Effects of trypsin on cytosolic calcium levels in the rat aortic endothelium

The effect of trypsin on vascular tone and the cytosolic calcium concentration (Ca(2+)](i)) of endothelial and smooth muscle cells were examined in the rat aorta. A calcium indicator, fura-PE3, was used to measure Ca(2+)](i) simultaneously with vascular tone. In the endothelium-intact rat aorta, carbachol and trypsin increased Ca(2+)](i) in a dose-dependent manner. In the endothelium-denuded rat aorta, carbachol did not change Ca(2+)](i), but trypsin slightly increased it. Addition of trypsin to the norepinephrine-stimulated rat aorta relaxed the muscle with an additional increase in Ca(2+)](i). Under calcium-free conditions, trypsin induced a transient increase in Ca(2+)](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 Ca(2+)-ATPase blocker, and lanthanum, a nonselective Ca(2+) channel blocker. However, indomethacin, a nonselective cyclooxygenase inhibitor, and SKF-96365, a store-operated Ca(2+)-channel blocker, had no effect on the trypsin-induced relaxation. These results suggest that trypsin increases Ca(2+)](i) in the endothelial cells through SKF-96365-insensitive Ca(2+) channels and regulates the release of NO, which results in relaxation of the rat aorta.