The inhibitory effect of anandamide on luteinizing hormone-releasing hormone secretion is reversed by estrogen

Because Delta-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA (10(-9) M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v. also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v. instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats.     

On the presence of a nondopaminergic, peptidergic prolactin release-inhibiting factor in hypothalamic extracts of infantile rats

Crude hypothalamic extracts prepared from brains of 1-day-old rats produced a dose-dependent inhibition of prolactin (Prl) release by adult male hemipituitaries, and to a lesser extent by hemipituitaries of adult ovariectomized (OVX), estrogen-primed rats. These extracts contained 6-fold lower levels of dopamine than adult hypothalami. The inhibitory effect of the adult hypothalamic extracts, contrary to infantile hypothalamic extracts could be blocked by spiroperidol. Digestion of the infantile hypothalamic extracts with pronase totally abolished their Prl release-inhibiting activity, indicating the peptidic nature of this inhibitory substance. In contrast to their effect on Prl release by hemipituitaries, infantile hypothalamic extracts stimulated Prl release from dispersed anterior pituitary cells of OVX estrogen-primed rats, pointing to the importance of estrogen in modulating prolactin release-inhibiting factor (PIF) activity and the possibility that the PIF receptor is trypsin-sensitive.     

Plasma luteinizing hormone and prolactin levels and hypothalamic catecholamine synthesis in steroid-treated ovariectomized rats

The effects of estrogen (E) and progesterone (P) on synthesis rates and endogenous levels of hypothalamic norepinephrine (NE) and dopamine (DA) were determined in individual ovariectomized (OVX) rats. 3H-tyrosine (3H-T) was injected intra-arterially and the rate of its incorporation into 3H-DA and 3H-NE was determined at 10, 15, 30, and 45 min. Steroid treatment for 2 days effectively decreased plasma LH levels and elevated plasma prolactin (Prl) levels while endogenous levels of NE and DA were not affected. NE synthesis was not affected by steroid treatment. A significant increase in DA synthesis was correlated with low plasma LH levels, suggesting that DA inhibits LH release. The elevated plasma Prl levels seen in steroid-treated rats were correlated with increased DA synthesis in the hypothalamus. This may indicate that elevated plasma Prl levels stimulate dopaminergic neurons in a short-loop negative feedback fashion.     

Morphine and endorphins modulate dopamine turnover in rat median eminence.

The is evidence that some of the actions of both endogenous and exogenous opioids (e.g., stimulation of prolactin release) are mediated by interaction with catecholaminergic systems. Morphine (1.67, 5, and 15 mg/kg of body weight, intraperitoneally) altered dopamine turnover as measured by the alpha-methyl-p-tyrosine method in the median eminence, neostriatum, and frontal cortex of male Sprague-Dawley rats. The turnover rate of dopamine was reduced in the median eminence and frontal cortex but accelerated in the neostriatum. In the frontal cortex all doses were effective in decreasing dopamine turnover; however, in the median eminence the lowest dose of morphine did not significantly alter dopamine turnover. All three doses accelerated dopamine turnover in the neostriatum. Naloxone effectively reversed the effects of morphine at all doses in all brain areas, whereas it had no effect on turnover when given alone. In the median eminence, neostriatum, and frontal cortex, intraventricular injection of [D-Ala2,D-Leu5]-enkephalin (25 micrograms) or beta-endorphin (15 micrograms) produced the same effects on dopamine turnover as morphine. The actions of these peptides were blocked by naloxone. It is hypothesized that opiates and opioid peptides increase prolactin release by reducing the activity of the tuberoinfundibular dopaminergic system.     

Effect of sulpiride-induced hyperprolactinaemia on catecholamine turnover and LRH concentration in the medial basal hypothalamus of rats

In order to clarify the mechanism of hyperprolactinaemic anovulation, the medial basal hypothalamic (MBH) catecholamine (CA) turnover and LRH concentration, and the serum levels and pituitary contents of gonadotrophins and prolactin (Prl) in hyperprolactinaemic female rats were examined. Hyperprolactinaemia (HPrl) was produced by oral administration of sulpiride for 10 consecutive days; each measurement made on the sulpiride-treated rats was compared with that of control dioestrus rats. Prl, LH, FSH and LRH were determined by radioimmunoassay; CA turnover, as assessed by the accumulation of CA following monoamine oxidase inhibition, was assayed by high performance liquid chromatography with electrochemical detection. Sulpiride treatment induced (1) an increase in the serum Prl and a decrease in the serum LH, (2) an increase in the pituitary FSH and LH contents, (3) an increase in the MBH LRH concentration, and (4) an increase in the MBH dopamine (DA) turnover. These results suggest that HPrl may induce anovulation by impaired LH secretion which was caused by the suppression of LRH release due to an increase in DA turnover in the MBH.     

Prolactin secretion from human prolactinomas perifused in vitro: effect of TRH, prostaglandin E1, theophylline, dopamine and dopamine receptor blockers

To clarify the functional characteristics of prolactin (Prl)-producing adenoma cells, the effect of TRH, prostaglandin E1 (PGE1), theophylline, dopamine and dopaminergic antagonists on Prl secretion was examined in vitro in perifused pituitary adenoma tissues obtained at surgery from 8 patients with prolactinoma. Perifusion with TRH at a concentration of 10(-6) to 10(-5) M resulted in a significant increase in effluent Prl levels in 3 of the 8 adenoma tissues. In the remaining 5 adenomas, TRH produced no effect on Prl release in vitro. On the other hand, PGE1 (10(-5) M) stimulated Prl secretion in 2 of the 4 adenomas examined. Addition of theophylline (5.5 mM) caused a marked increase of effluent Prl levels in all 8 prolactinomas regardless of the reactivity to TRH or PGE1. Dopamine (5 X 10(-7) M) suppressed Prl secretion from adenoma tissue in 5 of 7 patients tested but had no effect in the remaining two adenomas. When perifused simultaneously with dopamine, sulpiride (D2-selective dopamine receptor blocker, 5 X 10(-7) M) blocked the inhibitory effect of dopamine on Prl release in 3 of the 4 dopamine-sensitive prolactinomas. In one adenoma responsive to dopamine but resistant to sulpiride, YM-09151-2 (relatively specific D1-dopamine receptor blocker, 5 X 10(-7) M) antagonized the dopaminergic inhibition of Prl release. When perifused alone, neither sulpiride nor YM-09151-2 affected Prl release from any of the adenoma tissues tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyproheptadine-mediated inhibition of growth hormone and prolactin release from pituitary adenoma cells of acromegaly and gigantism in culture

The effect of cyproheptadine on growth hormone (GH) and prolactin (Prl) secretion from cultured pituitary adenoma cells of acromegaly and pituitary gigantism was studied. When varying doses of cyproheptadine ranging from 0.01 to 1 microM were added to the incubation media, GH secretion was consistently inhibited and a dose-response relationship was observed between the cyproheptadine concentrations and the amounts of GH released into the media. In pituitary adenomas which concurrently produced and secreted Prl, cyproheptadine likewise suppressed Prl release in a dose-related manner. This effect of cyproheptadine was not blocked by coincubation with serotonin. Similarly, coincubation with a dopaminergic antagonist, haloperidol, failed to reverse the inhibitory action produced by cyproheptadine. When coincubated with dopamine, cyproheptadine further inhibited GH and Prl secretion. These results suggest that cyproheptadine possesses a direct action on human somatotroph adenoma cells to inhibit GH and Prl secretion by an unknown mechanism that is different from serotonergic and dopaminergic systems.     

Physiological role of alpha-melanocyte-stimulating hormone in modulating the secretion of prolactin and luteinizing hormone in the female rat.

Long-term ovariectomized (OVX) rats were injected in the third cerebral ventricle with 5 microliter of the globulin fraction of an antiserum raised against alpha-melanocyte-stimulating hormone (alpha-MSH) or an equal volume of the globulin fraction of normal rabbit serum (NRS). Immunoneutralization of brain alpha-MSH produced an increase in the area under the secretion curve of prolactin (Prl), the amplitude of Prl pulses, and mean plasma Prl (P less than 0.01). In animals that had received two injections of NRS or anti-MSH and were subjected to a 2-min ether stress, Prl levels significantly increased within 5 minutes in the NRS-injected rats, whereas Prl levels in the antiserum-injected rats did not increase any further from the initially high baseline levels. The administration of antibodies against alpha-MSH produced a small increase (P less than 0.05) in the area under the secretion of luteinizing hormone (LH) and mean plasma LH; however, the number of LH pulses was unaffected. We conclude that endogenous alpha-MSH of central origin is a physiological neuromodulator of release of Prl and LH in the OVX rat and is involved in the stress-induced release of Prl.     

Sex differences in [3H]-estradiol binding in brain and pituitary after acute dopaminergic treatment. In vivo studies in the rat

Responses to estrogen differ between the sexes, yet sex differences in specific binding of estradiol (E2) to its receptor are not observed consistently. Dopaminergic treatment has been shown to increase binding of 3H-E2 in selected brain areas and anterior pituitary in the female rat, and the dopaminergic system is sexually differentiated. In order to determine whether or not dopaminergic stimulation might induce sex differences in 3H-E2 binding, male and female gonadectomized-adrenalectomized rats were pretreated either with bromocriptine, a dopamine agonist, or with diethyldithiocarbamate (DDC), an inhibitor of dopamine beta-hydroxylase. DDC was used in order to increase endogenous release of dopamine. After such acute dopaminergic treatment, specific binding of 3H-E2 in nuclear and extranuclear fractions of 6 brain areas and pituitary in vivo was determined 1 h after intravenous injection of 3H-E2 (1 microgram/kg body weight). Administration of either bromocriptine or DDC increased specific 3H-E2 binding to nuclear and extranuclear fractions of basal hypothalamus and anterior pituitary from female but not from male rats, thus inducing sex differences in binding in these two tissues. Bromocriptine also increased specific binding in the pineal in females. Total binding was increased in a crude membrane fraction (P2) from pituitary of female but not of male rats after administration of DDC, but the percent of extranuclear specific binding found in the P2 fraction was decreased after DDC in both males and females. The findings suggest that dopaminergic stimulation may induce sex differences in 3H-E2 binding by increasing binding in some brain areas and anterior pituitary in females but not in males.     

Regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors by pulsatile GnRH in female rats: effects of estradiol and prolactin

GnRH has been shown to modulate the concentration of its own pituitary receptors (GnRH-R), and changes in GnRH-R during the rat estrous cycle may reflect changes in GnRH secretion. To examine the relationship between GnRH and GnRH-R in female rats, we measured GnRH-R and serum gonadotropin responses to pulsatile GnRH in restrained ovariectomized (OVX) and ovariectomized estradiol-implanted (OVX-E2) rats. In addition, we examined the effects of suppression of serum PRL. Pulsatile injections of GnRH (10-250 ng/pulse) given every 30 min for 24 or 48 h did not increase GnRH-R in OVX or OVX-E2 rats compared to that in saline controls (246 +/- 27 fmol/mg). Bromocriptine treatment (2 mg/day) had no effect on GnRH-R in OVX animals. In contrast, OVX-E2 rats treated with bromocriptine showed significantly increased GnRH-R (500 +/- 43 fmol/mg) in response to GnRH injections. When ovine PRL was administered to bromocriptine-treated OVX-E2 rats, the GnRH induced rise in GnRH-R was abolished. Gonadotropin responses to GnRH were not correlated with changes in GnRH-R. In OVX animals, LH was only elevated in response to 250-ng pulses of GnRH. In OVX-E2 animals, basal LH was increased by all doses of GnRH, and acute responses to 50- and 250-ng pulses were observed. Bromocriptine treatment resulted in increased LH sensitivity to GnRH in OVX rats, but did not further enhance the responses in OVX-E2 animals. We conclude that in female rats, the presence of both E2 and a low serum PRL level is necessary for GnRH to increase GnRH-R, and the interaction of these factors may be involved in the regulation of GnRH-R during the estrous cycle.     

Stimulatory role of substance P on gonadotropin release in ovariectomized rats.

Substance P (SP) has been shown to be present in the hypothalamus and anterior pituitary. To evaluate a possible physiological role of endogenous SP in the control of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release, specific antiserum against SP (anti-SP) was injected intraventricularly (3 microliters into the third ventricle) or intravenously (50 or 200 microliters) into conscious, ovariectomized (OVX) rats. Third ventricular injection of the antiserum induced a significant decrease in both plasma LH and FSH levels when compared to values in control animals injected with normal rabbit serum (p less than 0.01 and p less than 0.025, respectively). The effect was observed within 10 mi and levels remained suppressed for 60 min. In contrast, intravenous injection of large doses of anti-SP had no effect on the release of both hormones. In order to confirm the stimulatory effect of SP itself, synthetic SP was injected intravenously and intraventricularly into estrogen-primed (E-primed), OVX rats. Synthetic SP dramatically stimulated LH release, but not FSH release when injected either intravenously or intraventricularly at doses of 10 and 50 micrograms (p less than 0.001, p less than 0.005 vs. control, respectively). To investigate any direct action of SP on gonadotropin release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats. SP did not affect the release of gonadotropins in vitro. These results indicate that endogenous hypothalamic SP exerts a tonic stimulatory hypothalamic control of basal gonadotropin release in OVX rats.     

Inhibition by prolactin of post-castration rise in LH.

Castration of male and female rats resulted in a marked rise in serum LH. The rise in serum LH was partially or completely prevented by injection of prolactin (Prl), by implantation of a small amount of Prl in the median eminence (ME), by grafting 2 anterior pituitaries (APs) underneath the kidney capsule, or by transplantation of a Prl-secreting pituitary tumor underneath the skin. The larger pituitary tumor transplants secreted more Prl and were more effective in reducing LH release than the smaller tumors which secreted less Prl. Suppression of LH release generally was greater during the earlier than in the later phases of the different treatments. The pituitary LH response to synthetic LH-RH was the same in ovariectomized rats with or without pituitary grafts, and the decrease in hypothalamic LH-RH after orchidectomy was prevented by pituitary grafts. These results indicate that Prl can depress LH release after castration and that these effects are mediated via the hypothalamus.     

Ovariectomy permits progesterone to increase the binding of [3H]spiperone to the anterior pituitary gland in estrogen-primed rats

The binding of the dopamine (DA) D2 antagonist spiperone to DA receptors in the anterior pituitary gland was evaluated in intact and ovariectomized (OVX) estrogen-primed rats in which circulating levels of progesterone (P) were varied. Nembutal (PB; 35 mg/kg, ip) was administered at 1130 h to intact proestrous animals to prevent the LH-stimulated release of ovarian P. Two hours later some of these intact rats were QVX to remove the endogenous ovarian steroids. Both intact and OVX rats were given exogenous P. All rats were killed on the following morning, and adenohypophysial binding of [3H]spiperone was evaluated at a saturating concentration. The binding in intact PB-blocked rats that received P was only 52% of that in PB-blocked rats that were OVX and received P. The binding of [3H]spiperone in PB-treated rats that were OVX and given P did not differ statistically from that in normal estrous rats. A parallel experiment was conducted on other rats that had been OVX 7 days before the sc implantation (on day 0) of Silastic capsules containing estradiol (E2). Two days later (day 2), some of these rats were injected with Nembutal at 1130 h. Two hours later, the E2 capsules of some rats were removed to simulate ovariectomy; Silastic capsules containing P were implanted into both E2-bearing and non-E2-bearing rats. All rats were killed the following morning (day 3), and the density of adenohypophysial DA receptors was assessed. Binding densities were greatest in rats bearing both E2 and P capsules and in rats from which E2 capsules were removed and replaced with P capsules. We conclude that treatment of OVX rats with the sequential combination of E2 and P increases the density of DA receptors in the anterior pituitary gland. However, in intact rats an additional ovarian product prevents the P-induced increase in the density of adenohypophysial DA receptors.     

In vitro effect of leptin on LH release by anterior pituitary glands from female rats at the time of spontaneous and steroid-induced LH surge.

OBJECTIVE: The purpose of this work was to study the direct effect of leptin on LH release by anterior pituitary glands from female rats at the time of spontaneous and steroid-induced LH surge. METHODS: LH responsiveness to leptin by pituitaries from rats killed in the afternoon (1500 h) at different stages of the 4-day estrous cycle (diestrus-1: D1; diestrus-2: D2; proestrus; estrus), ovariectomized (OVX; 15 days post-castration) and ovariectomized steroid-primed (OVX-E(2)/Pg; pretreated with 5 microg estradiol and 1 mg progesterone), was evaluated in vitro. Hemi-adenohypophyses were incubated in the presence of synthetic murine leptin for 3 h. RESULTS: Addition of increasing concentrations of leptin (0.1-100 nmol/l) to the incubation medium of proestrus pituitaries produced a dose-related stimulation of LH release; the maximal increase to 315% of control was obtained with 10 nmol/l leptin. Leptin (10 nmol/l) enhanced LH release at all days of the estrous cycle, the greatest response occurring in proestrus (318%) and the lowest at D1 (123%). In order to evaluate the role of nitric oxide (NO) in the action of leptin on LH release, glands from proestrus rats were incubated in the presence of 10 nmol/l leptin with or without 0.3 mmol/l N(G)-monomethyl-l-arginine (NMMA), a competitive inhibitor of NO synthase (NOS). NMMA completely suppressed the stimulation of LH release induced by leptin. Leptin also stimulated LH release by pituitaries from OVX rats, and treatment with steroid hormones led to a marked increase in the response (OVX: 162% compared with OVX-E(2)/Pg: 263%; P<0.05). For comparative analysis, a similar experimental procedure was carried out using GnRH (10 nmol/l). Leptin acts at the pituitary level in a similar manner as GnRH, although with significantly lower potency. CONCLUSIONS: These results confirm and extend previous reports regarding a direct action of leptin at the pituitary level, stimulating LH release by anterior pituitaries from female rats at the time of spontaneous and steroid-induced LH surge. In the female rat pituitary this leptin action is controlled by gonadal steroids and mediated by NO.     

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine

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.     

Endocannabinoid control of sperm motility: the role of epididymus

Endocannabinoids are endogenous ligands for plasma membrane receptors (CB1 and CB2), belonging to the superfamily of G-protein-coupled receptors. They mimic some of the effects played by D9-tetrahydrocannabinol (THC), the active principle isolated from Cannabis sativa. N-arachidonoylethanolamine (anandamide, AEA) is the main endocannabinoid described to date in the testis and in human seminal plasma. However, the activity of AEA in controlling male reproduction is still poorly understood. In this study we report on physiological activity of endocannabinoids in the male reproductive tract. Using wild type (WT) and CB1 knock out mice (CB1KO) we show that endocannabinoids act in the epididymus. Here, through CB1, they inhibit sperm motility measured as the percentage of motile spermatozoa (SPZ). In particular, while in WT mice, as expected, the percentage of motile SPZ (measured in caput and cauda of epididymus) was significantly lower in the caput as compared with the cauda, in CB1KO mice a strong increase of motile SPZ in the caput was measured.     

Progesterone receptor and dopamine receptors are required in Delta 9-tetrahydrocannabinol modulation of sexual receptivity in female rats

Ovarian steroids, estrogen and progesterone, influence the sensitivity of certain neural processes to cannabinoid treatment by modulation of brain dopaminergic activity. We examined the effects of the active ingredient of cannabis, Delta(9)-tetrahydrocannabinol (THC), on sexual behavior in female rats and its influence on steroid hormone receptors and neurotransmitters in the facilitation of sexual receptivity. Our results revealed that the facilitatory effect of THC was inhibited by antagonists to both progesterone and dopamine D(1) receptors. To test further the idea that progesterone receptors (PR) and/or dopamine receptors (D(1)R) in the hypothalamus are required for THC-facilitated sexual behavior in rodents, antisense and sense oligonucleotides to PR and D(1)R were administered intracerebroventricularly (ICV) into the third cerebral ventricle of ovariectomized, estradiol benzoate-primed rats. Progesterone- and THC-facilitated sexual behavior was inhibited in animals treated with antisense oligonucleotides to PR or to D(1)R. Antagonists to cannabinoid receptor-1 subtype (CB(1)), but not to cannabinoid receptor-2 subtype (CB(2)) inhibited progesterone- and dopamine-facilitated sexual receptivity in female rats. Our studies indicate that THC acts on the CB(1) cannabinoid receptor to initiate a signal transduction response that requires both membrane dopamine and intracellular progesterone receptors for effective induction of sexual behavior.     

Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the N-methyl-D-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: beta-endorphin and gamma-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10(-9)M) on N-methyl-D-aspartic acid-induced LHRH release, but naltrexone, a micro-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on beta-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not beta-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10(-1)M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.     

Vasoactive intestinal peptide (VIP) mediates the effect of estrogens on the dopaminergic tone in the hypothalamic-pituitary axis of ovariectomized (OVX) rats

The role of vasoactive intestinal peptide (VIP) in the regulation of dopamine (DA) concentration in mediobasal hypothalamus (MBH), posterior and anterior pituitary of ovariectomized (OVX) estrogenized rats was studied using passive immunization against VIP with a specific antiserum (a-VIP). Chronic estradiol administration decreased DA concentration in MBH, and in posterior and anterior pituitary, compared to OVX control rats. DA tissue concentration increased following a-VIP administration to control and estrogenized OVX rats. In vitro study of VIP and a-VIP on DA release from MBH in chronically estrogenized OVX rats showed that estrogens decreased DA evoked-release from MBH; a-VIP increased DA evoked-release from MBH of control OVX and estrogenized rats. VIP decreased DA evoked-release from MBH of OVX rats, but had no effect on estrogenized rats. VIP decreased DA tissue concentration in MBH of OVX control but not of estrogenized rats. It is suggested that VIP decreases DA synthesis and release from hypothalamic neurons in female rats, and that VIP partially mediates the inhibitory effect of long-term estrogen administration on DA release from MBH.     

Immunoneutralization of prolactin prevents stimulatory feedback of prolactin on hypothalamic neuroendocrine dopaminergic neurons

We have found that exogenous prolactin (PRL) stimulates all three populations of hypothalamic neuroendocrine dopaminergic neurons. In this study, we investigated the effects of immunoneutralization of endogenous PRL on the activity of these neurons. Injection of 17β-estradiol (E₂) (20 μg subcutaneously) 10 d after ovariectomy induced a proestrus-like increase in PRL in peripheral plasma the following afternoon. At 1000 h the day after E₂ injection, rats received either rabbit antirat PRL antiserum (PRL-AS) (200 μL) or normal rabbit serum (NRS, 200 μL, controls) intraperitoneally. Groups of rats were then decapitated every 2 h from 1100 h to 2100 h. Trunk blood was collected and serum extracted with protein A to remove the PRL-AS/PRL complex, and the remaining free PRL was measured by radio immunoassay. Sites of neuroendocrine dopaminergic nerve terminals, the median eminence (ME), and intermediate and neural lobes of the pituitary gland were excised and stored for determination of dopamine (DA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) concentrations by high-performance liquid chromatography electrochemical detection (EC). In addition, the anterior lobe of the pituitary gland, the locus of DA action, was collected. The concentration of PRL in NRS-treated animals increased by 1500 h, peaked by 1700 h, and returned to low levels by 2100 h. PRL-AS prevented the increase in PRL secretion in response to E₂. The turnover of DA (DOPAC:DA ratio; an index of dopaminergic neuronal activity) in the ME of NRS-treated animals increased at 1500 h and rapidly returned to basal levels. Treatment with PRL-AS prevented the increase in DA turnover in the ME. DA turnover in the intermediate lobe increased coincident with the peak of PRL in serum of NRS-treated rats. PRL-AS administration prevented increased DA turnover in the intermediate lobe. The turnover of DA in the neural lobe increased by 1300 h and decreased steadily through 2100 h. However, administration of PRL-AS minimally suppressed the turnover of DA in the neural lobe. Moreover, administration of PRL-AS attenuated the rise of DA in the anterior lobe associated with the waning phase of the E₂-induced PRL surge. These results clearly indicate that endogenous PRL regulates its own secretion by activating hypothalamic neuroendocrine dopaminergic neurons.