solution contained (mm): 107 N-methyl-d-glucamine chloride (NMDG-Cl), 1.5 MgCl2, 2.5 MnCl2, 0.5 CdCl2, 0.05 GdCl3, 10 glucose, and 10 Hepes, pH 7.4 adjusted with NMDG. This solution osmolarity measured by a freezing point depression osmometer (OSMOMAT030, Germany) was 230 mosmol/kg·H2O. A 300 mosmol/kg·H2O isotonic bath solution was made by adding 70 mm d-mannitol to the hypotonic solution. A 370 mosmol/kg·H2O hypertonic bath solution was prepared by adding 140 mm d-mannitol to the hypotonic solution. In the experiment for Cl- dependence, Cl- in the hypotonic medium was replaced by equimolar aspartate- to obtain the hypotonic solution containing 39 mm [Cl-]o. In the experiment for anion selectivity, the hypotonic bath solution contained (mm): 115 NaX, 10 glucose, and 10 Hepes, pH 7.4 adjusted by NaOH. X- denotes I-, Cl- or aspartate-. The pipette solution contained (mm): 95 CsCl, 20 TEACl, 5 ATP-Mg, 5 EGTA, 5 Hepes, and 80 d-mannitol, pH 7.2 adjusted by CsOH. The osmolarity of this solution was 300 mosmol/kg·H2O. In the intracellular dialysis experiments, anti-ClC-3 antibody was diluted to 300 μg/ml by distilled water, and added to the pipette solution (final concentration was 5 μg/ml.). For preabsorbed anti-ClC-3 antibody, antibody and antigen were dissolved by distilled water separately, mixed in a ratio of 1:10, then stored at 4 °C overnight. The mixed solution was added to the pipette solution. Finally, the pipette solution contained 5 μg/ml antibody and 50 μg/ml antigen. The osmolarity of the pipette solutions was not significantly altered by adding antibody alone or antigen-presorbed antibody. Based on the shift of the reversal potential (ΔErev), the permeability ratios (PX/PCl) were calculated by the modified Goldman-Hodgkin-Katz equation shown in Equation 1, Px/PCl=([Cl−]nexp(−ΔErevF/RT)−[Cl−]s)/[X−]s(Eq. 1) where [Cl-]n and [Cl-]s are the Cl- concentrations in the normal and substituted extracellular solutions. [X-]s is the concentration of the substituting anion. F is the Faraday constant, R is the gas constant, and T is absolute temperature. Statistical Analysis—All data are expressed as mean ± S.E. Statistical analyses were performed using the Student's t test or analysis of variance. Values of p < 0.05 were considered significant. Anti-ClC-3 antibody and antigen were supplied by Alomone Labs (Jerusalem, Israel). Trypsin, CsCl, CsOH, l-aspartate acid, EGTA, TEAC, Hepes, ATP-Mg, DIDS, 4-α-phorbol 12-myristate 13-acetate (4α-PMA), PDBu, genistein, vanadate, Tris, Me2SO, and Dulbecco's modified Eagle's medium/F12 were obtained from Sigma. Expression of ClC-3 cDNA in A10 Cells—Expression of ClC-3 protein in A10 cells was determined by Western blot with the use of a polyclonal antibody directed against ClC-3. The anti-ClC-3 antibody recognized a major band at 80–90 kDa. The ClC-3 protein expression was significantly increased in ClC-3-A10 cells (Fig. 1). Effect of ClC-3 Antisense on Expression of ClC-3 Protein—To determine the uptake of oligonucleotide by A10 cell, the oligonucleotides were labeled with fluorescence. As shown in Fig. 2A, under resting conditions the fluorescence in the cells was negligible, but the fluorescence in cells treated with antisense, sense, or missense were greatly increased, which confirmed the uptake of oligonucleotides by these cells. Fig. 2B shows that 100 μg/ml ClC-3 antisense oligonucleotide decreased ClC-3 protein expression in a time-dependent manner. After 48 h of ClC-3 antisense treatment, the decrease in ClC-3 protein expression reached a maximum. Fig. 2C shows that the cells in the quiescent state were transfected with oligonucleotides by incubation for 48 h with Lipofectamine2000, the ClC-3 protein was decreased by 12.7 ± 1.6%, 26.8 ± 2.8%, 57.8 ± 2.4%, and 58.2 ± 2.3% (data from six different experiments; p < 0.01) in 5 μl/ml Lipofectamine2000 plus 25, 50, 100, and 200 μg/ml ClC-3 antisense, respectively. 100 μg/ml ClC-3 antisense reached a maximal effect. Sense, missense oligonucleotides, and Lipofectamine2000 did not alter ClC-3 protein expression (n = 6 for each group; p > 0.05 versus control). The Characteristics of Cl-Currents—In A10 cells, the patch clamp whole cell currents were very small when the cells were exposed in the isotonic solution. If the bath solution was changed from isotonic solution to hypotonic solution, it evoked the large outward rectifying currents (ICl.vol) with a reverse potential of -2.0 ± 1.5 mV. In ClC-3-A10 cells, hypotonic solution also evoked an outward rectifying current (ICl,ClC-3) with a reverse potential of -2.6 ± 1.2 mV. Compared with ICl.vol, ICl.ClC-3 was larger. The current densities of ICl.vol (n = 12) and ICl.ClC-3 (n = 19) were -23.5 ± 1.5 and -55.9 ± 2.7 pA/pF (p < 0.05 versus ICl.vol) at -80 mV, respectively, and 41.8 ± 2.8 and 100.6 ± 5.6 pA/pF (p < 0.01 versus ICl.vol)at +80 mV, respectively (Fig. 3, A and B). When the [Cl-]o in the bath solution was changed from 116 to 39 mm, the current was significantly decreased, with a change in the reverse potential from -1.6 ± 2.0 to 27.9 ± 2.6 mV in A10 cells (n = 6) and from -2.3 ± 1.7 to 26.6 ± 1.9 mV in ClC-3-A10 cells (Fig. 3, C and D). Both ICl.vol and ICl.ClC-3 were inhibited by DIDS. After exposure to DIDS, the current densities of ICl.vol (n = 8) and ICl.ClC-3 (n = 7) were reduced from -21.8 ± 2.4 to -14.3 ± 1.6 pA/pF and -56.0 ± 4.1 to -34.1 ± 2.5 pA/pF at -80 mV, respectively, and reduced from 36.4 ± 3.0 to 16.4 ± 1.6 pA/pF and 101.1 ± 9.1 to 41.1 ± 4.9 pA/pF at +80 mV, respectively. DIDS had the same inhibition in ICl.vol and ICl.ClC-3. However, DIDS had more inhibition on currents at +80 mV than that at -80 mV (Fig. 3, E and F). This indicates that the effects of DIDS on ICl.vol and ICl.ClC-3 were voltage-dependent. 100 μg/ml ClC-3 antisense remarkably decreased ICl.vol by 54.3 ± 4.6% at +80 mV and 56.0 ± 5.8% at -80 mV, which were similar to the decreased magnitude of ClC-3 protein expression induced by 100 μg/ml ClC-3 antisense, whereas sense, missense, and Lipofectamine2000 failed to inhibit this current. Based on results from the ClC-3 antisense experiments, we further examined the inhibitory effects of intracellular dialysis of an anti-ClC-3 antibody on ICl,vol and ICl,ClC-3. Fig. 4 shows that intracellular dialysis of an anti-ClC-3 antibody could completely block the ICl,vol (Fig. 4, A and B) and ICl,ClC-3 (Fig. 4, C and D). To exclude nonspecificity of the anti-ClC-3 antibody, the effect of preabsorbed anti-ClC-3 antibody on the current was determined. It was found that intracellular dialysis of preabsorbed anti-ClC-3 antibody did not inhibit ICl,vol and ICl,ClC-3 (Fig. 4, A and B). Table I illustrates the anion selectivity of both the ICl,vol channel in A10 cell and ICl,ClC-3 channel in ClC-3-A10 cell. The reverse potential induced by I- was more minus than that by Cl-, whereas aspartate- produced a positive reverse potential. The anion selectivity order of these channels was as follows: I- > Cl- > aspartate-. There was no significant difference in reverse potentials induced by anions and the anion permeability between ICl,vol and ICl,ClC-3.Table IShifts in reversal potential (mV) induced by different anions and their relative permeability (permeability ratio of anions) in A10 and ClC-3-A10 cellsnA10nClC-3-A10Cl-6-1.0 ± 0.97-2.0 ± 0.9I-6-7.5 ± 0.57-7.9 ± 0.8Asp-636.8 ± 2.8737.6 ± 2.1PI/PCl61.29 ± 0.0671.26 ± 0.08PAsp/PCl60.21 ± 0.0570.22 ± 0.05 Open table in a new tab The Change in [Cl-]i Induced by Hypotonic Solution—In [Cl-]i measuring experiments, change of the medium from isotonic solution to hypotonic solution significantly decreased [Cl-]i in A10 from 31.1 ± 2.1 to 24.9 ± 1.2 mm (n = 30; p < 0.01 versus isotonic solution), whereas in ClC-3-A10 cells, the hypotonic solution induced more of a decrease in [Cl-]i than that in A10 cells. The [Cl-]i in ClC-3-A10 cells was reduced from 30.6 ± 1.1 to 19.9 ± 1.0 mm (n = 30; p < 0.05 versus A10 cell in hypotonic solution). In contrast, 100 μg/ml ClC-3 antisense significantly inhibited the decrease in [Cl-]i induced by hypotonic solution, and made [Cl-]i have a lower response to hypotonic challenge. [Cl-]i was diminished from 31.1 ± 2.1 to 28.5 ± 1.9 mm (n = 30; p < 0.05 versus A10 cell in hypotonic solution; Fig. 5). Effects of PDBu, Genistein, and Vanadate on ICl,vol, ICl,ClC-3, and [Cl-]i—Fig. 6A illustrates that 100 nm PDBu completely blocked ICl,vol (A, top trace in a) and ICl,ClC-3 (A, bottom trace in a). The decrease in [Cl-]i induced by hypotonic solution in A10 and ClC-3-A10 cells was completely reverted to normal levels by PDBu (Fig. 6D), whereas, 4α-PMA (negative control for phorbol ester activation of PKC) had no significant effect on ICl,vol (A, top trace in b), ICl,ClC-3 (A, bottom trace in b), and [Cl-]i induced by hypotonic solution in A10 and ClC-3-A10 cells (Fig. 6D). Fig. 6B shows that 30 μm genistein completely inhibited ICl,vol (top trace) and ICl,ClC-3 (bottom trace). The levels of [Cl-]i induced by hypotonic solution in A10 and ClC-3-A10 cells were almost elevated to the normal level by genistein (Fig. 6E). Whereas, the protein-tyrosine phosphatase inhibitor, sodium orthovanadate, potentiated ICl.vol (Fig. 6C, top trace) and ICl,ClC-3 (Fig. 6C, bottom trace) in a voltage-independent manner. The densities of currents for ICl,vol before and after exposure to 500 μm vanadate were -24.1 ± 2.3 and -34.2 ± 3.1 pA/pF at -80 mV (n = 8), respectively, and 40.2 ± 2.9 and 55.6 ± 2.7 pA/pF at +80 mV (n = 8), respectively. For ICl,ClC-3, the densities of currents were -63.5 ± 5.2 and -94.7 ± 5.0 pA/pF at -80 mV (n = 8), respectively, and 96.7 ± 5.4 and 141.4 ± 6.5 pA/pF at +80 mV (n = 8), respectively. The increases in ICl.vol and ICl.ClC-3 induced by vanadate were 45.7 ± 4.4 and 48.5 ± 5.0% at -80 mV, and 47.1 ± 4.8 and 49.3 ± 4.7% at +80 mV, respectively. This was consistent with the effect of vanadate on [Cl-]i. This protein-tyrosine phosphatase inhibitor further significantly reduced [Cl-]i level, which was in lower level induced by hypotonic solution, from 24.8 ± 1.6 to 21.2 ± 1.4 mm (n = 30; p < 0.05) in A10 cells, and from 20.9 ± 1.3 to 16.2 ± 1.1 mm (n = 30; p < 0.05) in ClC-3-A10 cells (Fig. 6F). In the present study, we first demonstrated that hypotonic solution significantly reduced [Cl-]i and simultaneously activated an outward rectifying ICl,vol in A10 cells. Both ClC-3 antisense and intracellular dialysis of anti-ClC-3 antibody significantly inhibited the hypotonicity-induced decrease in [Cl-]i and activation of ICl,vol. We then found that overexpression of ClC-3 in A10 cells (ClC-3-A10 cells) yielded a larger ICl,vol with the same reverse potential, sensitivity to voltage-dependent inhibition by DIDS, and anion selectivity (I- > Cl- > aspartate-) as that of endogenous ICl.vol. in A10 cells under hypotonic conditions. Furthermore, we showed that the hypotonic cell swelling induced more decrease in [Cl-]i in ClC-3-A10 cells, and activation of ICl.ClC-3 also was remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Finally, we found that hypotonic cell swelling-induced changes in [Cl-]i are not only intimately linked to the action of ICl.vol and ICl.ClC-3 but also, as having been reported for ICl.vol and ICl.ClC-3 in other cells (16von Weikersthal S.F. Barrand M.A. Hladky S.B. J. Physiol. 1999; 516: 75-84Crossref PubMed Scopus (74) Google Scholar, 17Co-ca-Prados M. Sanchez-Torres J. Peterson-Yantorno K. Civan M.M. J. Membr. Biol. 1996; 150: 197-208Crossref PubMed Scopus (74) Google Scholar, 18Jin N.G. Kim J.K. Yang D.K. Cho S.J. Kim J.M. Koh E.J. Jung H.C. So I. Kim K.W. Am. J. Physiol. 2003; 285: G938-G948PubMed Google Scholar), well controlled by endogenous PKC and proteintyrosine phosphorylation. These data provide compelling evidence that ClC-3 channels are responsible for the Cl- transportation and ICl.vol during hypotonic perturbations in A10 cells. The molecular identification of the protein responsible for ICl.vol has been particularly difficult to resolve. The main reason for this difficulty is that endogenous ICl.vol is expressed practically in all types of cells, which can be always superimposed with membrane currents because of transgenic expression of candidate genes. Several molecular candidates responsible for ICl.vol were previously proposed: 1) the multidrug transporter, P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) family of transporters (11Valverde M.A. Diaz M. Sepulveda F.V. Gill D.R. Hyde S.C. Higgins C.F. Nature. 1992; 355: 830-833Crossref PubMed Scopus (524) Google Scholar), 2) pICln, which encodes a small 235 amino acid protein with little homology to any known anion channel structure (12Paulmichl M. Li Y. Wickman K. Ackerman M. Peralta E. Clapham D. Nature. 1992; 356: 238-241Crossref PubMed Scopus (310) Google Scholar), 3) ClC-2, a member of the large ClC superfamily of voltage-dependent Cl- channels (13Grunder S. Thiemann A. Pusch M. Jentsch T.J. Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar), and 4) ClC-3, also a member of the ClC superfamily (4Duan D. Winter C. Crowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). Although both P-gp and pICln expression appeared to yield chloride currents with many of the properties of native ICl.vol, it now seems clear that the currents observed in these studies were not due to expression of either P-gp or pICln but likely due to contamination by endogenous ICl.vol (14Clapham D.E. J. Gen. Physiol. 1998; 111: 623-624Crossref PubMed Scopus (57) Google Scholar). Although ClC-2 represents a bona fide anion channel regulated by cell volume, expressed ClC-2 currents differ significantly in voltage sensitivity, anion selectivity, and pharmacology from conventional, outwardly rectifying ICl.vol found in most cells (15Jentsch T.J. Gunther W. Bioessays. 1997; 19: 117-126Crossref PubMed Scopus (170) Google Scholar). The ClC-3 hypothesis was based on the fact that stable or transient transfection of a full-length ClC-3 cDNA cloned from guinea pig ventricle (gpClC-3) into NIH/3T3 cells yielded a basally active chloride conductance that was strongly modulated by cell volume (4Duan D. Winter C. Crowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). Many properties of the expressed IgpClC-3 resemble those reported for native ICl.vol in heart and other tissues, including an outwardly rectifying unitary slope conductance of 40 pS, an anion selectivity of I- > Cl- > Asp-, inactivation at positive potentials, increase by extracellular hypotonicity, and inhibition by hypertonicity, by extracellular nucleotides, by phorbol esters, by stilbene derivatives, and by tamoxifen. Furthermore, site-directed mutagenesis of an asparagine near the end of the transmembrane-spanning domains (N579K) altered rectification and anion selectivity of the expressed IgpClC-3. These observations were initially confirmed by other independent studies of several groups (16von Weikersthal S.F. Barrand M.A. Hladky S.B. J. Physiol. 1999; 516: 75-84Crossref PubMed Scopus (74) Google Scholar, 17Co-ca-Prados M. Sanchez-Torres J. Peterson-Yantorno K. Civan M.M. J. Membr. Biol. 1996; 150: 197-208Crossref PubMed Scopus (74) Google Scholar, 18Jin N.G. Kim J.K. Yang D.K. Cho S.J. Kim J.M. Koh E.J. Jung H.C. So I. Kim K.W. Am. J. Physiol. 2003; 285: G938-G948PubMed Google Scholar). Thus, ClC-3 represented a viable molecular candidate responsible for native ICl.vol in heart or any other mammalian cell type (19Valverde M.A. Curr. Opin. Cell Biol. 1999; 11: 509-516Crossref PubMed Scopus (40) Google Scholar). Several reports (20Li X. Shimada K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; 275: 35994-35998Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar, 22Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), however, failed to support a role for exogenously expressed ClC-3 as a viable candidate for native ICl.vol. The magnitude of swelling-activated Cl- current is not significantly different between nontransfected and human ClC-3 cDNA-transfected HEK293 cells (22Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Overexpression of ClC-3 in HEK293 cells (22Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and X. oocytes (23Steinmeyer K. Schwappach B. Bens M. Vandewalle A. J. Biol. Chem. 1995; 270: 31172-31177Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 24Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) did not produce outward rectifying ICl.vol. In ClC-3 cDNA-transfected CHO-K1 cells, the ClC-3 channel current was not identified in the endogenous swelling-activated channel current, and not activated by cell swelling (20Li X. Shimada K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; 275: 35994-35998Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Some of these inconsistencies might be attributed to failure to successfully express functional ClC-3 protein and/or difficulties in effectively separating transgenic currents from endogenously expressed Cl- currents present in expression cell systems such as X. oocytes and some mammalian cells (25Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1070) Google Scholar). Furthermore, the controversy surrounding the actual physiological role of ClC-3 Cl- channels was additionally fueled by the report that disruption of the Clcn3 gene did not change the volume-regulated chloride current in hepatocytes, pancreatic acinar cells (26Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bosl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), and salivary acinar cells (27Arreola J. Begenisich T. Nehrke K. Nguyen H.V. Park K. Richardson L. Yang B. Schutte B.C. Lamb F.S. Melvin J.E. J. Physiol. 2002; 545: 207-216Crossref PubMed Scopus (89) Google Scholar) from Clcn3-/- mice. In these and another studies (28Li X. Wang T. Zhao Z. Weinman S.A. Am. J. Physiol. 2002; 282: C1483-C1489Crossref PubMed Scopus (117) Google Scholar), ClC-3 was primarily localized to intracellular membranes where it was proposed to function primarily in vesicular acidification, although other studies have clearly demonstrated plasma membrane localization of heterologously expressed ClC-3 (22Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 29Huang P. Liu J. Di A. Robinson N.C. Musch M.W. Kaetzel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 30Ogura T. Furukawa T. Toyozaki T. Yamada K. Zheng Y.J. Katayama Y. Nakaya H. Inagaki N. FASEB J. 2002; 16: 863-865Crossref PubMed Scopus (74) Google Scholar, 31Schmieder S. Lindenthal S. Ghrenfeld J. Biochem. Biophys. Res. Commun. 2001; 286: 635-640Crossref PubMed Scopus (34) Google Scholar) and endogenous ClC-3 (32Isnard-Bagnis C. Da Silva N. Beaulieu V. Yu A.S. Brown D. Breton S. Am. J. Physiol. 2003; 284: C220-C232Crossref PubMed Scopus (32) Google Scholar, 33Olsen M.L. Schade S. Lyons S.A. Amaral M.D. Sontheimer H. J. Neurosci. 2003; 23: 5572-5582Crossref PubMed Google Scholar) in various cell types. Recently, it has been shown that volume-sensitive osmolyte and anion channels (VSOACs) currents activated by hypotonic medium in atrial myocytes and pulmonary arterial smooth muscle cells from Clcn3-/- and Clcn3+/+ mice were remarkably similar in activation kinetics, steady-state current densities, slight outward rectification, and anion selectivity. However, there also are significant differences in sensitivity to PKC regulation, inhibition by intracellular dialysis with a new anti-ClC-3 antibody, [ATP]i depletion, and high free [Mg2+]i between them (34Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W. Duan D. Howowitz T.L.B. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (84) Google Scholar). These authors have suggested that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. It appears that differential sensitivity of native VSOACs to anti-ClC3 antibody and phorbol esters in different cell types in normal animals is consistent with the possible expression of distinct VSOACs subtypes. In fact, the responses of volume-regulated chloride current to PKC regulation in vascular smooth muscle cells from different blood vessels are not the same. Activation of PKC by PDBu increased the amplitude of swelling-activated chloride current in rabbit portal vein (35Ellershaw D.C. Greenwood I.A. Large W.A. J. Physiol. 2002; 542: 537-547Crossref PubMed Scopus (39) Google Scholar), whereas PKC activation inhibited ICl.vol in mouse pulmonary arterial smooth muscle cells (34Yamamoto-Mizuma S. Wang G.X. Liu L.L. Schegg K. Hatton W. Duan D. Howowitz T.L.B. Lamb F.S. Hume J.R. J. Physiol. 2004; 557: 439-456Crossref PubMed Scopus (84) Google Scholar). As shown in this study, PDBu significantly inhibited ICl.vol and increased [Cl-]i under hypotonic condition. Our results provide evidence to support that ClC-3 channels are responsible for volume-regulated Cl- transportation and Cl- currents and may play an important role in volume regulation in A10 vascular smooth muscle cells. Alternatively, it is possible that contradictory data about ClC-3 in mediating cell volume regulation are caused by differences in [Cl-]i and ClC-3 expression levels in different types of cells. In vascular smooth muscle cells, [Cl-]i is generally higher than other tissues, and the Cl- equilibrium potential (ECl) is approximately -20 mV, which is more positive than the resting membrane potential. Therefore, activation of the Cl- channels will lead to a net Cl- efflux followed by the membrane depolarization (36Martens J.R. Genband C.H. PSEBM. 1998; 218: 192-203Crossref PubMed Scopus (62) Google Scholar, 37Chipperfield A.R. Harper A.A. Prog. Biophys. Mol. Biol. 2000; 74: 175-221Crossref PubMed Scopus (148) Google Scholar). It is currently not known why [Cl-]i is higher in vascular smooth muscle. In sharp contrast to vascular smooth muscle cells, some neurons have passively distributed intracellular Cl-, and it sets the resting membrane potential closer to ECl (38Maduke M. Miller C. Mindell J.A. Annu. Rev. Biophys. Struct. 2000; 29: 411-438Crossref PubMed Scopus (155) Google Scholar). It is interesting that the ClC-3 expression level in vascular smooth muscle cells, including aorta smooth muscle cells is very high (39Lamb F.S. Clayton G.H. Liu B.X. Smith R.L. Barna T.J. Schutte B.C. J. Mol. Cell Cardiol. 1999; 31: 657-666Abstract Full Text PDF PubMed Scopus (44) Google Scholar), whereas only a small fraction of ClC-3 is expressed on the surface membrane in hepatocytes CHO-K1 cells and human hepatoma cell line Huh-7 (28Li X. Wang T. Zhao Z. Weinman S.A. Am. J. Physiol. 2002; 282: C1483-C1489Crossref PubMed Scopus (117) Google Scholar), Our results may, therefore, shed a new light on understanding the regulation of intracellular Cl- concentrations in different cell types. We thank Dr. Joseph R. Hume and Dr. Dayue Duan for providing us with the full-length gpClC-3 cDNA in this study and also for helpful discussions." @default.
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• W2046728004 title "Regulation of Intracellular Cl- Concentration through Volume-regulated ClC-3 Chloride Channels in A10 Vascular Smooth Muscle Cells" @default.
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