Kinetic Studies of K-CL Cotransport in Cultured Rat Vascular Smooth Muscle Cells

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During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl−-dependent Rb+ influx at different external Rb+ and Cl− ion concentrations, [Rb+]o and [Cl−]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity (Vm), and apparent Cl− and Rb+ affinities (Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl−-dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl− correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl−]o were strikingly bimodal with 60–80 mM in early, ~20–30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.



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