Exogenous and endogenous opioid-induced enhancements of naloxone's effects on serum luteinizing hormone levels in the male rat.

We have previously shown that brief periods of exposure to opiate alkaloid drugs markedly enhance the subsequent effects of the opiate antagonist, naloxone, on serum luteinizing hormone (LH) levels in the male rat. In the present studies, we have found that this phenomenon is not simply a property of opiate drugs, but can be produced by a metabolically stable analog of an endogenously occurring opioid peptide, methionine enkephalin (FK 33-824). These findings suggest that alterations in the sensitivity of those opioid receptors involved in LH-releasing hormone (LHRH) generated in our experimental paradigm may occur under in vivo conditions, particularly since it now appears that endogenous opioids are released in an episodic manner like most neurotransmitter/neuromodulators. We also attempted to more fully characterize the factors responsible for the development of opiate-induced enhancements of naloxone's effects on LH. We found that this effect was produced only by those doses of morphine which initially suppressed serum LH levels, followed by a "rebound" increase in the gonadotropin 6-8 h later. A modest facilitation of LHRH-evoked increases in serum LH was also observed, but our data suggest that this represents only a minor component of opiate-induced enhancements of naloxone's effects. These data indicate that hypothalamic or suprahypothalamic sites are the major loci involved, but no differences in the uptake or regional distribution of naloxone in brain have been previously found, as a function of morphine pretreatment, nor were we able to demonstrate any alterations in opiate binding sites in the hypothalamus or whole brain. Thus, the mechanisms involved in this effect remain unclear.

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.

Development of acute tolerance to the effects of naloxone on the hypothalamic-pituitary-luteinizing hormone axis in the male rat.

Although the development of acute and chronic tolerance to opiate agonists, mixed agonists-antagonists and endogenous opioid peptides is well established, tolerance to naloxone, a "pure" opiate antagonist, has never been reported. In the present studies, the acute development of tolerance to the effects of naloxone on the hypothalamic-pituitary-luteinizing hormone (LH) axis was demonstrated in the male rat. Naloxone causes rapid, dose-dependent increases in serum LH levels in the male, apparently by blocking the actions of an endogenously occurring opioid peptide which normally inhibits the release of LH. We found that naloxone (2 mg/kg) pretreatment markedly reduced naloxone-induced increases in serum LH after a second identical injection if the interval between the first and second injections was 2 to 4 hr. Serum levels had returned to control levels long before (approximately 60 min) the second injection. Normal naloxone-induced increases in serum LH were obtained if the interval between the first and second injections was 6 hr or more. This diminished LH-response to a second naloxone injection satisfied the two pharmacological criteria for the establishment of tolerance: a parallel shift to the right in the dose-response curve; and a reduced response to naloxone at the same brain concentration of the antagonist in "tolerant" vs. nontolerant rats. Furthermore, a number of factors, unrelated to tolerance per se, which could have contributed to the effects observed were ruled out. It appears, therefore, that the results presented may provide the first evidence for the development of acute tolerance to a pure opiate antagonist.

Structural changes of ribosome by the action of ethylene glycol.

The denaturation of ribosome and RNA by ethylene glycol (EG) has been studied in an attempt to further understand the conformation and stability of the ribosome. At high concentrations of EG, the ribosome, its subunits, and 16S RNA undergo drastic structural changes as shown by circular dichroism, ultraviolet absorption spectroscopy, and sedimentation velocity. Two separate conformational transitions were observed for the 30S subunit; one from 30 to 50% EG and another from 60 to 90% EG. This observation suggests the presence of two "domains" in the 30S subunit which differ in their stability. However, the 50S subunit undergoes a single sharp transition at 60 to 90% EG, consistent with the notion of a highly cooperative conformation. Association of the subunits stablizes part of the 30S subunit since the transition curve for the 70S ribosome does not exhibit significant change at the low EG concentration region as seen for the 30S subunit. Removal of proteins from the 30S subunit broadens the transition curve to lower EG concentrations and suggests the role of proteins in stabilizing the conformation of the 16S RNA.

Purification and conformation of ribosomal protein L25 from E. coli ribosome.

Ribosomal protein L25 from the large subunit of E. coli ribosomes has been purified using a new procedure involving a 2M LiCl extraction followed by phosphocellulose chromatography in 6 M urea elution buffer. The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer. The analysis of the far u.v. circular dichroism spectrum of L25 indicates L25 contains approximately 16% alpha-helix and approximately 19% beta-structure. The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga. Both of these methods predict approximately three times the percent alpha-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum. A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25. The large difference in the % alpha-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 M urea used in the preparation of ribosomal proteins.

Morphine-induced supersensitivity to the effects of naloxone on luteinizing hormone secretion in the male rat.

The effects of morphine pretreatment on naloxone-induced increases in serum luteinizing hormone (LH) levels were examined in male rats. After a single morphine injection (10 mg/kg), naloxone (0.5 mg/kg)-induced increases in serum LH levels were initially suppressed (0-3 hr), but returned to normal by 4 hr. Five to 24 hr after morphine pretreatment, however, naloxone-precipitated increases in LH were markedly exaggerated with the peak effect occurring at 6 hr (4 times greater than saline-pretreated controls). In morphine-implanted rats (75-mg pellet, 48 hr earlier), the enhanced sensitivity to naloxone was considerably more dramatic than that found after acute administration. Naloxone-induced increases in serum LH levels were more than 40 to 50 times greater in morphine-implanted animals than they were in placebo-implanted rats at the respective ED50 dose. Naloxone dose-response curves revealed that the naloxone ED50 was reduced by either morphine pretreatment regimen, but was much more pronounced in pellet-implanted animals [181.6 micrograms/kg in controls; 116.4 micrograms/kg in acutely morphinized animals (P less than .05); and 4.39 micrograms/kg in morphine-implanted rats (P less than .001)]. The only distinction between acute and chronic morphine administration was in the magnitude of the shift in the naloxone ED50 as identical peak increases in LH were obtained in both groups. Finally, our data indicate that naloxone-induced increases in serum LH levels can serve as a useful and sensitive means to assess opiate dependence. The mechanisms underlying the morphine-induced enhancement of the effects of naloxone on serum LH levels are not fully understood, but we found no differences in the uptake of naloxone into brain as a function of morphine pretreatment.

Opiate-induced enhancement of the effects of naloxone on serum luteinizing hormone levels in the male rat: specificity for Mu agonists.

We have shown previously that acute morphine administration markedly enhances naloxone-induced increases in serum luteinizing hormone (LH) levels in the male rat. The purposes of the present studies were to determine whether this effect was opiate-specific and, if so, whether it was mediated by mu, kappa or sigma opiate receptors. In agreement with our previous reports, we found that naloxone-induced increases in serum LH levels were markedly enhanced (greater than 400%) in morphine-pretreated rats, relative to controls, 6 to 8 hr after a single injection; furthermore, similar effects were observed with all mu agonists assessed with the order of potency being etorphine greater than levorphanol greater than morphine greater than methadone greater than codeine. In contrast, we were unable to demonstrate any enhancement of the effects of naloxone on serum LH levels by ketocyclazocine, cyclazocine or SKF 10,047, prototypic ligands for kappa and sigma binding sites in brain. Finally, we observed that neither ethanol nor Nembutal induced a period of supersensitivity to the effects of naloxone on LH, even though both compounds transiently depressed serum LH levels over a time course similar to that observed for the opiates. On the basis of these results, it appears that the phenomenon of opiate-induced enhancement of the effects of naloxone on serum LH levels is opiate specific and, most importantly, is a unique feature of mu opiate agonists. The mechanisms underlying this phenomenon are unclear, but our results suggest that as yet unidentified events occurring within the hypothalamus must be responsible.