Importance of arachidonic acid metabolites in regulating ATP-induced calcium fluxes in thyroid FRTL-5 cells

Abstract

Stimulating rat thyroid FRTL-5 cells with the purinergic agonist ATP activates both the inositol phosphate signal-transduction pathway and the phospholipase A 2 pathway. In the present study we wanted to investigate the possible inter-relationships between these two systems during ATP-induced changes in intracellular free calcium ([Ca 2+] i). Pretreatment of Fura-2 loaded cells with 4-bromophenylacyl, an inhibitor of phospholipase A 2, had no effect on the ATP-induced entry of Ca 2+ but inhibited the release of sequestered Ca 2+. Nordihydrogualaretic acid (NDGA), a lipoxygenase inhibitor, and 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of cytochrome P-450 enzymes, attenuated the ATP-evoked transient increase in [Ca 2+] i. Furthermore, the capacitative entry of Ca 2+ was also attenuated in NDGA- and ETYA-treated cells stimulated with ATP. Similar results were obtained using econazole, an inhibitor of cytochrome P-450 enzymes. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the ATP-evoked response in [Ca 2+] i. We also showed that stimulation of intact or permeabilized FRTL-5 cells with arachidonic acid released sequestered calcium. This calcium originated, at least in part, from an IP 3 sensitive calcium pool. In addition, arachidonic acid rapidly acidified the cytosol. The results suggest that metabolism of arachidonic acid by a non-cyclooxygenase pathway is of importance in supporting agonist-induced calcium fluxes evoked via stimulation of the inositol phosphate pathway in FRTL-5 cells. Furthermore, arachidonic acid per se may modify agonist-induced calcium fluxes in these cells.