Human Diploid Fibroblast Cell Culture Medium Contains a Factor That Increases Cytosolic Ca²⁺ and Stimulates Prostaglandin Synthesis by Cultured Bovine Aortic Endothelial Cells

Previously, we demonstrated that conditioned medium (CM) from cultures of human diploid fibroblast cells contains a factor that stimulates the production of prostacyclin (PGI₂) by cultured bovine aortic endothelial cells (BAEC). To study the mechanism by which CM stimulates PGI₂ production, we measured the effect of removal of extracellular calcium (Ca²⁺) on the concentration of cytosolic Ca²⁺ and on the production of 6-keto-prostaglandin F_1α, (6-keto-PGF_1α), a stable metabolite of PGI₂. The CM-induced production of 6-keto-PGF_1α was dependent on extracellular Ca²⁺ and did not require nascent protein synthesis. Application of CM to BAEC induced a transient increase in cytosolic Ca²⁺ concentration that was dependent on extracellular Ca²⁺. Bradykinin induced the production of 6-keto-PGF_1α by BAEC. However, bradykinin induced an increase in cytosolic Ca²⁺ concentration in the presence or absence of extracellular Ca²⁺. Voltage dependent Ca²⁺ channel blocker (verapamil, diltiazem) did not inhibit either the CM-induced increase in cytosolic Ca²⁺ or the production of 6-keto-PGF_1α by BAEC. These data suggest that CM increases the cytosolic Ca²⁺ concentration and stimulates PGI₂ production by BAEC. The increase in cytosolic Ca²⁺ concentration occurred via the influx of extracellular Ca²⁺ independent of L-type Ca²⁺ channels blocked by verapamil or diltiazem.

Fura2 - Spectrofluorometry - PGI₂ - 6-Keto-Prostaglandin F_1α - L-Type Ca²⁺ Channels