Delayed suppression of serum luteinizing hormone after naloxone treatment in neonatal female rats

In female neonatal rats, opiate receptor blockade markedly raises serum luteinizing hormone (LH) levels. The LH effect of acute treatment with opiate antagonists is apparently brief in older rats; however, age-related differences in antagonist pharmacokinetics may result in different LH response patterns. The duration of LH response to naloxone (NAL) and naltrexone (NTX) was examined in 5 day-old (d.o.) female rats and compared to the duration of analgesia blockade. The rise in serum LH following opiate receptor blockade in 5 d.o. rats was of similar duration to that previously observed in older animals and much briefer than blockade of analgesia. Furthermore, neonatal rats exhibited a delayed suppression of LH 6 hr following NAL, but not NTX, treatment. Stimulation and later suppression of LH were still observed after five repetitive NAL treatments at 6 hr intervals.     

Evidence that opiate receptors mediate suppression of hypertonic saline-induced drinking in the mouse by narcotic antagonists

The effects of naloxone, its $\text{dextro}$-stereisomer, and five other narcotic antagonists were determined on water intake induced by intracellular dehydration in the mouse. The intraperitoneal administration of a 2M sodium chloride solution served as the model for intracellular dehydration. $\text{1}$-Naloxone (0.01-10 mg/kg) reduced drinking in a dose-dependent fashion with an ED₅₀ of 0.55 mg/kg. In contrast, $\text{d}$-naloxone failed to suppress water consumption at doses up to 10 mg/kg. The other narcotic antagonists tested --- naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine --- also produced dose-dependent decreases in water consumption. The order of potency of these narcotic antagonists in suppressing water intake was highly correlated with their orders of potency in other procedures involving the opiate receptor. The stereoselectivity and order of potency suggest that the suppressant effects of the narcotic antagonists on drinking induced by hypertonic saline administration in the mouse are mediated through an opiate receptor-dependent mechanism.     

Narcotic antagonist-induced hypotension in the spontaneously hypertensive rat

Intravenous naloxone or naltrexone produced transient, dose-related reductions in the mean arterial pressure (MAP) and heart rate (HR) of urethane-anesthetized spontaneously hypertensive rats (SHRs). Yet these same doses of narcotic antagonists reduced HR but not MAP of normotensive Wistar-Kyoto rats (WKYs). Such effects were not observed upon administration to SHRs of increasing doses of methylnaltrexone, which possesses no central activity. (+)-Naloxone, which does not block opiate receptors, reduced HR but not MAP of both SHRs and WKYs. These findings indicate that SHRs and WKYs differ in their MAP and HR responses to narcotic antagonists. The high doses required for effect plus the brevity of the responses suggest that these drug effects are perhaps not mu-opiate receptor-mediated; however, the methylnaltrexone and (+)-naloxone findings clearly implicate a central specificity of action. We conclude that narcotic antagonist-induced changes in MAP and HR in SHRs are possibly specific and central in origin yet not mediated by mu-opiate receptors.     

Quaternary narcotic antagonists' relative ability to prevent antinociception and gastrointestinal transit inhibition in morphine-treated rats as an index of peripheral selectivity

Single doses of naloxone (0.025 to 0.5 mg/kg) or of one of four quaternary narcotic antagonists (i.e. nalorphine allobromide, nalorphine methobromide, naloxone methobromide or naltrexone methobromide, 1 to 60 mg/kg) were given s.c. to rats before morphine, 5 mg/kg i.v. In the absence of antagonists morphine reduced G.I. transit of a charcoal meal to about 15% of drug-free controls and consistently delayed nociceptive reactions (55°C hot plate) in all animals. Doses of antagonists slightly reducing morphine antinociception (centrally effective = A) and restoring G.I. transit to about 50% of drug-free rats (peripherally effective = B) were estimated. The A:B ratio, indicating peripheral selectivity, was at least 8 for any of the quaternary antagonists given 10 min before morphine, but prolonging this interval may have resulted in a lower figure (i.e. less peripheral selectivity) because of reduced A and increased B. This was definitely so for naltrexone methobromide (A:B, > 60 at 10 min, about 1 at 80 min) and was not apparent for nalorphine methobromide according to available data, which for nalorphine allobromide and to a lesser extent for naloxone methobromide showed only an increase in B at intervals longer than 10 min. Both morphine-induced antinociception and inhibition of G.I. transit were reduced by naloxone at the lower doses tested and were fully prevented at the higher. These findings indicate that, unlike naloxone, the investigated quaternary narcotic antagonists are interesting prototype drugs for selective blockade of opiate receptors outside the CNS, although certain critical aspects, possibly biological N-dealkylation to the corresponding tertiary antagonists, condition peripheral selectivity.     

Effects of opioid antagonists and their quaternary analogs on temperature changes in morphine-dependent rats

Subcutaneous administration of three opioid antagonists; naloxone, naltrexone and nalmefene, produced a significant rise in tail skin temperature and a subsequent fall in rectal temperature in morphine dependent rats. However, subcutaneous administration of equimolar concentrations of the quaternary derivatives of these opioid antagonists (naloxone methobromide, naltrexone methobromide and n-methylnalmefenium iodide) failed to produce any significant alterations in either tail skin or rectal temperatures in the morphine dependent rat. At doses of naloxone methobromide 6 to 9 times greater than naloxone, there was a slight reduction of rectal temperatures with no significant elevation of skin temperature. However, the fall in rectal temperature was still significantly less than that achieved with administration of naloxone. When each of these six agents were administered centrally (20 micrograms/5 microliter, icv) in the morphine dependent rat, similar increases in tail skin temperature and decreases in rectal temperature were observed. These temperature changes were similar to those observed following systemic administration of the opioid antagonist. Previously, we have suggested that acute withdrawal in the morphine-dependent rat may serve as an animal model for the mechanism of the menopausal hot flush. Collectively, these results suggest that the temperature changes associated with morphine-withdrawal in our rat model for studying the mechanisms of the menopausal hot flush are centrally mediated.     

3-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals

A fluoro-analogue of the potent narcotic antagonist, naltrexone, was synthesized and shown to bind with high affinity to opiate receptors in vitro. 3-[18F]acetylcyclofoxy was prepared via a one-step triflate displacement reaction with the positron emitting 18F ion from tetraethylammonium [18F] fluoride. 3-[18F]acetylcyclofoxy accumulation in opiate receptor rich brain regions of both rat and baboon is shown to be completely displaced by the active enantiomer of naloxone [-)-naloxone) while the identical dose of the pharmacologically inert (+)-naloxone has no detectable effect. Moreover, both rat and baboon brain showed the well documented, typical opiate receptor distribution so that basal ganglia and thalamus are clearly visible in the living baboon brain up to 95 min after intravenous injection of 3-[18F] acetylcyclofoxy. We expect that 3-[18F )acetylcyclofoxy will be a useful probe for visualizing opiate receptors in living humans.     

Naloxone administration to female hamsters advances puberty by enhancing luteinizing hormone release

In the female hamster, a daily rhythm of gonadotropin release begins almost 3 weeks prior to the initiation of 4-day estrous cycles. A temporal relationship exists between the onset of this cyclic release of gonadotropin and age at puberty. We hypothesized that since opiate agonists depress circulating gonadotropins and antagonists increase them in both adult and immature rodents, endogenous opiates may influence the mechanism controlling cyclical gonadotropin release in the prepubertal female hamster and thus affect rate of sexual maturation and hence the age at puberty. This proposal was tested by chronic administration of naloxone (NAL), an opiate receptor antagonist. We predicted that NAL might induce the early initiation of daily surges of luteinizing hormone (LH) if endogenous opiates inhibit sexual maturation. Naloxone was injected daily (50 mg/kg body wt) at about 1300 hr from Days 1 through 17 of age. The NAL injections increased serum LH and significantly advanced the age at which first estrus vaginal discharge was observed (32 vs 38 days for saline-injected controls in Experiment I and 31 vs 37 days in Experiment II). However, the NAL injections did not correspondingly advance the age of initiation of endogenously generated daily cycles of circulating LH. We conclude that blockade of opiate receptors accelerates sexual maturation by directly inducing the release of LH and not by advancing the age of initiation of endogenous gonadotropin surges.     

Effects of naloxone and naltrexone on receptive and proceptive behaviors in female rats.

The sexual receptive and proceptive behaviors induced by opiate antagonists, naloxone and naltrexone in estrogen-primed ovariectomized rats were observed under the presence of sexually active males. The females were treated intraperitoneally with naloxone or naltrexone at doses ranging from 0.5 to 4.0 mg/kg and the sexual behavior of females was tested before and after the injection of drug. The results obtained suggest that the opiate antagonists play a role in the regulation of lordosis behavior, but not proceptive behavior in female rats.     

Enhancement of opiate-induced depressions in serum luteinizing hormone levels by the prior administration of naloxone in the male rat

The effects of naloxone pretreatment on opiate agonist-induced depressions in serum luteinizing hormone (LH) levels were examined in male rats. Our results demonstrated a pronounced enhancement of morphine's actions 6 hours after the administration of naloxone (0.5 mg/kg). This effect was characterized by a 10 fold reduction in the ED50 (1.26 mg/kg versus 0.13 mg/kg in saline- and naloxone-pretreated rats, respectively) and much greater depressions in serum LH levels at each dose of morphine. The actions of naloxone were not confined to morphine, since similar increased potencies were found for opioid agonists with selectivity for a variety of opioid receptor subtypes. Because naloxone did not alter the uptake of subsequently administered morphine into brain, our results cannot be explained on the basis of an increased availability of the agonist. Rather, it appears that naloxone pretreatment induces a change in the sensitivity of those receptors involved in the effects of opioid agonists on LH.     

Failure of naloxone to stimulate luteinizing hormone secretion during pregnancy and steroid treatment of ovariectomized beef cows

The response of serum luteinizing hormone (LH) to naloxone, an opiate antagonist, and gonadotropin-releasing hormone (GnRH) was measured in cows in late pregnancy to assess opioid inhibition of LH. Blood samples were collected at 15-min intervals for 7 h. In a Latin Square arrangement, each cow (n = 6) received naloxone (0, 0.5, and 1.0 mg/kg BW, i.v.; 2 cows each) at Hour 2 on 3 consecutive days (9 +/- 2 days prepartum). GnRH (7 ng/kg body weight, i.v.) was administered at Hour 5 to all cows on each day. Mean serum LH concentrations (x +/- SE) before naloxone injection were similar (0.4 +/- 0.1 ng/ml), with no serum LH pulses observed during the experiment. Mean serum LH concentrations post-naloxone were similar (0.4 +/- 0.1 ng/ml) to concentrations pre-naloxone. Mean serum LH concentrations increased (p less than 0.05) following GnRH administration (7 ng/kg) and did not differ among cows receiving different dosages of naloxone (0 mg/kg, 1.44 +/- 0.20; 0.5 mg/kg, 1.0 +/- 0.1; 1.0 mg/kg, 0.9 +/- 0.1 ng/ml). In Experiment 2, LH response to naloxone and GnRH was measured in 12 ovariectomized cows on Day 19 of estrogen and progesterone treatment (5 micrograms/kg BW estrogen: 0.2 mg/kg BW progesterone) and on Days 7 and 14 after steroid treatment. On Day 19, naloxone failed to increase serum LH concentrations (Pre: 0.4 +/- 0.1; Post: 0.4 +/- 0.1 ng/ml) after 0, 0.5, or 1.0 mg/kg BW.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced ability of naloxone to stimulate LH and testosterone release in aging male rats; possible relation to increase in hypothalamic met5-enkephalin

Blood luteinizing hormone (LH) and testosterone levels are lower in old than in young male rats. The specific opiate antagonist, naloxone, previously shown to increase serum LH in mature male rats, exhibited relatively little ability to raise serum LH and testosterone levels in old (18–20 mo) as compared to young (4–5 mo) male rats. The brain opiate, met⁵-enkephalin, which depresses LH, was found to be significantly higher in the hypothalamus of old than of young male rats. These observations suggest that hypothalamic opiates may be partially responsible for the lower serum LH and testosterone levels in old male rats, and for reduced release of these hormones in response to naloxone administration.     

Endogenous opioids and opiate antagonists modulate the blood pressure of the spontaneously hypertensive rat

Endogenous opioids have been implicated as modulators of the central nervous system regulation of blood pressure and heart rate. Whether these neuropeptides participate in blood pressure regulation in hypertension is unknown. To begin to study this question, we examined the response to opiate antagonists and agonists in the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY) rat. The long-acting opiate antagonist naltrexone, 2.5 micrograms/kg, was injected into the lateral ventricle of the brain in awake, freely-moving SHR and produced a significant 19 mmHg decrease in mean arterial blood pressure compared to basal blood pressure (p less than 0.01); a decrease was not observed at a two logarithm lower dose. In contrast, naltrexone had no effect on the blood pressure of normotensive Wistar-Kyoto (WKY) rats. To evaluate a possible regulatory role for the predominantly kappa receptor active opioids, alpha- and beta-neo-endorphin, 10 micrograms each, was administered to SHR on separate days by intracerebroventricular injection. alpha- and beta-neo-endorphin caused significant decreases in mean arterial blood pressure of 11 and 9 mmHg respectively, effects reversed by pre-treatment with the opiate antagonist, naloxone. Heart rate was unaffected by any of the injected opioids or antagonists. Our naltrexone results support the hypothesis that an endogenous opioid(s) contributes to the hypertensive state of the SHR. Additionally, alpha- and beta-neo-endorphin can lower blood pressure in this model.     

Functional opioid pathways are necessary for hypocretin-1 (orexin-A)-induced feeding

We investigated the interaction of the orexigenic neuropeptide, hypocretin-1 (Hcrt-1, also known as orexin-A), with endogenous opioids (also orexigenic neuropeptides). Rats were injected with naltrexone (NTX, nonspecific opioid antagonist) i.p., i.c.v., in the lateral hypothalamus (LH), and in the accumbens shell (AcbSh), and naloxone methiodide (nonspecific opioid antagonist unable to cross the blood brain barrier) was injected i.p. Rats were then injected with Hcrt-1 in the LH. Food intake was measured for up to 4h thereafter. Rats were also pretreated with NTX in the LH, with Hcrt-1 injected in the AcbSh. NTX suppressed Hcrt-1-induced feeding only when injected i.p., i.c.v., and in the AcbSh. These studies reveal the necessity for functional central opioidergic pathways involving the AcbSh, but not the LH in Hcrt-1-induced feeding.     

Unexpected effects of nalmefene, a new opiate antagonist, on the hypothalamic-pituitary-gonadal axis in the male rat

In order to gain additional information on the role of brain opioid peptides in the regulation of the hypothalamic-pituitary-gonadal axis, we studied the effects of nalmefene, a new opiate antagonist, on gonadotropin and testosterone secretion in male rats. The results were compared with those obtained with naloxone, a well-studied antagonist. Acute injections of either nalmefene or naloxone (2 mg/kg) produced 4-fold increases in LH and testosterone secretion. In castrated male rats treated with testosterone propionate (TP), nalmefene (10 mg/kg) reversed the androgen negative feedback on LH secretion; surprisingly, when higher doses (25 and 50 mg/kg) were injected, the compound lost its ability to antagonize the testosterone-induced inhibition of LH levels. In contrast, naloxone was able to increase LH levels in TP-treated castrated rats even at the highest dose tested (50 mg/kg). Chronic administration of these antagonists resulted in suppression of the acute release of LH and T secretion in nalmefene-treated but not in naloxone-injected animals. These data are consistent with previous observations suggesting that opioid peptides a) exert a tonic inhibitory effect on LH and testosterone production and b) participate in the negative androgen-induced feedback control of LH secretion. Our results also show that the antagonistic action of nalmefene, but not naloxone, is reversed when higher doses are used or following chronic administration.     

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.     

Narcotic antagonists attenuate drinking induced by water deprivation in a primate

Pure narcotic antagonists such as naloxone and naltrexone have consistently been shown to attenuate drinking in the rat after periods of water deprivation. One objective of this study was to extend observations to a primate species, the squirrel monkey. Whereas naloxone and naltrexone have a greater relative affinity for opiate receptors preferentially binding morphine and other opiate alkaloids than for those with high affinity for the endogenous opioid peptides, diprenorphine, another pure opiate antagonist, binds with equally high affinity to both receptor subtypes. Therefore, a second objective was to determine the actions of diprenorphine on drinking in water-deprived rats and squirrel monkeys and to compare the effects of this drug to those of naloxone and naltrexone. All three narcotic antagonists suppressed water consumption of monkeys and rats deprived of water for 18 and 24 hr, respectively. Diprenorphine was the most potent compound tested in both species, producing significant reductions in water consumption of monkeys and rats at systemic doses as low as 0.01 and 0.1 mg/kg respectively. Moreover, diprenorphine was the longest acting of the three drugs in the monkey. These results demonstrate that the narcotic antagonists attenuate drinking in primates as well as in rodents and support the hypothesis that these drugs reduce water intake by interrupting the activity of endogenous opioid pathways mediating drinking behavior.     

Gonadal steroid modulation of brain opioid systems

It is becoming increasingly clear that the effects of the opioids and their synthetic analogs on anterior pituitary function largely depend on the steroid milieu present in the animal at time of drug administration. However, it is still unclear whether gonadal steroids regulate the opioid-modulated mechanisms by affecting the number of opiate receptors in the brain. To further investigate these issues, the effects of opiate agonists and antagonists on LH, FSH and prolactin (Prl) secretion have been studied in: (a) normal and castrated male rats, and (b) normally cycling female rats. The binding characteristics of the brain subclass of mu opiate receptors have been analyzed in the same group of experimental animals; this type of receptors seems to be particularly involved in the control of gonadotropin and Prl release. When injected intraventricularly into normal male rats, morphine (200 micrograms/rat) induced in a significant elevation of serum LH levels at 10 and 20 min. In long-term castrated animals the administration of the drug significantly reduced LH secretion at 40 and 60 min after the injection, the inhibition lasted up to 180 min. Morphine, when given intraventricularly to normal males, induced a conspicuous and significant elevation of serum Prl levels at 10, 20, 40 and 60 min after treatment. However, when the drug was administered to castrated rats, it did not significantly affect Prl release at any time interval considered. Morphine intraventricular injections did not modify serum FSH levels either in normal or in castrated male rats. The concentration of mu opiate receptors was found to be similar when measured in the whole brain of normal and orchidectomized rats. In adult cycling female rats, s.c. injections of naloxone (2.5 mg/kg) stimulated LH release in every phase of the estrous cycle; the magnitude of the responses was highly variable, being particularly elevated at 16.00 h of the day of proestrous and at 10.00, 12.00 and 14.00 h of the day of estrous. Conversely, LH response to naloxone was totally obliterated at 18.00 and 20.00 h of the day of proestrous, when the preovulatory LH surge was found to occur. The concentration of brain opiate receptors of the mu type showed significant variations during the different phases of the estrous cycle, with higher levels at 12.00 h of the day of proestrous and at 18.00 h of the day of estrous.(ABSTRACT TRUNCATED AT 400 WORDS)     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Opiate antagonist treatment reinstates estrous cycles in middle-aged persistent-estrous rats

Middle-aged female rats display luteinizing hormone (LH) surge deficits and cycle irregularity followed by the onset of persistent estrus (PE). The central nervous system has been identified as a primary locus of failure in PE rats, but the particular neural elements involved have not been determined. The goal of the present study was to identify a role for endogenous opioid peptides (EOP) in age-related acyclicity by evaluating the effect of opiate antagonist treatment on vaginal cytology in PE rats. PE rats were administered, s.c., saline (SAL), naloxone (NAL) or naltrexone (NTX) once daily for 20 days, or repetitively on Day 1 and on successive proestrus days if cyclicity was resumed. Single NTX (50 mg/kg), but not NAL (2 mg/kg), treatment interrupted the PE state in almost half of treated animals. Daily or repetitive proestrus NTX (10 mg/kg) treatment interrupted PE more frequently, and many animals displayed repeated estrous cycles and ovulation. Afternoon LH surges were observed after initial NTX treatment in animals displaying PE interruption. This demonstration that LH surges and ovulatory cycles can be reinitiated in PE rats with NTX suggests that dysfunction in the 'brake' on EOP secretion during proestrus may be one of the neuroendocrine impairments mediating acyclicity in aging female rats.     

Effects of narcotic antagonists on L-dopa reversal of reserpine-induced catalepsy and blepharoptosis in mice

Reserpine (1 mg/kg, i.p.) induced catalepsy and blepharoptosis in mice which were readily reversed by the administration of L-dopa (300 mg/kg, i.p.). The administration of the pure narcotic antagonists naloxone (10 mg/kg, i.p.) and naltrexone (1 mg/kg, i.p.) significantly potentiated L-dopa reversal of reserpine-induced catalepsy. Lower doses of the narcotic antagonists did not significantly alter this reversal. The L-dopa reversal of blepharoptosis was not significantly altered by either naloxone or naltrexone. These results indicate that while opiate receptors may be involved in L-dopa reversal of catalepsy, they may not have a role in the alteration of blepharoptosis.