Christopher A. Leadem

Effects of endogenous opioid peptides and opiates on luteinizing hormone and prolactin secretion in ovariectomized rats

Adult female rats were implanted with permanent cannulae in the third ventricle of the brain and ovariectomized. 3 weeks later, blood samples were withdrawn every 5 min from intraatrial cannulae placed the previous day. After a control sampling period of 30 min, the rats received an intraventricular bolus injection of saline (2 microliter) or beta-endorphin (beta E; 10 micrograms); sampling was continued for an additional 2 h. Saline injection caused no effect on luteinizing hormone (LH) and prolactin (PRL) secretion. beta E stimulated PRL secretion within 5-10 min, the values peaked in the next 10 min. Thereafter, as PRL levels fell, a suppression of LH secretion became apparent. Inhibition of LH release started 20-35 min after beta E injection and lasted for 35-65 min. The antecendent PRL secretion was apparently not responsible for the observed delayed LH response, since blockade of PRL response with bromocriptine failed to affect the beta E-induced LH suppression. Further, continuous intraventricular infusion of beta E (5 or 10 micrograms/h) for 3 h markedly suppressed the amplitude and frequency of LH episodes in long-term ovariectomized rats. Bolus intraventricular injection of other endogenous opioid peptides and opiate receptor agonists produced different PRL and LH responses. Dynorphin (10 micrograms) similarly suppressed LH release but was only moderately effective in stimulating PRL. Leucine enkephalin (50 micrograms) stimulated LH and inhibited PRL release, while methionine-enkephalin (50 micrograms) selectively stimulated PRL release. The methionine-enkephalin analogs, FK-33824 (50 ng) and DALAMID (50 micrograms), evoked sequential PRL and LH responses similar to those seen after beta E injection. Interestingly, morphiceptin (a specific mu receptor agonist; 10 micrograms) markedly suppressed LH release, but only sparingly stimulated PRL release. Delta receptor peptide (a specific delta receptor agonist; 10 micrograms) selectively suppressed LH release. Bremazocine (a specific kappa receptor agonist; 0.5 mg/kg) administered intravenously suppressed LH release selectively. These studies show that of the four endogenous opioid peptides tested beta E was most effective in evoking sequential PRL and LH responses, and these effects may be mediated by either epsilon receptors or multiple opiate receptor subtypes; stimulation of kappa receptors by bynorphin or bremazocine suppressed LH release, and further studies would be needed to understand the mode of action of the two enkephalins and the delta opiate receptors in eliciting disparate PRL and LH responses.

Effects of Specific Activation of Mu-, Delta- and Kappa-Opioid Receptors on the Secretion of Luteinizing Hormone and Prolactin in the Ovariectomized Rat

With the recent development of highly specific ligands for the mu, delta and kappa opioid receptors it was of interest to define the effects of activation of each of these receptor types on LH and prolactin (PRL) secretion. The compounds were infused (10 microliters/h) at various concentrations into the third cerebroventricle of unanesthetized, ovariectomized rats. The mu agonist, DAGO, at both 1 and 10 micrograms/h caused a significant suppression of LH secretion and a significant stimulation of PRL release. DPDPE, the delta agonist, had no effect on either hormone at 1 microgram/h but inhibited LH secretion at 10 micrograms/h. There was still no effect of this high dose of DPDPE on PRL release. The kappa agonist, U50,488H, had no effect on either hormone at 10 micrograms/h, but at 100 micrograms/h produced a significant suppression of LH release and a highly variable increase in PRL. Coinfusion of 100 micrograms/h of naloxone with the high dose of each of the agonists completely blocked the responses of both hormones to each of the agonists with one exception: the highly variable stimulation of PRL by U50,488H was not affected, thus indicating a nonspecific effect of U50,488H on PRL secretion. These results demonstrate that: activation of the mu receptors produces an inhibition of LH secretion and a stimulation of PRL release; activation of the delta receptors produces an inhibition of LH secretion but has no effect on PRL release, and activation of the kappa receptors produces an inhibition of LH release and a variable stimulation of PRL secretion.

Effects of naloxone on catecholamine and LHRH release from the perifused hypothalamus of the steroid-primed rat

The purpose of the present investigation was to determine the effects of naloxone on LHRH and catecholamine release from preoptic area-medial basal hypothalamic (POA-MBH) fragments in vitro. Ovariectomized rats were treated with estradiol benzoate, followed 2 days later by progesterone. The rats were killed 2 after progesterone administration and the POA-MBH dissected out and incubated in a perifusion system. After preincubation, medium with or without naloxone (1 mg/ml) was infused into the perifusion chambers and LHRH, norepinephrine, epinephrine and dopamine release were monitored. Naloxone concurrently released LHRH and the three catecholamines during the entire perifusion period. LHRH and catecholamine output returned to the control range within 30-45 min after cessation of naloxone infusion. These data show that naloxone can promptly stimulate catecholamine release and concur with the view that LH release evoked by naloxone in vivo may be due to hypersecretion of LHRH and they further raise the possibility that norepinephrine and epinephrine released in the vicinity of peptidergic neurons in the POA-MBH may participate in LHRH hypersecretion.

Prolactin Cell Activity in Female and Male Syrian Hamsters: An Apparent Sexually Dimorphic Response to Light Deprivation and Pinealectomy

In order to determine the role of the pineal gland in mediating the effects of long-term light deprivation on prolactin (PRL) cell activity in a highly photosensitive species, PRL synthesis, storage and release were determined in both female and male Syrian hamsters that were either blind, blind and pinealectomized or left intact for 14 weeks. PRL release was determined in vivo by measuring the amount of immunoreactive (RIA) PRL in the serum of the animals in each group with a heterologous RIA for hamster PRL. Binding resulted in a 98 and 88% reduction in serum PRL levels in female and male hamsters, respectively. Pinealectomy largely prevented the suppressive effects of blinding on PRL release; however, PRL levels in blind pinealectomized animals were intermediate between those in intact and blind animals. PRL synthesis was evaluated by assessing the amount of 3H-leucine incorporated into PRL by anterior pituitaries in vitro. In female hamsters 14 weeks of light deprivation resulted in an 87% decrease in the incorporation of 3H-leucine into newly synthesized PRL whereas in males only a 40% reduction occurred. While pinealectomy completely prevented the inhibitory effects of blinding on PRL synthesis in males, it was less effective in female hamsters inasmuch as PRL synthesis was still nearly 50% lower in blind pinealectomized animals than in controls. Stored PRL, as represented by the total amount of RIA-PRL in vitro, was 96% lower in blind female hamsters as compared with intact controls; in blind male hamsters, stored PRL was reduced by 77%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Short Photoperiod on Hypothalamic Methionine-Enkephalin and LHRH Content and Serum β-Endorphin-Like Immunoreactivity (β-end LI) Levels in Golden Hamsters

Adult female golden hamsters were used to study the effect of short photoperiod on the endogenous opioid system and the effect of pinealectomy on the serum β‐endorphin‐iike immunoreactivity (β‐end LI) levels. Hamsters were housed under either long photoperiod (14L:10D) or short photoperiod (2L:22D) and the regularity of the estrous cycles was determined by daily vaginal exfoliative cytology. Hamsters under short photoperiod became acyclic after about 7 wk. At the end of 8 wk, all the hamsters were decapitated and medial basal hypothalamic (MBH) content of LHRH and methionine‐enkephalin (met‐enkephalin) were measured by specific radioimmunoassays (RIA). Both LHRH and met‐enkephalin levels of the MBH were significantly elevated in the short‐photoperiod hamsters as compared to the normally cycling control animals under long photoperiod. In a second experiment, the effect of pinealectomy (PNX) on the serum levels of β‐end LI in the short‐photoperiod hamsters was determined. The serum β‐end LI levels were increased approximately threefold in the noncyclic hamsters housed under 8 wk of short‐photoperiod conditions. Pinealectomized hamsters kept under 8 wk of short‐photoperiod exhibited lower serum β‐end LI levels similar to those of normally cycling hamsters kept under long photoperiod. These results indicate a possible functional relationship between increased pineal activity (as a result of short photoperiod) and increased MBH met‐enkephalin, LHRH, and serum β‐end LI levels.

Photoperiodic Sensitivity of Prepubertal Female Fisher 344 Rats

The laboratory rat is thought to be a poor model for study of the photoperiodic control of reproduction; however, this has only been investigated in a few rat strains. The purpose of the present investigation was to determine if the neuroendocrine‐reproductive system of the Fisher 344 (F344) rat, an inbreed strain, is sensitive to light deprivation. All treatments were performed on 28‐day‐old female F344 rats and the animals maintained for 8 weeks in a 14:10 light: dark cycle. Blinding resulted in a 65% (P < 0.01) reduction in uterine weight and a 25% (P < 0.01) decrease in ovarian weight. Accompanying these reductions in blinded animals were significant inhibitions of anterior pituitary weight, serum prolactin levels, and pituitary prolactin synthesis as measured in vitro. Pinealectomy of the blinded animals prevented all of these effects. Additionally, when olfactory bulbectomy, a procedure known to sensitize rats to the effects of photoperiod, was combined with blinding, the results did not differ significantly from that found with blinding alone. From these data we conclude that 1) the neuroendocrine‐reproductive system of the prepubertal F344 female rat is highly sensitive to light deprivation; 2) light deprivation produces its antigonadotrophic effect through the pineal gland; and 3) olfactory bulbectomy does not potentiate the effects of blinding in the F344 rat.

Diurnal variation in norepinephrine-stimulated release of pineal serotonin in vitro

Adult, male rats were maintained under 12L∶12D with lights on at 06.00 h. Their pineal glands were incubated at 37‡C in the presence or absence of 10−4 M norepinephrine (NE). 5-HT and various metabolites were quantitated in post-incubation media and pineal glands by high performance liquid chromatography coupled with electrochemical detection. No differences were observed in the quantities of 5-HT released by pineal glands in four hour incubations starting at either 06.00, 13.00 or 18.00 h; however, a highly significant decrease below these levels was observed at 01.00 h. NE significantly stimulated 5-HT release at 13.00 and 18.00 h, but was ineffective at 01.00 and 06.00 h. These results confirm recently reported stimulatory effects of NE on the release of 5-HT into pineal gland incubation medium and further suggest a diurnal rhythm of pineal gland sensitivity to NE in vitro with maximum stimulation of 5-HT release at midphotophase.

Evidence for an inhibitory influence of the pineal on prolactin in the female rat.

The effect of the pineal gland on prolactin (PRL) synthesis, storage and release was tested in female rats. To do this, we incubated in vitro hemi-anterior pituitaries from 80-day-old female rats that had been rendered, 8 weeks previously, either blind-anosmic, blind-anosmic and pinealectomized or left intact. Reproductive organ weights, pituitary weights and pituitary DNA content were decreased in animals rendered both blind and anosmic. These effects were reversed by removal of the pineal gland. Additionally, the serum levels of PRL were diminished in blind-anosmic rats from that of intact controls. The synthesis of PRL in vitro was dramatically reduced in the pituitaries of blind-anosmic rats as evidenced by a 63% decrease in [3H]leucine incorporation into PRL from that observed in the intact group. Likewise, the total amount (medium + pituitaries) of radioimmunoassayable PRL in vitro was depressed by 26% in the blind-anosmic group as compared with intact controls. Pinealectomy reversed the reductions in de novo synthesized and total immunoreactive PRL in vitro. From these studies we conclude that in the blind-anosmic female rat the pineal chronically inhibits PRL synthesis and storage in the pituitary and, possibly, its release into the blood. These pineal-induced effects could be accounted for by a reduction in the pituitary mammotroph as might be indicated by the decrease in pituitary DNA content observed in dual-sensory deprived rats.

Nutritional Status, Time of Day and Pinealectomy: Factors Influencing the Sensitivity of the Neuroendocrine-Reproductive Axis of the Rat to Melatonin

Afternoon but not morning injections of melatonin (Mel) resulted in significant 68, 50 and 20% reductions in seminal vesicle, ventral prostate and testicular weights, respectively, as compared with underfed controls. As in the animals injected in the morning, reproductive organ weights in underfed-pinealectomized rats were unaffected by afternoon injections of Mel. Both pituitary and serum prolactin levels were significantly depressed in underfed rats receiving Mel in the afternoon; however, Mel was ineffective when given either in the morning or to pinealectomized rats. These data indicate that there is a diurnal rhythm of sensitivity to Mel in undernourished rats which is abolished by pinealectomy.

Preliminary evidence that a dopamine receptor antagonist blocks the prolactin-inhibitory effects of melatonin in anosmic male rats

Previous studies have shown that daily afternoon injections of melatonin in anosmic male rats result in depressed accessory sex organ weights and serum prolactin levels. The present data indicate that the prolactin-inhibitory effect of melatonin may be mediated via the dopaminergic system.