We investigated the involvement of arachidonate in the PRL secretory process using three experimental systems: hemipituitary glands incubated in vitro, cultured pituitary cells, and dispersed anterior pituitary cells perifused in columns. Arachidonate (100 microM) significantly (P less than 0.05) stimulated PRL release in the former system and stimulated PRL secretion in a dose-related manner in cultured cells. In hemipituitary glands, indomethacin, a cyclooxygenase inhibitor, potentiated the arachidonate-mediated stimulation, whereas nordihydroguaiaretic acid or BW755c abolished it. The latter two agents, but not indomethacin, abolished the effect of phospholipase A2 on PRL release in vitro. BW755c also inhibited the stimulatory effect of TRH on PRL release in both experimental systems. Conversely, the stimulation of PRL release by phorbol myristate acetate (PMA), although significantly reduced, was not abolished by either nordihydroguaiaretic acid or BW755c. Quinacrine, a phospholipase A2 inhibitor, also abolished the stimulatory effect of phospholipase A2 or TRH on PRL release. In cultured cells, quinacrine inhibits basal PRL release, but does not affect PRL release induced by arachidonate or (Bu)2 cAMP. These results more firmly establish a role for arachidonate as an intracellular mediator of PRL release and suggest the involvement of an arachidonate metabolic pathway(s) (lipoxygenase and epoxygenase) other than prostaglandin or thromboxane formation. The effect of PMA on PRL release may be attributable only in part to an increase in the production of arachidonate metabolites, and most of PMA's effect on PRL release may relate to its activation of protein kinase C.

This study was designed to investigate basal and thyrotropin-releasing hormone (TRH)-stimulated prolactin release in the presence of agents that influence arachidonic acid metabolism. Agents that decrease its production by blocking phospholipase A2 activity, i.e., quinacrine and 4-bromophenacylbromide, significantly decreased prolactin secretion from anterior pituitary glands in vitro and from dispersed pituitary cells in a perifusion column. Phospholipase A2 and phorbol myristate acetate, substances that increase intracellular concentrations of arachidonic acid, markedly stimulated prolactin release by dispersed pituitary cells and by anterior pituitary glands incubated in vitro. The involvement in prolactin secretion of arachidonic acid metabolic products produced via the lipoxygenase pathway was investigated indirectly using nordihydroguaiaretic acid (NDGA), a specific inhibitor of this enzyme. NDGA progressively (dose-related) inhibited the release of prolactin in vitro and blocked the stimulating effect of 50 nM TRH on prolactin release from hemipituitary glands. Indomethacin, a specific inhibitor of the cycloxygenase pathway, had no significant effect on basal and TRH-stimulated prolactin release. The results suggest that arachidonic acid metabolism is involved in basal and TRH-stimulated prolactin secretion and that lipoxygenase pathway products are at least partially responsible for these effects.

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.

The in vitro effect of dopamine, haloperidol and pimozide on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine by anterior pituitary glands was studied. Dopamine decreased the incorporation of 32Pi into phosphatidylinositol in a dose-related manner without affecting phosphatidylcholine and phosphatidylethanolamine. Haloperidol and pimozide did not modify 32Pi incorporation into these phospholipids by themselves but completely reversed the inhibitory effect of dopamine on phosphatidylinositol. The inhibition of 32Pi incorporation into phosphatidylinositol synthesis in the presence of 500 nM dopamine was significant at 20 min of incubation and maximal at 30 min. The possibility that a decrease in phosphatidylinositol cleavage and turnover may be involved in the inhibitory regulation of prolactin secretion by DA is suggested.