Binding characteristics of hypothalamic mu opioid receptors throughout the estrous cycle in the rat.

This study presents a detailed analysis of the binding characteristics of hypothalamic mu opioid receptors during the different phases of the estrous cycle of the female rat. Different groups of adult female rats with a regular 4-day estrous cycle were killed by decapitation at 10.00 h of diestrus days 1 and 2, at 10.00, 12.00, 14.00, 16.00, 18.00 and 20.00 h of the day of proestrus and at 10.00, 12.00, 14.00, 16.00 and 18.00 h of the day of estrus. At sacrifice, the hypothalami of the animals were dissected out, plasma membrane preparations were obtained and the binding characteristics (Bmax, Kd) of the specific mu opioid ligand dihydromorphine (DHM) on mu opioid receptors were evaluated. Blood has been collected from the trunk vessels to monitor with specific radioimmunoassays serum levels of LH, estradiol and progesterone. The data obtained indicate that in the hypothalamus of female rats with a regular 4-day estrous cycle, the binding characteristics of DHM for mu receptors show important variations during the different phases of the estrous cycle. In general, the number of mu opioid receptors is elevated during the morning of diestrus day 2 and during the day of proestrus being maximal at 14.00 h and declining significantly at 18.00 and 20.00 h of the same day. At estrus, the number of mu receptors appears high at 10.00 and 16.00 h and low at 12.00, 14.00 and 18.00 h. All these variations take place without any significant change of the affinity (Kd) of DHM for the mu receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the binding characteristics of hypothalamic mu opioid receptors in rats by gonadal steroids.

Recent evidence suggests that the effects of the opioids on gonadotropin release may depend on the endocrine status existing in the experimental animal. In the brain, the effects of the opioids are exerted through the interaction with different classes of opioid receptors (mu, delta, kappa, etc.). Among these, the mu receptors appear to be particularly relevant to the control of gonadotropin secretion. Different groups of experiments have been performed in the rat in order to analyze whether changes of circulating levels of sex steroids may have an impact on the binding characteristics of hypothalamic mu opioid receptors, as evaluated by a receptor binding assay performed on plasma membrane preparations, using [3H]dihydromorphine as a mu ligand. In a first series of experiments, it has been observed that the ontogenesis of hypothalamic mu opioid receptors is different in male and in female rats: the concentration of mu sites, similar in animals of the two sexes at 16 days of age, increases in females, but not in males, between day 16 and day 26 of life. This sexual difference persists in 60-day old animals, when the brain is fully mature. It has also been observed that the pattern of maturation of hypothalamic mu receptors can be reversed by neonatal castration of males and by neonatal testosterone treatment of females. In a second series of experiments, it has been shown that in the hypothalamus of regularly cycling female rats the concentration of mu receptors varies during the different phases of the estrous cycle. In particular, a rather high density of mu sites during diestrus day 2 and the morning of the day of proestrus was found; this is followed by a progressive decline during the afternoon of the day of proestrus and the day of estrus, with a minimum value of the concentration of mu receptors being recorded in the first day of diestrus. These fluctuations seem to be linked to the physiological changes of serum levels of ovarian steroids: in fact, in a third series of experiments, it has been found that the positive feedback effect on LH release, exerted by the treatment of ovariectomized female rats with estrogens plus progesterone, is accompanied by a significant decrease of the concentration of hypothalamic mu opioid receptors; treatments with estrogens alone, able to induce a negative feedback effect on LH secretion, are not associated with modifications of hypothalamic mu receptors. These data seem to indicate that hypothalamic mu receptors may be involved in the positive but not in the negative feedback control of LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of brain alpha 2-adrenoceptor and mu-opioid receptor densities during morphine dependence and spontaneous withdrawal in rats.

The densities of brain alpha 2-adrenoceptors and mu-opioid receptors, quantitated by means of the binding of the agonists [3H]clonidine and [3H]dihydromorphine, respectively, were studied during the development of morphine dependence and spontaneous withdrawal in the rat. The oral administration of morphine (12-130 mg/kg for 3-21 days) led to inconsistent changes in alpha 2-adrenoceptor density while the density of mu-opioid receptors was down-regulated. In contrast, spontaneous opiate withdrawal (3-72 h) significantly increased the density of alpha 2-adrenoceptors while the density of mu-opioid receptors was rapidly up-regulated to control values. In the hypothalamus, but not in other brain regions, the increase in alpha 2-adrenoceptor density after withdrawal followed a time course (3-72 h) related to the severity of the abstinence syndrome. Thus, there was a positive and significant correlation between the severity of withdrawal and the density of alpha 2-adrenoceptors in the hypothalamus. Short-term treatment with clonidine (2 x 0.5 mg/kg, i.p.) prevented the morphine withdrawal-induced increases in alpha 2-adrenoceptor density in various brain regions, but not in the hypothalamus. The main results suggest that modulation of hypothalamic alpha 2-adrenoceptor density during morphine withdrawal is a relevant physiological mechanism by which the opiate abstinence syndrome is counteracted.

Visualization of mu1 opiate receptors in rat brain by using a computerized autoradiographic subtraction technique.

We have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of mu1 sites, a common very-high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala2, D-Leu5]enkephalin selectively inhibit the mu1 binding of [3H]dihydromorphine, leaving mu2 sites, while low morphine concentrations eliminate the mu1 binding of [3H][D-Ala2, D-Leu5]enkephalin, leaving delta sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent mu1 binding sites. The regional distributions of mu1 sites labeled with [3H]dihydromorphine were quite similar to those determined by using [3H][D-Ala2, D-Leu5]enkephalin. High levels of mu1 binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of mu1 sites in analgesia. Other regions with high levels of mu1 binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to mu1 sites varied among the regions, ranging from 14% to 75% for [3H][D-Ala2, D-Leu5]enkephalin and 20% to 52% for [3H]dihydromorphine.

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.

Reduction of opioid binding in neuroblastoma x glioma cells grown in medium containing unsaturated fatty acids.

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of 3H-labelled [DAla2]-[DLeu5]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [3H]dihydromorphine and [3H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To evaluate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 microM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligand-receptor recognition. Whether this can offer a satisfactory explanation for our observations remains to be established.

Opiate and muscarinic ligand binding in five limbic areas after bilateral olfactory bulbectomy.

Bilateral olfactory bulbectomy (BBX) in mice leads to a variety of neutrochemical changes in 5 limbic areas associated with the bulbs. Within one week after BBX, opiate ligand binding declined by 73% in the amygdala, rose by 82% in the hypothalamus and then returned to sham levels by 4 weeks in both areas. Opiate binding also declined by 47% in the olfactory tubercle and to essentially zero in the piriform cortex and the olfactory peduncle after 16 weeks. Muscarinic cholinergic binding declined in the amygdala and hypothalamus at 16 weeks after BBX, but reductions in muscarinic binding were observed in the piriform cortex and the olfactory peduncle by two weeks postsurgery. Muscarinic binding in the olfactory tubercle was unaffected by BBX, as was binding of beta-adrenergic and benzodiazepine ligands in the limbic areas. Binding of [3H]spiroperidol rose 61% in the olfactory tubercle two weeks after surgery and then declined to normal levels. Choline acetyltransferase activity rose by 64% within one week after BBX in the piriform cortex and remained elevated throughout the study. Activity of this enzyme also rose in the olfactory peduncle and the olfactory tubercle after surgery. BBX had only moderate effects on glutamic acid decarboxylase in the limbic areas, and enzyme activity increased 25% in the olfactory tubercle and the piriform cortex 4 weeks after BBX. BBX also resulted in a moderate decrease (22%) in DOPA decarboxylase activity in the olfactory tubercle two weeks after BBX. The implications of these neurochemical changes are discussed in terms of what is known about bulb-limbic system connections.