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+ (Cae2+) 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+ (Cai2+) release, followed by a sustained plateau phase of ACTH release that is mediated by protein kinase-C-induced Cae2+ influx via L-type VSCC. Pretreatment for 15 min with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 microM), but not the cyclooxygenase inhibitor indomethacin (10 microM) or the epoxygenase inhibitor SKF525A (100 microM) 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 dioctanolglycerol, which directly activates protein kinase-C; and by 59% the spike-type response to ionomycin, which releases Cai2+ by an inositol 1,4,5-trisphosphate-independent mechanism. These results suggest that NDGA either inhibits the production of a lipoxygenase metabolite involved in Cae2+ influx and/or Cai2+ release or acts other than by inhibiting lipoxygenase, such as by directly blocking membrane transport of Cae2+. 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 microM, 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 Cae2+ and 54% by both removing Cae2+ and depleting Cai2+, indicating either that 12(S)-HETE facilitates transmembrane Ca2+ transfer or that increased cytosolic Ca2+ is necessary for 12(S)-HETE's action.(ABSTRACT TRUNCATED AT 400 WORDS)