Elliot F. Hahn

Changes in rat brain opiate receptor content upon castration and testosterone replacement

Abstract The saturable binding of naltrexone- 3 H in the brains of castrated male rats exceeds that found in intact animals by a factor of two. This increase is androgen dependent since testosterone replacement reduced the binding to control levels. Scatchard analysis of the saturation curves revealed that the change in binding reflects increased available binding sites and is not due to increased binding affinity. This relationship between testosterone and brain opiate receptors provides for the participation of endorphins in the regulation of pituitary gonadotropins by gonadal hormones. The increased content of opiate receptors in the brains of castrated rats correlates with the greater brain N-demethylation of morphine establishing a further link between this biotransformation and agonist action.

Naloxonazine, a potent, long-lasting inhibitor of opiate binding sites

Naloxazone, the hydrazone derivative of naloxone, has proven useful in studies of opiate binding site heterogeneity both in vivo and in vitro based on its long-acting inhibition of high affinity, or mu1, binding sites. However, the need for high doses of naloxazone to inactivated the mu1 sites raised the possibility that its actions might result from lower concentrations of a more active compound. We now present evidence suggesting that this more active compound is the azine derivative of naloxone. In acidic solutions, approximately 35% of naloxazone, spontaneously rearranges to the azine, naloxonazine. Unlike naloxazone, naloxonazine is relatively stable in solution. It does not appreciably dissociate into naloxone and naloxazone and no additional compounds can be detected. Under assay conditions under which no azine formation can be detected, no inhibition of binding of either 3H-dihydromorphine or 3H-DADL is found after incubating tissue with naloxazone at concentrations up to 2000 nM followed by extensive washing. Naloxonazine, on the other hand, produces a potent, dose-dependent inhibition of binding which is resistant to washing. Despite the washes, naloxonazine at 50 nm abolishes high affinity binding with some inhibition seen at concentrations down to 10 nM.

Castration affects male rat brain opiate receptor content.

We previously reported that saturable stereospecific binding of [3H]-naltrexone in rat brain homogenates prepared from castrated male rats was greater than the corresponding binding in intact animals. We now report that we have replicated these results and that the difficulty of other investigators in observing these differences is due to methodological factors. Specifically, when samples were filtered individually and rapidly, differences between castrated and intact rats were maintained. The increase in binding was also observed when tissues were washed to remove endogenous opioids prior to incubation, when [3H]-naloxone was used as the ligand, and when various antagonists were used as displacers in the radioreceptor assay.

Comparison of effects of chronic administration of naloxone and naloxonazine upon food intake and maintainance of body weight in rats.

A comparison of the effects of the short-acting opioid antagonist naloxone, with the irreversible and highly-specific mu-1 antagonist naloxonazine, has categorized the mediation of opioids in some forms of feeding into mu-1 and non-mu-1 components. The mu-1 sites have been implicated in free-feeding, deprivation-induced feeding and morphine-induced hyperphagia, based upon their sensitivity to both naloxone and naloxonazine. However, the ability of naloxone, but not naloxonazine to inhibit feeding, induced by either 2-deoxy-D-glucose glucoprivation, ethylketocyclazocine, dynorphin or (D-ala2., D-leu5.)-enkephalin implies the existence of non-mu-1 opioid receptor mechanisms in these responses. The present study compared the effects of the daily administration of naloxone and naloxonazine (10 mg/kg, i.v.) in rats in three different types of maturational or dietary situations. In adult rats, naloxonazine and naloxone significantly reduced body weight (7% and 4%, respectively) and food intake (21% and 13%, respectively) over 14 days. These effects were more pronounced in adolescent rats where naloxonazine and naloxone significantly reduced the gain in body-weight (53% and 33%, respectively) and food intake (24% and 15%, respectively) over 14 days. In the adolescent rats, the effects of naloxonazine were significantly greater than those of naloxone. In contrast, chronic treatment with neither naloxone nor naloxonazine altered body weight or food intake of rats made obese by dietary manipulations and left on that diet during treatment with antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Opiate Receptor Concentrations are Increased in Adult Spontaneously Hypertensive Rats

The saturable binding of 3H-naltrexone in the brains of eight week old spontaneously hypertensive rats (SHR) is about twice that measured in corresponding normotensive WKY rats. This increase is dependent on age since in three and four week old SHR and WKY rats no difference in binding is observed. Scatchard analysis of the saturation curves for the adult animals revealed that the change in binding is due to an increase in the number of binding sites and does not reflect a difference in binding affinity. The increase in opiate receptor content of SHR rats coincides with the appearance of elevated blood pressure in these animals, and supports a concept in which an interaction between the endorphins and the endocrine system may be involved in the mechanisms controlling hypertension.

Irreversible opiate agonists and antagonists: V. Hydrazone and acylhydrazone derivatives of naltrexone

We have synthesized a series of hydrazones and acylhydrazones of naltrexone. These substitutions had modest effects on competition of mu binding but many greatly enhanced the relative potency of the compounds for delta receptors. Increased delta affinity was most prominent with the acylhydrazones. Many of the derivatives elicited a wash-resistant inhibition of binding which was restricted to mu, not delta, binding sites. This wash-resistant inhibition of binding did not correlate with affinity, as determined by IC50 values, implying that the inhibition could not be explained simply by slow rate of dissociation due to increased affinity.

Differential sensitivity of opioid-induced feeding to naloxone and naloxonazine

The high-affinity mu-1 opioid binding site has been implicated in some opioid responses (e.g., supraspinal analgesia) but not others (e.g., respiratory depression) by comparing the actions of naloxone, a short-acting, non-selective antagonist, and naloxonazine, an irreversible and selective mu-1 antagonist. The mu-1 site has been implicated in the opioid component modulating free feeding and deprivation-induced feeding, but not glucoprivic feeding. The present study compared naloxone and naloxonazine antagonism of hyperphagia induced by morphine, ethylketocyclazocine (EKC), dynorphin and d-ala2,d-leu5-enkephalin (DADL) in rats. Morphine produced a dose-dependent (0.01–5 mg/kg) hyperphagia in midly food-deprived rats that was blocked by naloxone (0.01–10 mg/kg). Naloxonazine (10 mg/kg) shifted the morphine hyperphagia dose-response curve to the right. These effects could not be fully accounted for by the intrinsic hypophagic properties of these antagonists. EKC produced a dose-dependent (0.5–5 mg/kg) hyperphagia which was blocked by naloxone (10 mg/kg) only at low effective EKC doses. Naloxonazine (10 mg/kg) failed to affect EKC hyperphagia. Naloxone, but not naloxonazine also blocked dynorphin and DADL hyperphagia. These results indicate that feeding induced by opiate and opioid agonists are differentially mediated by the mu-1 and other opioid binding sites; these data contrast with the modulation by the mu-1 site of the supraspinal analgesia induced by each of these agonists.

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.

Early exposure to naloxone increases blood pressure in normotensive and hypertensive rats

Abstract Continuous in utero and postpartum exposure of SH and WKY rats to naloxone results in a significant increase in their systolic blood pressure relative to respective control animals. After six weeks of age, however, naloxone was no longer effective in sustaining this increase in blood pressure. Chronic exposure to naloxone beginning at three weeks of age failed to produce any significant differences in blood pressure between treated and control animals. Although naloxone has been shown to elevate blood pressure in hypotensive states, this report represents the first example of an increase produced by the narcotic antagonist in the normotensive state.

Opiate target site N-demethylase enzymes: Differences from the liver N-demethylase

Abstract Mixtures of morphine-63H and morphine-N-14CH3 were incubated with rat brain subcellular fractions. Isotope ratio measurements served as the marker for identification, purification and quantitation of N-nor products which were shown to consist almost solely of N-normorphine. The microsomal, synaptosomal and mitochondrial, but not the supernatant brain preparations yielded N-normorphine. The microsomal incubations were then repeated in the presence of cytochrome P-450 inhibitors which suppressed the liver reaction but did not affect the brain biotransformation. The brain N-dealkylase is therefore different from the one in the liver and is not a cytochrome P-450 linked enzyme.