Michael S. Blank

Antagonist of Gonadotropin-Releasing Hormone Blocks Naloxone-induced Elevations in Serum Luteinizing Hormone

Administration of a gonadotropin-releasing hormone (GnRH) antagonist, [D-Phe, D-Trp3.6]-GnRH, to immature female rats blocks the equivalent elevations in serum luteinizing hormone (LH) which are provoked by exogenous, natural GnRH (8 ng/100 g BW) or naloxone (0.25 mg/100 g BW), a specific opiate antagonist. A significant inhibition of GnRH- or naloxone-induced release of LH is obtained when rats are pretreated for 0, 15, 30 or 60 min with 5,000 ng/100 g BW of GnRH antagonist but no inhibition is evident when the antagonist is injected 180 min before either stimulant of LH secretion. A similar time-course is observed for GnRH antagonist inhibition of basal LH levels. The minimally effective dose of GnRH antagonist for suppressing both GnRH- and naloxone-induced LH release is 1,000 ng/100 g BW. More than 80% of the LH response to GnRH or naloxone is blocked by the highest tested dose (10,000 ng/100 g BW) of GnRH antagonist. Since naloxone has no direct influence on pituitary release of LH, these similar influences of GnRH antagonist on the LH-releasing properties of natural GnRH and naloxone, strongly suggest that the systemic administration of the opiate antagonist, naloxone, stimulates the release of endogenous GnRH.

Suppression by naloxone of water intake induced by deprevation and hypertonic saline in intact and hypophysectomized rats

Abstract Naloxone, an opiate antagonist, was administered to intact and hypophysectomized male rats following hypertonic saline pretreatment or 12 hr water deprivation. Water intake following hypertonic saline or water deprevation was reduced by 0.01 – 10 mg/kg of naloxone in a dose-related fashion in both intact and hypophysectomized rats. Water consumption induced by hypertonic saline administration appeared to be more susceptible to the suppressant effects of naloxone than did that evoked by water deprevation. These results demonstrate that naloxone reduces water intake in the rat following intracellular dehydration by hypertonic saline administration, as well as after general dehydration induced by water deprevation. Furthermore, the suppressant effects of naloxone on water intake do not appear to involve pituitary endorphins, although a possible involvement of antidiuretic hormone in these effects cannot be excluded.

Influence of adrenocorticotropin and adrenalectomy on gonadotropin secretion in immature rats.

We examined the effects and mechanisms of action of ACTH and ACTH fragments on gonadotropin secretion in immature rats. ACTH administered by daily injection or continuous infusion (osmotic minipumps) attenuated the postcastration rise in serum LH. Pituitary LH concentration was either unchanged or increased in ACTH-treated rats and pituitary sensitivity to gonadotropin-releasing hormone (GnRH) was reduced by ACTH treatment. A fragment of ACTH (ACTH 4-10), which is less steroidogenic, did not alter levels of serum LH, and ACTH did not reduce LH secretion in adrenalectomized castrates. Serum and pituitary concentrations of prolactin were normal in ACTH-treated animals. These studies demonstrate that the suppression of gonadotropin secretion by ACTH is mediated by the adrenal gland. This mechanism causes a decreased pituitary sensitivity to GnRH, but LH synthesis does not appear to be affected. Prolactin does not play a role in this mechanism.

Endogenous opioids and hypogonadism in human obesity

Massively obese males often show symptoms of hypogonadism, but the mechanism for this is unclear. Increased endogenous opioid inhibition of the hypothalamic GnRH pulse generator resulting in insufficient stimulation of the pituitary gonadotroph has been proposed as a possible mechanism. If this hypothesis is correct, obese males should be more sensitive to the LH-elevating effects of the opiate antagonist, naloxone, than men of normal weight and gonadal status. This study investigated the etiology of obesity-related hypogonadism by examining luteinizing hormone (LH) and follicle stimulating hormone (FSH) responses to gonadotropin-releasing hormone (GnRH) and to infusions of saline or naloxone. Subjects were five obese (201 +/- 14% IBW) and five normal weight (control) (97 +/- 4% IBW) males. Before treatment, obese males had significantly (p < 0.05) lower testosterone levels than control subjects (307 +/- 72 vs. 597 +/- 49 ng/dl), whereas estradiol, androstenedione, and dehydroepiandrosterone levels were not different between the two groups. Both groups showed equivalent elevations in LH (fourfold to sixfold) in response to GnRH stimulation, but obese patients had significantly lower basal (p < 0.05) and GnRH-stimulated (p < 0.01) FSH levels. Infusions of naloxone (but not saline) led to significant (p < 0.01) increases in LH above preinfusion baseline levels (20.5 +/- 2.8% in obese and 28.6 +/- 6.3% in controls). In control subjects, integrated LH levels during naloxone infusion were not significantly elevated above those found during saline infusion, while obese subjects exhibited a 43% augmentation of integrated LH (31.0 +/- 5.3 ng/ml during naloxone vs. 21.7 +/- 1.8 ng/ml during saline, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone sensitivity to naloxone in maturing male chimpanzees

We have systematically investigated the involvement of endogenous opioids in gonadotropin secretion during primate sexual maturation by examining LH/FSH responses to gonadotropin-releasing hormone (GnRH) and changes in LH secretion during infusions of saline or naloxone, an opiate antagonist, in ten male chimpanzees between one and nine years of age. Animals were anesthetized with ketamine (10 mg/kg) and injected or infused IV with GnRH, naloxone or saline. Circulating levels of serum LH were elevated to the same extent (approximately 400%) in response to GnRH (100 micrograms) in animals 1-5 years old (juvenile) and in animals 6-9 years old (pubertal). No differences were noted between the two groups in GnRH-stimulated levels of serum FSH. During treatment with naloxone (0.14 mg/kg bolus followed by 0.2 mg/kg/h maintenance infusion for 3 h), serum LH levels in pubertal animals were significantly (p less than 0.05) elevated by as much as 95% over LH levels found during treatment with saline. Juvenile animals, on the other hand, failed to demonstrate significant increases in serum LH following naloxone at the doses tested. A strong correlation (r = .84) was found between circulating testosterone and serum LH levels during naloxone treatment. These data indicate that opioid inhibition of LH secretion can be reversed by naloxone only when puberty is reached in chimpanzees and suggest an alteration in opioid regulation of GnRH near the time of puberty. The strong correlation between testosterone levels and LH responses to naloxone suggests that steroids may participate in the maturation of opioid control of LH during puberty of nonhuman primates.

Endocrine changes induced by venipuncture in rhesus monkeys

An animal-worn, remotely activated blood collection device was used to obtain estimates of baseline levels of testosterone (T), growth hormone (GH) and prolactin (PRL) from unrestrained and active male rhesus monkeys. As soon as possible after this baseline period, the males were captured and a final venipuncture sample was obtained. Venipuncture produced elevated GH levels compared to values obtained using the blood collection device, but was without detectable effect upon levels of T and PRL. Cortisol values, available only from a single animal, displayed an increase in response to venipuncture, much like that seen for GH.

Diurnal Influences on Serum Luteinizing Hormone Responses to Opiate Receptor Blockade with Naloxone or to Luteinizing Hormone-Releasing Hormone in the Immature Female Rat

Abstract The major objective of these studies was to determine whether there is a temporal pattern in the gonadotropin response of immature rats to luteinizing hormone-releasing hormone (LHRH) or the opiate antagonist, naloxone. Thirty-day-old female rats were injected at 3-hr intervals over a 24-hr period with either naloxone (2.5 mg/kg body wt) or LHRH (8 ng/100 g body wt). Animals were decapitated 15 min later and serum samples were assayed for luteinizing hormone (LH) by radioimmunoassay. The serum LH response to naloxone and LHRH varied significantly with the time of day. Naloxone administration had no statistically significant (P > 0.05) effect on levels of serum LH at 1500 and 1800 hr compared to levels in saline-injected controls, but induced a significant rise in serum LH at all other times. Naloxone had its greatest effect during the late evening and early morning hours (2100 to 0900 hr). A similar, but not identical, pattern of LH responsiveness to LHRH was observed, with the two rhythms being truly divergent only during the late afternoon when LH sensitivity to LHRH was high but low to naloxone. These data indicate that there is a diurnal pattern of pituitary sensitivity to both naloxone and LHRH in the immature rat and suggest, for the most part, that temporal variations in the LH response to opiate antagonists may result from altered pituitary sensitivity to endogenous LHRH. However, the enhanced response of the pituitary to LHRH during the late afternoon, when opioid inhibition of hypothalamic LHRH secretion appears to be at a nadir could provide a mechanism in the immature rat whereby adult-like LH surges can be stimulated. The early afternoon LH response to various doses of naloxone was examined in intact and ovariectomized 30-day-old rats.

Central, stereoselective receptors mediate the acute effects of opiate antagonists on luteinizing hormone secretion☆

Although a central site of acute opiate action in regulating luteinizing hormone (LH) secretion has been suggested by the ability of centrally implanted opiate antagonists to increase LH levels, opiate antagonists are lipophilic and could influence the pituitary in situ. Also, the physiological significance of opiate receptor blockade with antagonists rests on the assumed, but untested, stereoselectivity of these receptors. Therefore, a lipophobic quaternized derivative of naltrexone (MRZ 2663-Naltrexone methobromide) and dextro- (+) and levo- (-) stereoisomers of naloxone were used to study the site- and stereoselectivity of gonadotropin responses to opiate antagonists in vivo. Male rats were injected intracerebroventricularly (icv) or intravenously (iv) with the quaternary or tertiary congeners of naltrexone and subcutaneously (sc) with (-) or (+)-naloxone. Rats injected icv with 20 ug of quaternary naltrexone displayed significant increases in serum luteinizing hormone (LH). The onset of the response was rapid with serum LH levels being significantly elevated 15 minutes after the injection and returning to basal levels 30 minutes later. Rats injected iv with 10 mg/kg of quaternary naltrexone failed to show significant LH responses. Rats injected either centrally or periphally with equivalent doses of tertiary naltrexone showed LH responses that were similar to those found in animals injected icv with quaternary naltrexone. As little as 0.5 mg/kg of (-)-naloxone resulted in significant elevations in serum LH that were higher than those elicited by up to 10 mg/kg of (+)-naloxone, indicating that this effect of naloxone is stereoselective. These data support the argument that opioids can acutely modulate LH secretion through actions at stereoselective opioid receptors in the central nervous system.

Monoaminergic antagonists which block naloxone-induced release of luteinizing hormone bind selectively to hypothalamic opiate receptors

The possibility that adrenergic receptor antagonists which prevent naloxone-induced release of luteinizing hormone (LH) in vivo exert their action by direct competition with naloxone for hypothalamic opiate receptors was investigated in vitro in immature female rats. First, 26-day-old rats were injected with prazosin, an alpha 1-adrenergic blocker, or yohimbine, an alpha 2-adrenergic blocker, before receiving naloxone (2.5 mg/kg body wt.). Both adrenergic antagonists prevented naloxone-provoked LH secretion in a dose-dependent manner with yohimbine exhibiting a slightly greater potency. In a separate experiment hypothalami from 26-day-old rats were removed, membrane pellets prepared and incubated with [3H]naloxone in the presence of increasing concentrations of naloxone or various monoamine-active substances. Phentolamine, prazosin and yohimbine were the most effective competitors for naloxone binding sites while pronethalol, methysergide and metergoline were far less effective. These findings parallel the relative inhibitory potencies of these compounds in vivo for preventing naloxone-induced LH release as shown here and in a previous report. Clonidine and L-phenylephrine, both alpha-adrenergic agonists, also showed activity in the binding assay. Surprisingly, alpha-methyl-p-tyrosine and 5-hydroxytryptophan, substances which substitute for monoamine precursors early in the biosynthetic pathway, also displaced [3H]naloxone from hypothalamic receptors. These results offer a mechanism for the modulating effects of monoamine-active drugs on opiate antagonist-induced LH release and may have significance for inhibition of LH secretion by endogenous opiates.

Prolactin and Blood Pressure Responses to Perphenazine in Human Subjects: Comparison of the Oral and Intramuscular Routes

Although several phenothiazines are known to stimulate prolactin (PRL) secretion, only chlorpromazine is in general use for this purpose in humans. However, chlorpromazine has severe sedative and hypotensive effects. Therefore, the effects of perphenazine on human PRL release and on blood pressure were evaluated. Perphenazine was administered orally (8mg) and intramuscularly (5mg and 2mg) to determine the optimal route and dose for evaluating PRL release. The postural hypotensive effect of perphenazine was evaluated with the 2mg intramuscular (IM) dose. The mean time of peak PRL response (hr ± SD) was significantly shorter (p<0.05) for the 5mg IM (1.7 ± 0.4) than the oral (4.5 ± 0.6) rout. Also, the mean ratio of peak/baseline PRL was significantly greater for the 5mg IM (8.87 ± 5.69) than the oral (5.12 ± 2.90) route. The major side-effect produced by perphenazine was drowsiness, which was moderate to severe with the 5mg IM dose. A lower IM dose (2 mg) retained PRL releasing activity, reduced drowsiness, and did not produce hypotension. For clinical testing, intramuscular perphenazine is preferred over oral perphenazine because of the shorter latency period and the higher PRL levels. Intramuscular perphenazine (2mg) is preferred to chlorpromazine since it did not produce a clinically significant hypotensive effect. This is the first report on the dynamic responses of PRL and blood pressure to intramuscular perphenazine in humans.