Role of eicosanoids in vasopressin-induced calcium mobilization in A7r5 vascular smooth muscle cells

The role of arachidonic acid (AA) and its metabolites in vasopressin (AVP)-induced calcium mobilization in A7r5 aortic smooth muscle cells was explored by intracellular calcium monitoring, [¹⁴C]AA labeling, and high- performance liquid chromatography (HPLC) techniques. In fura 2-loaded A7r5 cells, AA potentiated AVP-stimulated increase in intracellular free Ca²⁺ ([Ca²⁺](i)). The cyclooxygenase inhibitor indomethacin reduced both the AA- and AVP-induced influx of extracellular Ca²⁺. AVP-induced [Ca²⁺](i) transients were not altered by lipoxygenase inhibitors but were reduced in a dose-dependent fashion by ketoconazole, an inhibitor of cytochrome P-450 monooxygenases. Among several epoxygenase metabolites of AA tested, 5,6- epoxyeicosatrienoic acid potentiated AVP-induced [Ca²⁺](i) transients. Reverse-phase HPLC analysis of lipid extracts from A7r5 cells prelabeled with [¹⁴C]AA isolated a radioactive peak that did not coelute with established products of cyclooxygenase-, lipoxygenase-, or cytochrome P-450-catalyzed oxidations of AA. This peak was significantly increased after AVP stimulation and was completely blocked by preincubation with ketoconazole. Thus the stimulation of V₁-vascular AVP receptors of A7r5 cells triggers several cytoplasmic signaling pathways involving AA metabolite formation through the cyclooxygenase and epoxygenase pathways.