Edward R. Meyer

Characterization and possible opioid modulation of N-methyl-D-aspartic acid induced increases in serum luteinizing hormone levels in the developing male rat

It has been previously reported that the excitatory amino acid, N-methyl-D-aspartic acid (NMDA), elicits prompt increases in serum luteinizing hormone (LH) levels in young male rats. The present studies were carried out to determine whether the effects of NMDA on LH were mediated by the release of LHRH from the hypothalamus. We also examined whether NMDA-sensitive neuronal pathways interacted with the endogenous opioid system regulating LHRH release and the ontogeny of NMDA-evoked increases in serum LH. We found that the age-response curve for NMDA-induced increases in LH was an inverted U; at early ages (10 and 15 days) the amino acid was marginally effective in increasing LH levels, it became maximally effective from post-natal days 20-40 and thereafter rapidly lost its efficacy such that it was virtually inactive in adult animals. Dose-response curves revealed that adult animals were more than 10-fold less sensitive to NMDA than their younger counterparts. Our studies also demonstrated that NMDA increased LH via a direct effect on the hypothalamic release of LHRH since a potent LHRH antagonist competitively inhibited the effects of NMDA. Finally, we observed that morphine competitively inhibited the effects of NMDA on LH release, suggesting a relationship between NMDA-sensitive neuronal pathways and those endogenous opioid-containing systems which are known to regulate LH release.

Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester

The hypothesis that an arginine-nitric oxide (NO) synthase-NO system mediates the morphine abstinence syndrome was tested in adult male rats implanted subcutaneously for 3 days with one morphine (75 mg) pellet followed by naloxone-precipitated withdrawal (0.5 mg/kg). Injection with a NO synthase inhibitor, NG-nitro-L-arginine methyl ester (NAME, 100 mg/kg subcutaneous), shortly before naloxone-induced withdrawal significantly inhibited abstinence signs by 25-80%. Continuous infusion of NAME via subcutaneous osmotic pumps during the development of morphine physical dependence and during naloxone-precipitated withdrawal also inhibited morphine abstinence signs. In addition, treatment with isosorbide dinitrate, a NO donor, induced a quasi morphine-abstinence syndrome (QMAS) that was significantly suppressed by implantation of a morphine pellet 3 days before isosorbide dinitrate treatment. These results indicate that NO mediates part of the expression of the morphine abstinence syndrome.

Gender Differences in the Reinforcing Properties of Morphine

The purpose of the present studies was to examine whether gender differences could be observed in an important aspect of morphines pharmacology: its reinforcing properties. Our results showed that morphine served as a positive reinforcing agent in both male and female rats in a place conditioning paradigm, but that the dose-response curves displayed marked sex-related differences. At doses from 0.2 up to 10.0 mg/kg, morphine induced an equally strong preference for the drug-associated chamber in males and females. However, as the dose was increased from 10-17.5 mg/kg, morphine ceased to act as a positive reinforcer in males. In contrast, a very strong preference for the morphine-associated chamber was still observed in females at doses up to 30 mg/kg. No gender differences in the blood and brain levels of morphine were observed subsequent to morphine administration during the conditioning phase, suggesting that pharmacokinetic factors were not involved in the sex-related differences observed. Consequently, these results suggest that there are intrinsic sex-linked differences in the doses of morphine that can induce a preference for the drug-associated chamber in a place-conditioning paradigm that are most likely related to differences in the sensitivity of the central nervous system to morphines reinforcing properties in males and females.

Morphine exerts testosterone-like effects in the hypothalamus of the castrated male rat

Previous research has indicated that endogenous opioids participate in the regulation of activity in the hypothalamic-pituitary-luteinizing hormone (LH) axis and mediate the negative feedback control exerted by testosterone. If this assumption is correct, then two predictions can be made. First, the effects of testosterone should be competitively inhibited by narcotic antagonists; and, second, opiates should mimic the acute and chronic effects of testosterone in the castrated male rat. The results of the present investigations support both of these predictions. We found that naloxone competitively antagonized the depressive effects of testosterone on serum LH in the castrated rat and, conversely, that testosterone competitively antagonized the LH-releasing properties of naloxone. In addition, morphine and testosterone both depressed serum LH levels in a dose-dependent fashion in the acutely castrated animal. Moreover, morphine was just as effective as testosterone in reversing the castration-induced fall in hypothalamic-LH-releasing hormone (LH-RH), which occurs in the chronically castrated male rat. On the other hand, morphine failed to reverse the long-term changes in pituitary LH content and increase in serum LH, which is consistent with prior observations that morphine affects only the hypothalamic aspect of the hypothalamic-pituitary-LH axis in the male rat. These results, thus, support the concept that an as yet unidentified opioid-containing neuronal system regulates activity in the hypothalamic-pituitary-LH axis and mediates the effects of testosterone on this axis.

Gender-linked differences in the expression of physical dependence in the rat.

In earlier studies, it was shown that there were gender differences in several aspects of the pharmacological profile of morphine, including its antinociceptive activity, discriminative stimulus properties and its reinforcing effects. The purpose of the present studies was to examine whether there might also be gender-related differences in the development of physical dependence, as reflected in the expression of an opiate withdrawal syndrome upon cessation of chronic morphine administration. We found that a more severe spontaneous withdrawal syndrome was produced by chronic morphine injections or morphine pellet implantation in male rats than in females. The duration of the withdrawal syndrome was also longer. In contrast to our observations with spontaneous withdrawal, we found no gender differences in the naloxone-precipitated withdrawal syndrome induced by chronic morphine administration. These observations suggest that there are gender differences only in the expression of the spontaneous withdrawal syndrome, but not in the neuro-adaptive changes associated with the generation of physical dependence as reflected by naloxone-precipitated withdrawal.

Testosterone does not influence opiate binding sites in the male rat brain

It has been reported previously that castration produces testosterone-reversible increases in the density of 3H-naltrexone binding sites in the male rat brain. Unfortunately, we were unable to replicate these observations in a comprehensive series of studies. Specifically, we found that castration failed to produce changes in the Kd or Bmax of opiate binding sites in whole male rat brain, or in the hypothalamus, utilizing 3H-dihydromorphine (a mu receptor ligand), 3H-D-alanine, D-leucine enkephalin (delta) or 3H-naltrexone (ubiquitous). Furthermore, we found that the relative proportion of mu and delta binding sites in brain was unchanged by castration. The reasons for the discrepancy between the present results and those previously reported are unclear, but it appears that the provocative hypothesis that testosterone influences opioid receptors in brain must be carefully reevaluated.

Luteinizing hormone releasing hormone mediates naloxone's effects on serum luteinizing hormone levels in normal and morphine-sensitized male rats

Naloxone produces large increases in serum luteinizing hormone (LH) levels in normal males and females, supporting a role for endogenous opioids (EOP) in the tonic inhibition of LH. Since the antagonist apparently exerts no important effects on the pituitary, the reasonable assumption has been made that it elevates gonadotropin levels by affecting the release of LH-releasing hormone (LHRH) from the hypothalamus. However, at present there is no direct in vivo evidence supporting this widely-held view. In an attempt to directly demonstrate that naloxone increases the secretion of LHRH, and thereby elevates serum LH levels, we examined whether a potent synthetic antagonist of LHRH ( [D-p Glu1, D-Phe2, D-Trp3,6]-LHRH, GPT-LHRH) blocked the effects of naloxone in male rats with a normal response to naloxone and in those with a markedly enhanced sensitivity to the drug induced by a brief period of morphine pellet implantation. Our results demonstrated that GT-LHRH antagonized equipotent doses of LHRH (100 ng/kg) and naloxone (0.5 mg/kg) over a similar time course with approximately the same AD50. Most importantly, however, we showed that the GPT-LHRH produced equivalent, parallel shifts to the right in the dose-response curves for LHRH and naloxone, indicative of competitive inhibition. We also found that GPT-LHRH completely abolished the enhanced response to naloxones effects on LH which occurs in morphine-pretreated rats. Since we observed no competition between LHRH and naloxone for their binding sites in pituitary or brain, the only viable interpretation of our results is that naloxone increases LH by inducing the release of LHRH.

Chronic opiate exposure in the male rat adversely affects fertility

We examined whether morphine administration to adult male rats adversely affected pregnancy outcome after mating with drug-naive females and at what point in the complex series of steps leading to viable offspring it exerted its actions. The results indicate that chronic paternal morphine exposure markedly influenced fertility measures in a number of important ways. There was a pronounced increase in pseudopregnancies in females mated with males treated chronically with morphine (40%) when compared to controls (<6%), indicating that vaginal penetration occurred, but successful impregnation failed; only 33% of matings between drug-naive females and morphine-treated males resulted in pregnancies, as compared to 74.5% in controls. In addition, there were fewer implantation sites in gravid females mated with morphine-treated males than in controls. Taken together, these observations suggest that morphine-exposed male rats were apparently able to copulate, but there was a failure in successful impregnation of the females. These findings suggest a primary defect in either the quality of male sexual behavior or a complete failure of the fertilization or conception processes in females mated with morphine-exposed males. This potentially important effect of paternal morphine administration on conception and/or preimplementation loss of embryos has not been previously noted and deserves more systematic study.

Ethanol-induced inhibitions of testicular steroidogenesis in the male rat: Mechanisms of actions

Abstract Ethanol markedly inhibits the biosynthesis of testosterone in the male of several species. Since several in vitro studies have suggested that ethanol per se is not a gonadal toxin and that it must be metabolized to exert its effects, we examined this possibility under in vivo conditions in the present studies. We found that the administration of the alcohol dehydrogenase inhibitor, pyrazole, to adult male rats significantly elevated blood ethanol levels. However, rather than resulting in a potentiation of the effects of ethanol on testicular steroidogenesis, pyrazole-induced elevations in blood ethanol concentrations produced a significant attenuation of ethanols effects. In view of these observations, it is difficult to maintain that ethanol itself is responsible for inhibiting the production of testosterone. On the contrary, our results may provide the first in vivo support for the hypothesis that ethanol must be metabolized to exert its effects on testicular steroidogenesis.

Effects of phenoxybenzamine on the narcotic withdrawal syndrome in the rat

Abstract The effects of various α- and β-adrenergic blocking agents on the narcotic withdrawal syndrome in rats were determined. α-Adrenergic blockers caused a dose-dependent suppression of two behavioural responses characteristic of precipitated narcotic withdrawal in the rat: diarrhoea and, most notably, wet-dog shakes. The β-adrenergic blocker, propranolol, did not effect the expression of the narcotic withdrawal syndrome. The effects of α-blockers on withdrawal behaviour did not appear to be due to the slight degree of sedation produced by the highest doses of the drugs, because pentobarbital and promethazine, in doses sufficient to induce marked sedation and anaesthesia, did not decrease the severity of withdrawal. In fact, pentobarbital appeared to exacerbate the abstinence syndrome.