Modulation of cytosolic Ca 2+ concentration by thapsigargin and cyclopiazonic acid in human aortic endothelial cells

Abstract

To clarify the agonist-induced Ca 2+ entry mechanism, effects of thapsigargin and cyclopiazonic acid, selective inhibitors of endoplasmic reticulum Ca 2+-ATPase, on intracellular free Ca 2+ concentration ([Ca 2+] i) were studied in cultured human aortic endothelial cells loaded with the fluorescent Ca 2+ indicator fura-2. Thapsigargin (1–1000 nM) and cyclopiazonic acid (0.1–100 μM) produced a biphasic change in [Ca 2+] i, which consisted of a transient peak elevation followed by a long-lasting decline of [Ca 2+] i in a concentration-dependent manner. In the presence of thapsigargin or cyclopiazonic acid, the rapid transient elevation of [Ca 2+] i elicited by histamine was attenuated in a time-dependent manner. The slow declining phase of the response to thapsigargin and cyclopiazonic acid was completely eliminated by removal of extracellular Ca 2+, and it was also prevented by reduction of the extracellular Cl − concentration to 40 mM or by the Cl − channel blocker N-phenylanthranilic acid. These findings suggest that the initial transient rising phase and the slow declining phase of [Ca 2+] i in response to thapsigargin and cyclopiazonic acid reflect a blockade of Ca 2+ uptake into the endoplasmic reticulum and the Cl −-sensitive Ca 2+ entry activated by the depletion of agonist-sensitive intracellular Ca 2+ stores, respectively, in human aortic endothelial cells.