Effects of prostaglandin E 2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus

Abstract

The present study attempts to elucidate the possible role of adenosine 3′,5′-monophosphate (cAMP) and prostaglandin E 2 (PGE 2) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH-RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P 2 membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 μg/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step-wise increment in several doses of PGE 2 (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE 2 induced significant ( P<0.01) increments in LH-RH release at doses of 5.68 × 10 −7, 5.68 × 10 −6, and 5.68 × 10 −5 M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH-RH release was also obtained; forskolin and cholera toxin significantly ( P<0.01) stimulated LH-RH release at doses of 1 × 10 −4 and 5.4 × 10 −10 M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium. An intermittent infusion of dibutyryl cAMP (dbcAMP: 1 × 10 −7 M, 10-min on, 20-min off) resulted in rhythmic LH-RH release from median eminences superfused in vitro. In separate experiments, to examine adenylate cyclase activity, P 2 membrane fractions from the mediobasal hypothalamus were preincubated with appropriate test agents. Adenylate cyclase reaction was initiated by adding adenosine triphosphate. After a 15-min incubation, the reaction was terminated by boiling, the supernatant recovered and subjected to cAMP determination by RIA. The following results were obtained: (1) in vivo E priming of ovariectomized animals significantly increased basal adenylate cyclase activity of P 2 membrane preparations as compared to those from unprimed rats; (2) known adenylate cyclase activators, such as forskolin and cholera toxin clearly produced a dose-related increase in cAMP production; and (3) PGE 2 at the concentration of 5.68 × 10 −6 M stimulated cAMP production. It appears that cAMP and PGE 2 may be involved in the activation of the LH-RH neural apparatus. It is tempting to postulate that PGE 2 may stimulate adenylate cyclase to increase intracellular cAMP levels which, in turn, trigger the release of LH-RH from the median eminence nerve terminals.