Regulation of intracellular Cl- concentration through volume-regulated ClC-3 chloride channels in A10 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 I(Cl.vol) in A10 cells. The responses of [Cl(-)](i) and I(Cl.vol) to hypotonic challenge were enhanced by expression of ClC-3, and inhibited by ClC-3 antisense. The currents in A10 (I(Cl.vol)) and in ClC-3-A10 cells (I(Cl.ClC-3)) were remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Reduction in [Cl(-)](i) and activation of I(Cl.vol) and I(Cl.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 I(Cl.vol) and I(Cl.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.
Zhou
Jia-Guo
JG
Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, People's Republic of China.
Ren
Jing-Li
JL
Qiu
Qin-Ying
QY
He
Hua
H
Guan
Yong-Yuan
YY
D000818:Animals; D002460:Cell Line; D048429:Cell Size; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D016372:RNA, Antisense / Q000494:pharmacology; D051381:Rats; D014162:Transfection
D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D016372:RNA, Antisense