REGULATION OF CALCIUM INFLUX AND CATECHOLAMINE SECRETION IN CHROMAFFIN CELLS BY A CYTOCHROME-P450 METABOLITE OF ARACHIDONIC-ACID

These studies were designed to determine the role of arachidonic acid metabolites in catecholamine secretion from adrenal chromaffin cells. Inhibitors of the cytochrome P450-dependent metabolism of arachidonic acid were shown to interfere with stimulus-secretion coupling in cultu red chromaffin cells. Ketoconazole (10 mu M), clotrimazole (20 mu M) a nd piperonyl butoxide (50 mu M) inhibited carbachol-dependent catechol amine secretion by 44%, 83%, and 100%, respectively; histamine-depende nt secretion by 25%, 60%, and 81%, and secretion induced by 59 mM KCl depolarization by 25%, 55%, and 89%. Uptake of Ca-45(2+) into the cell s in response to carbachol was inhibited 63% by ketoconazole, 86% by c lotrimazole, and 95% by piperonyl butoxide; KCl-dependent uptake was i nhibited 7%, 56%, and 85%, respectively. However, cytochrome P450 inhi bitors did not inhibit catecholamine secretion when cells were stimula ted with the calcium ionophores ionomycin or lasalocid. These results indicated the involvement of a cytochrome P450 product in controlling Ca2+ influx in response to membrane depolarization. Cells prelabeled w ith [H-3]arachidonic acid formed a H-3-labeled metabolite which comigr ated with authentic 5,6-epoxyeicosatrienoic (5,6-EET) acid on reverse phase and normal phase HPLC. Pretreatment with clotrimazole inhibited the production of this H-3-labeled metabolite. Addition of synthetic 5 ,6-EET (1 nM) to cells pretreated with piperonyl butoxide resulted in catecholamine secretion. These data suggest a role for a cytochrome P4 50 metabolite of arachidonic acid in agonist-stimulated catecholamine secretion.