Responsiveness of Immature versus Adult Male Rat Hypothalami to Dibutyryl Cyclic AMP- and Forskolin-Induced LHRH Release in vitro

Abstract

In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcAMP, 5 × 10^–8 M), butyrate (5 × 10^–8 M) and forskolin (10^–4 M) on immunoreactive luteinizing hormone-releasing hormone (LHRH) release from superfused hypothalamic fragments from intact male rats of age 25, 30, 45, or 60–75 day (adult). The results indicate that at 25 days of age, male rat hypothalami were most responsive to cyclic AMP (162% of preinfusion basal LHRH release); by 30 days of age, dbcAMP also elicited increased LHRH release (120% of basal). By 45 days of age, the dbcAMP effect on in vitro LHRH release was slightly inhibitory (78% of basal); however, by adulthood, the effect of this cyclic nucleotide on LHRH release was minimal (92% of basal). Butyrate also induced age-dependent modifications in in vitro LHRH release from male rat hypothalami, with slight increases following butyrate delivery at 25 days of age (115%), slight decreases at 30 (82%) and 45 days of age (68%), and little change in adulthood (94%). This latter finding emphasizes the importance of using butyrate as a control for butyryl derivatives of cyclic AMP, which are known to liberate butyric acid as a product of hydrolysis of parent compounds. To address the possibility that the lack of effectiveness of dbcAMP in the older animal preparations was solely due to an increasing sensitivity of male rat medio-basal hypothalamus fragments to butyrate, we examined the effect of forskolin (10^–4 M), an adenylate cyclase stimulator, on LHRH release. In the 30-day-old male rat preparation, continuous infusion of forskolin evoked marked increases in LHRH release, characterized by episodic surges; in contrast, this compound was relatively ineffective in inducing LHRH surges in the adult male rat condition. This study also shows that while hypothalamic LHRH concentration reached maximal levels by 45 days of age, spontaneous in vitro release of LHRH increased in a near-linear fashion as a function of age of male rat donor (from 25 days of age to adult), thus indicating a dissociation between spontaneous LHRH neuronal activity and dbcAMP-stimulated LHRH release. In sum, it can be concluded that the responsivity of the hypothalamic neural LHRH apparatus to cyclic AMP (either exogenously applied or generated through stimulation of adenylate cyclase) decreases as the rats mature, with no response close to puberty and during adulthood. In addition, maturational changes in LHRH release and LHRH concentration in superfused medio-basal hypothalamus fragments from intact male rats can be inferred using this in vitro superfusion system.

Copper amplification of prostaglandin E2 stimulation of the release of luteinizing hormone-releasing hormone is a postreceptor event.

“…Nevertheless, one should be cautious in drawing the conclusion that copper does not amplify cAMP action. Lack of responsiveness of in vitro incubated hypothalamic fragments to dibutyryl cAMP has been observed by others (12,32), and we noted that of the three derivatives of cAMP tested, the 8-bromo derivative was the only one that stimulated LH-RH release and, even at a concentration of 1 mM, it elicited only a marginal response (Fig. 1).…”

“…PGE2 is known to stimulate the activity of adenylate cyclase in many tissues (7)(8)(9). Forskolin, which directly activates the catalytic subunit of adenylate cyclase (10,11), stimulates LH-RH release from hypothalamic tissue incubated under in vitro conditions (12,13). Moreover, the stimulation of LH-RH release by forskolin and by PGE2 is not additive (13), suggestive of a common mechanism of action-namely, cAMP.…”

Activation of two different but complementary biochemical pathways stimulates release of hypothalamic luteinizing hormone-releasing hormone.

“…LHRH release can be evoked either with dibutyrylcAMP or by stimulation of endogenous cAMP formation (2,3,16). In addition, evidence has been presented that part of the mechanism by which PGE2 induces LHRH secretion may involve cAMP (2).…”

“…LHRH and PGE2 released into the incubation medium were assayed as described (1, 13) using specific antisera (16,17). The LHRH assay was initiated immediately after each incubation to avoid having to acidify the medium (18).…”

Receptors and neurosecretory actions of endothelin in hypothalamic neurons.

“…Fractions were collected every 5 min and stored at -20°C prior to RIA. GnRH assay was performed as described (24) old primary cultures was more potently inhibited by ET-1 (IC50 = 1.5 nM) than by ET-3 (IC50 = 27 nM). 1251-labeled ET-1 also bound specifically to the GT1-1 and GT1-7 neuronal cell lines.…”