Role of intracellular calcium in volume regulation by rabbit medullary thick ascending limb cells

Previous studies demonstrated that Ca²⁺-activated K⁺ channels in luminal membrane of rabbit medullary thick ascending limb cells (MTAL) are activated on exposure of the cells to hyposmotic solutions [J. Taniguchi and W.B. Guggino. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F347-F352, 1989]. In this study, we investigated the mechanism of activation of Ca²⁺-activated K⁺ channels in MTAL cells exposed to hyposmotic solutions. MTAL cells swell in hyposmotic medium and regulate volume back toward the starting volume. This regulatory volume decrease (RVD) is inhibited at high medium K⁺ concentrations or by presence of quinine or Ba²⁺ in extracellular medium, suggesting involvement of K⁺ channels. Measurements of intracellular Ca²⁺ concentrations with fura-2 show that intracellular Ca²⁺ rises in hyposmotic solutions and that this rise does not occur in absence of extracellular Ca²⁺. Nifedipine and verapamil also inhibit rise in intracellular Ca²⁺. Decreasing intracellular Ca²⁺ by removal of external Ca²⁺ in presence of EDTA or by chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibits RVD. We conclude that hypotonic solutions activate K⁺ efflux probably via K⁺ channels and Ca²⁺ influx via a nifedipine- and verapamil-sensitive pathway. Lowering intracellular Ca²⁺ removes the ability of MTAL cells to regulate volume in hyposmotic solutions.