Quin2 microfluorometry and effects of verapamil and diltiazem on calcium release from rat aorta smooth muscle cells in primary culture

Abstract

We investigated the effects of the Ca2+ antagonists diltiazem and verapamil on release of Ca2+ from intracellular store sites of rat aorta vascular smooth muscle cells in primary culture. Using the microfluorometry of Ca2+-indicator dye quin2, relative changes in cytosolic Ca2+ concentration could be measured. In the presence of 1 mM extracellular Ca2+, both diltiazem (IC50, 0.31 microM) and verapamil (IC50, 0.47 microM) dose-dependently inhibited elevations in the cytosolic Ca2+, as induced by depolarization of the plasma membrane with high extracellular K+. In the absence of extracellular Ca2+, caffeine and high extracellular K+ induced transient and dose-dependent elevations of the cytosolic Ca2+, and these elevations were not inhibited by either diltiazem or verapamil. Norepinephrine also induced a transient and dose-dependent elevation of cytosolic Ca2+ in the absence of extracellular Ca2+. However, this elevation was inhibited by verapamil and diltiazem (when the norepinephrine concentration was 10(-5) M, IC50 for verapamil and diltiazem was 4.0 and 24.9 microM, respectively). Thus, while verapamil and diltiazem may have no direct effect on the release of Ca2+ from the depolarization- and the caffeine-sensitive intracellular Ca2+ storage sites, the agents do seem to inhibit the adrenoceptor-mediated Ca2+ release mechanism in vascular smooth muscle cells.