IMPORTANCE OF ARACHIDONIC-ACID METABOLITES IN REGULATING ATP-INDUCED CALCIUM FLUXES IN THYROID FRTL-5 CELLS

Stimulating rat thyroid FRTL-5 cells with the purinergic agonist ATP a ctivates both the inositol phosphate signal-transduction pathway and t he phospholipase A(2) pathway. In the present study we wanted to inves tigate the possible inter-relationships between these two systems duri ng ATP-induced changes in intracellular free calcium ([Ca2+](i)). Pret reatment of Fura-2 loaded cells with 4-bromophenylacyl, an inhibitor o f phospholipase A(2), had no effect on the ATP-induced entry of Ca2+ b ut inhibited the release of sequestered Ca2+. Nordihydroguaiaretic aci d (NDGA), a lipoxygenase inhibitor, and 5,8,11,14-eicosatetraynoic aci d (ETYA), an inhibitor of cytochrome P-450 enzymes, attenuated the ATP -evoked transient increase in [Ca2+](i). Furthermore, the capacitative entry of Ca2+ was also attenuated in NDGA- and ETYA-treated cells sti mulated with ATP. Similar results were obtained using econazole, an in hibitor of cytochrome P-450 enzymes. However, treatment of the cells w ith indomethacin, a cyclooxygenase inhibitor, had no effect on the ATP -evoked response in [Ca2+](i). We also showed that stimulation of inta ct or permeabilized FRTL-5 cells with arachidonic acid released seques tered calcium. This calcium originated, at least in part, from an IP3 sensitive calcium pool. In addition, arachidonic acid rapidly acidifie d the cytosol. The results suggest that metabolism of arachidonic acid by a non-cyctooxygenase pathway is of importance in supporting agonis t-induced calcium fluxes evoked via stimulation of the inositol phosph ate pathway in FRTL-5 cells. Furthermore, arachidonic acid per se may modify agonist-induced calcium fluxes in these cells.