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The level of cytosolic free calcium ([Ca2+]i) and the production rate of prostacyclin were simultaneously measured in perfused monolayers of cultured vascular smooth muscle (VSM) cells. After loading of fura-2 (a fluorescent calcium indicator), the monolayer of VSM cells (cultured on a cover glass) was fixed in the perfusion cuvette and the cuvette was placed in a fluorometer to monitor the change in [Ca2+]i. The monolayer was perfused and the fractionated perfusion solution was collected to determine 6-keto-PGF1 alpha (a metabolite of prostacyclin) production found in the solution. Afterwards, the time-dependent changes in [Ca2+]i and 6-keto-PGF1 alpha synthesis were compared. Bradykinin (BK, 10(-6) M), angiotensin (Ang) II (10(-7) M) as well as ionomycin (10(-6) M) induced simultaneous increases in [Ca2+]i and 6-keto-PGF1 alpha production. An inhibitor against prostaglandin synthesis, acetylsalicylic acid (ASA, 10(-6) M) abolished BK-induced 6-keto-PGF1 alpha synthesis, whereas ASA did not affect the increase in [Ca2+]i. BK-induced increases in [Ca2+]i and 6-keto-PGF1 alpha production occurred in a dose-dependent manner and the half-maximal response was observed at the same concentration of BK (10(-7) M). These results indicate that an increase in [Ca2+]i is closely associated with BK as well as AngII-induced prostacyclin synthesis. It is suggested that an increase in [Ca2+]i plays a prior role in prostacyclin synthesis. Thus, an interaction between phospholipase A2 (prostaglandin synthesis) and phospholipase C (inositol trisphosphate-Ca2+ mobilization) is suggested.

tohoku journal of experimental medicine

Simultaneous Measurements of Cytosolic Free Calcium Level and Prostaglandin Synthesis Reveal a Correlation between Them in Perfused Monolayer of Cultured Rat Vascular Smooth Muscle Cells: Effects of Bradykinin and Angiotensin II.