Regulation of intracellular Cl- concentration through volume-regulated ClC-3 chloride channels in A10 vascular smooth muscle cells

Abstract

We previously found that antisense oligonucleotide specific to 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 (I Cl.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 I Cl.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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.