Effects of Trypsin on Cytosolic Calcium Levels in the Rat Aortic Endothelium

Abstract

The effect of trypsin on vascular tone and the cytosolic calcium concentration ([Ca²⁺]_i) of endothelial and smooth muscle cells were examined in the rat aorta. A calcium indicator, fura-PE3, was used to measure [Ca²⁺]_i simultaneously with vascular tone. In the endothelium-intact rat aorta, carbachol and trypsin increased [Ca²⁺]_i in a dose-dependent manner. In the endothelium-denuded rat aorta, carbachol did not change [Ca²⁺]_i, but trypsin slightly increased it. Addition of trypsin to the norepinephrine-stimulated rat aorta relaxed the muscle with an additional increase in [Ca²⁺]_i. Under calcium-free conditions, trypsin induced a transient increase in [Ca²⁺]_i. Trypsin-induced endothelium-dependent relaxation was inhibited by preincubation with l-NMMA, an endothelial NO synthase inhibitor, U-73122, a phospholipase C inhibitor, cyclopiazonic acid, a sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase blocker, and lanthanum, a nonselective Ca²⁺ channel blocker. However, indomethacin, a nonselective cyclooxygenase inhibitor, and SKF-96365, a store-operated Ca²⁺-channel blocker, had no effect on the trypsin-induced relaxation. These results suggest that trypsin increases [Ca²⁺]_i in the endothelial cells through SKF-96365-insensitive Ca²⁺ channels and regulates the release of NO, which results in relaxation of the rat aorta.