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Action of luteinizing hormone-releasing hormone in rat ovarian cells : hormone production and signal transduction Wang, Jian


The present study was conducted to investigate the hypothesis that membrane phosphoinositide breakdown may participate in the actions of luteinizing hormone-releasing hormone (LHRH) on hormone production in the rat ovary. In granulosa cells prelabeled with [³H]-arachidonic acid or [³H]-inositol, treatment with LHRH increased the accumulation of radiolabeled inositol lipids, diacylglycerol and free arachidonic acid, but luteinizing hormone (LH) or cholera toxin did not exert the same effect. Activation of protein kinase C by the phorbol ester, 12-0-tetradecanoyl phorbol-13-acetate (TPA) had a stimulatory action on membrane phosphoinositide breakdown. In addition, TPA did not alter arachidonic acid release but potentiated the A23187 stimulated liberation of arachidonic acid. Changes in the cytosolic free calcium ion concentrations, [Ca²⁺]i, induced by LHRH were studied in individual cells using fura-2 microspectrofluorimetry. The resting [Ca²⁺]i was 96.7 ± 2.9 nM (n= 115). The alterations in [Ca²⁺]i induced by LHRH were transient and returned to resting levels within 84±3 second (n=64). A potent LHRH antagonist completely blocked the effect of LHRH on [Ca²⁺]i. Some cells responded to LHRH alone, whereas others responded to angiotensin II, suggesting that there are different subpopulations of granulosa cells. Sustained perifusion of LHRH resulted in a desensitization of the [Ca²⁺]i response to LHRH but not to the calcium ionophore A23187. LHRH treatment accelerated [Ca²⁺]i depletion in granulosa cells perifused with Ca²⁺ free medium, indicating the involvement of intracellular Ca²⁺ pool(s) in [Ca²⁺]i changes induced by LHRH. The complex interactions between the signal transduction pathways involved in the regulation of progesterone and prostaglandin E₂ were also examined. LHRH increased basal progesterone level (5 and 24h culture) and attenuated progesterone production induced by follicle-stimulating hormone (FSH) or cholera toxin (24h). On the other hand, both basal and FSH or cholera toxin stimulated prostaglandin E₂ formation were increased by LHRH (5 and 24h) . A23187, TPA and melittin (an activator of phospholipase A₂) were used to examine the roles of Ca²⁺, protein kinase C and free arachidonic acid, respectively, in LHRH action. Melittin stimulated basal progesterone and prostaglandin E₂ production, and enhanced the stimulation of prostaglandin E₂ by LHRH, A23187 and TPA, indicating that LHRH alters cyclooxygenase activity. A23187 or TPA attenuated the formation of progesterone induced by FSH or cholera toxin (5 and 24h). In contrast, A23187 and TPA augmented cholera toxin or FSH induced prostaglandin E₂ formation. The stimulatory effects of A23187 and TPA on prostaglandin E₂ were synergistic, whether or not FSH or cholera toxin was present during the incubation. The role of arachidonic acid in the action of LHRH was further investigated. Arachidonic acid enhanced progesterone production in a dose dependent manner and potentiated TPA induced progesterone production. The stimulatory effect of arachidonic acid was blocked by nordihydroguaiaretic acid, whereas monohydroxyeicosatetraenoic acids and hydroperoxyeicosatetraenoic acid mimicked the effect of arachidonic acid, suggesting the involvement of lipoxygenase metabolites in LHRH action. In addition, arachidonic acid partially reversed the inhibitory action of LHRH and TPA on FSH induced progesterone production. Although arachidonic acid, TPA and LHRH stimulated progesterone production, arachidonic acid only slightly elevated 20-alpha-hydroxy- progesterone production as compared to that induced by LHRH and TPA. These results suggest that arachidonic acid or its metabolites have a stimulatory role in the action of LHRH on the de novo synthesis of ovarian steroid hormones. Collectively, these findings support the hypothesis that the actions of LHRH or LHRH like peptides on ovarian hormone production are mediated by multiple second messengers involving Ca²⁺, protein kinase C and arachidonic acid metabolites.

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