Arachidonic acid metabolites have been implicated in the regulation of ACTH secretion. To define further which eicosanoid(s) is primarily involved, we examined the effects of both inhibitors of the three arachidonate metabolic pathways (cyclooxygenase, lipoxygenase, and epoxygenase) and specific eicosanoid products on ACTH secretion by rat pituitary corticotrophs in a microperifusion system. CRF stimulates sustained ACTH release that is mediated by protein kinase-A-induced extracellular Ca2+ (Ca-e(2+)) influx via L-type voltage-sensitive calcium channels (VSCC). Arginine vasopressin (AVP) stimulates an initial spike phase of ACTH release that presumably is mediated by inositol 1,4,5-trisphosphate-induced intracellular Ca2+ (Ca-i(2+)) release, followed by a sustained plateau phase of ACTH release that is mediated by protein kinase-C-induced Ca-e(2+) influx via L-type VSCC. Pretreatment for 15 min with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 mu M), but not the cyclooxygenase inhibitor indomethacin (10 mu M) or the epoxygenase inhibitor SKF525A (100 mu M) inhibited the sustained response to CRF by 48% and the initial spike response to AVP by 38%. NDGA-induced inhibition was not reversed by indomethacin or SKF525A, alone or in combination, precluding arachidonate shunting into other pathways. However, the results suggested that epoxygenase metabolites may have a minor stimulatory and cyclooxygenase metabolites may have a minor inhibitory effect on ACTH secretion. Preexposure to NDGA suppressed by 43% the sustained response to 8-bromo-cAMP, which directly activates protein kinase-A; by 57% the sustained response to dioctanoylglycerol, which directly activates protein kinase-C; and by 59% the spike-type response to ionomycin, which releases Ca-i(2+) by an inositol 1,4,5-trisphosphate-independent mechanism. These results suggest that NDGA either inhibits the production of a lipoxygenase metabolite involved Ca-e(2+) influx and/or Ca-i(2+) release or acts other than by inhibiting lipoxygenase, such as by directly blocking membrane transport of Ca-e(2+). The three major lipoxygenase metabolites tested, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acid (HETE), all stimulated sustained ACTH release in a dose-dependent manner. At a concentration of 2 mu M, 12(S)-HETE was 4.7 and 2.5 times more potent than 5(S)- and 15(S)-HETE, respectively, and completely reversed NDGA inhibition of both CRF- and AVP-stimulated ACTH secretion. The ACTH-releasing activity of 12(S)-HETE was inhibited 26% by removing Ca-e(2+) and 54% by both removing Ca-e(2+) and depleting Ca-i(2+), indicating either that 12(S)-HETE facilitates transmembrane Ca2+ transfer or that increased cytosolic Ca2+ is necessary for 12(S)-HETE's action. Pretreatment with NDGA, which has been reported to block Ca-e(2+) influx via VSCC, had no effect on the sustained response to Bay K8644, which stimulates Ca-e(2+) influx via L-type VSCC; the sustained response to KCl, which induces Ca-e(2+) influx via all types of VSCC by depolarizing the cell membrane; or the sustained response to 12(S)-HETE. Thus, NDGA does not appear to act either by blocking Ca-e(2+) influx via L- or non-L-type VSCC or by directly inhibiting 12(S)-HETE's action. Furthermore, as NDGA blocked ACTH release stimulated by protein kinase-A- and -C-mediated activation of L-type VSCC, but not that stimulated by Ca-e(2+) influx induced by agents such as ionomycin, Bay K8644, and KCl, it seems unlikely that HETEs act distal to Ca-e(2+) influx. Rather, it suggests that they facilitate stimulated Ca-e(2+) membrane transport. We conclude that lipoxygenase metabolites of arachidonic acid, particularly 12(S)-HETE, are involved in the mechanism of ACTH secretion, probably acting to facilitate Ca-e(2+) influx via L-type VSCC.
