Effect of neonatal testosterone upon opioid receptors and the content of β-endorphin, neuropeptide Y and neurotensin in the medial preoptic and the mediobasal hypothalamic areas of the rat brain

The content of beta-endorphin, neuropeptide Y and neurotensin-like immunoreactivity (beta-End, NPY and NT), and the total number of opioid binding sites, were measured in the medial preoptic area (MPOA) and mediobasal hypothalamus (MBH) of ovariectomized adult rats which were oestrogen-primed. The rats had been injected neonatally with either testosterone propionate (TP) or vehicle (oil). NPY content was found to be higher in the MPOA of animals which received TP, whereas no significant difference was observed in the MBH NPY content. However, the NT concentration in the MBH of TP-treated rats was almost twice the amount detected in oil-treated rats and with this peptide no significant changes were detected in the MPOA. Finally, beta-End and the total number of opioid binding sites were reduced in both the MPOA and MBH of the rats which were exposed to TP neonatally. Since exposure to testosterone neonatally masculinises the rat hypothalamus, to the extent that female rats cannot generate oestrogen-stimulated prolactin and luteinizing hormone surges, we suggest that the neurochemical changes reported in this paper reflect some aspects of this sexual differentiation of the rat hypothalamus.     

Age-related changes of noradrenergic-NPY interaction in rat brain: norepinephrine, NPY levels and α-adrenoceptors

Noradrenergic-neuropeptide Y interaction, which is implicated in different physiological functions, was studied in senescent rats. Norepinephrine (NE) and neuropeptide Y (NPY) levels were measured in brainstem and hypothalamus, and α-adrenergic binding was investigated in brainstem in young (4 months) and old (34 months) Wistar rats. NE concentration was the same in senescent rats, whether NPY concentration was decreased both in brainstem and hypothalamus compared to levels in young rats. [³H]prazosin binding to α₁-adrenoceptors was not modified, but [³H]rauwolscine binding to α₂-adrenoceptors was altered with age. In fact, the density of α₂-adrenoceptors(B_max) was lower, while the binding affinity(K_d) was increased in old compared to young rats. These results suggest that the decrease of NPY levels could be one of the possible reasons for changes in [³H]rauwolscine binding to α₂-adrenoceptors in old rats. The G-protein-adenylate cyclase system, which is impaired in senescent rats, could be involved in the disorganization of noradrenergic-NPY interaction.     

α-Melanocyte stimulating hormone discloses a stimulatory effect of β-endorphin on somatostatin release

The influence of alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-END) on the secretion of somatostatin (SRIF) from the median eminence (ME) was studied using an in vitro incubation system. The MEs from adult male rats were first preincubated at 37 degrees C for 30 min with constant shaking in 0.4 ml of Krebs-Ringer bicarbonate-glucose buffer (pH 7.4) containing bacitracin in an atmosphere of 95% O2/5% CO2. Medium was discarded and replaced by medium containing different doses of alpha-MSH, beta-END, or a fixed dose of alpha-MSH (10(-7) M or 10(-9) M) plus beta-END at various concentrations. By themselves alpha-MSH and beta-END did not alter basal SRIF release, but in the presence of alpha-MSH (10(-7) M) beta-END stimulated somatostatin release. This effect was significant at concentrations of beta-END of 10(-8) M and higher. The permissive effect of alpha-MSH was observed at a concentration as low as 10(-9) M, but in this case the stimulatory effect of beta-END became evident only at higher doses tested (10(-7) M). It is suggested that alpha-MSH and beta-END participate in the modulation of SRIF release. By themselves beta-END and alpha-MSH did not affect basal release of SRIF but in the presence of alpha-MSH, beta-END had a stimulatory effect on SRIF release. The mechanism for this interaction is unknown. The results are consistent with the possibility that beta-END neurons have stimulatory and inhibitory effects on SRIF release and that alpha-MSH, by blocking the inhibitory components, discloses the stimulatory effect of beta-END on SRIF release.     

Pro-opiocortin fragments in human and rat brain: β-endorphin and α-MSH are the predominant peptides

The ‘pro-opiocortin’ fragments, β-lipotropin, β-endorphin, ACTH and α-MSH, were estimated in discrete areas of rat and human brain and pituitaries by means of radioimmunoassay in combination with gelfiltration. These peptides exihibited parallel patterns of distribution, but with β-endorphin and α-MSH predominant in the brain of rat and man, and, in contrast, their respective precursors, β-LPH and ACTH predominant in the adenohypophysis of rat and man. These data may be indicative of important differences in post-translational processing of ‘pro-opiocortin’ between these contrasting tissues.     

Effects of α-endorphin, β-endorphin and (des-tyr)-γ-endorphin on α-MPT-induced catecholamine disappearance in discrete regions of the rat brain

Following the intracerebroventricular administration of α-endorphin, β-endorphin and (des-tyrosine¹)-γ-endorphin in a dose of 100 ng, the α-MPT-induced catecholamine disappearance was found to be altered in discrete regions of the rat brain. In the regions in which α-endorphin exerted an effect, it without exception caused a decrease in catecholamine disappearance. Thus, in rats treated with α-endorphin the disappearance of noradrenaline was decreased in the medial septal nucleus, dorsomedial nucleus, central amygdaloid nucleus, subiculum, the ventral part of the nucleus reticularis medullae oblongatae and the A1 region, and that of dopamine in the caudate nucleus, globus pallidus, medial septal nucleus, nucleus interstitialis striae terminalis, paraventricular nucleus, zona incerta and central amygdaloids nucleus. β-endorphin was found to decrease noradrenaline disappearance in the ventral part of the nucleus reticularis medullae oblongatae, dopamine disappearance in the lateral septal nucleus and the disappearance of both amines in the rostral part of the nucleus tractus solitarii. Dopamine disappearance was increased in the medial septal nucleus and the zona incerta following β-endorphin treatment. Following treatment with (des-tyrosine¹)-γ-endorphin, noradrenaline disappearance was enhanced in the anterior hypothalamic nucleus, whereas dopamine disappearance was increased in the paraventricular nucleus, the zona incerta and the rostral part of the nucleus tractus solitarii. In addition to this the latter peptide also caused a decreased noradrenaline disappearance in the periventricular thalamus and the A7 region. The results fit well with the suggestion that endorphins act as modulators of catecholamine neurotransmission in particular brain regions. The pattern of effects of the endorphins differ from that previously observed following intracerebroventricular administration of methionine-enkephalin. This is in keeping with the notion that the enkephalin containing network in the brain and that containing β-LPH represent two independent systems with distinct differences in their projections to various brain regions.     

Stimulation of hypothalamic β-endorphin and dynorphin release by corticotropin-releasing factor (in vitro)

Corticotropin-releasing factor (CRF) at doses of 10⁻¹²–10⁻⁸ M significantly stimulated the release of β-endorphin and dynorphin from superfused rat hypothalamic slices. These effects were shown to be mediated by the CRF receptor since they were antagonized by the CRF receptor antagonist α-helical CRF_9–41 (10⁻⁶ M). The two opioid peptides showed different time courses of response and in the case of β-endorphin, an attenuation of the response upon continued exposure to CRF was observed.     

Effects of β-endorphin and its fragments on inhibitory avoidance behavior in rats

The effects on retrieval of a one-trial learning inhibitory avoidance response of β-endorphin, α-endorphin, and γ-endorphin, given prior to test have been studied in rats. β-Endorphin (β-LPH_61–91) in a relatively low dose (1.5 μg sc. or 50 ng icv.) facilitated inhibitory avoidance behavior, while a higher dose (10 μg sc. or 100 ng icv.) caused bimodal changes (facilitation in 50% of the animals and attenuation in another 40%. Peripheral injection of γ-endorphin attenuated inhibitory avoidance behaviour in a dose-dependent manner. The C-terminus of β-endorphin (β-LPH_78–91) was ineffective. α-Endorphin facilitated inhibitory avoidance behavior in a dose-dependent manner. Naltrexone pretreatment antagonized the bimodal effect of β-endorphin: following pretreatment with the opiate antagonist the low latency component disappeared, but the facilitatory effect of the neuropeptide remained the same.It is suggested that β-endorphin carries more than one bit of behavioral information. Inherent activities either related or unrelated to naltrexone-sensitive opiate receptors as well as biotransformation into α- and γ-endorphin may contribute to the multiple behavioral effects of this neuropeptide.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

Upregulation of brain somatostatin and neuropeptide Y following lidocaine-induced kindling in the rat

Male Sprague-Dawley rats received a daily injection of 60 mg/kg of lidocaine (> 30 days). Twenty percent of rats developed convulsions (kindled rats) and remaining rats did not show convulsions (non-kindled rats). The level of immunoreactive somatostatin (IR-SRIF) in kindled rats was significantly increased in amygdala than that in non-kindled rats and control rats. Immunoreactive neuropeptide Y (IR-NPY) contents in kindled rats were significantly increased in amygdala, hippocampus, cortex and striatum compared to non-kindled and control rats. The expression of SRIF mRNA in kindled rats produced a significant increase in amygdala, while NPY mRNA in kindled rats showed an elevated expression in both amygdala and hippocampus. These results coincide with the previous findings with the elevated expression of SRIF and NPY mRNA in electrically and pharmacologically kindled models, suggesting the important role of these peptides in the kindling phenomenon.     

Effects of serotonin depleters on the contents of β-endorphin, α-melanotropin and adrenocorticotropin in rat brain and pituitary

Serotonin depleters, 5,7-dihydroxytryptamine (5,7-DHT) andp-chlorophenylalanine (PCPA), were injected into adult male rats and β-endorphin (β-EP), α-melanotropin (α-MSH) and adrenocorticotropin (ACTH) levels in rat brain and pituitary were each estimated by radioimmunoassay combined with a gel column chromatography. (1) 5,7-DHT, injected intracerebroventricularly combined with pargyline, decreased the levels of immunoreactive-β-EP, -α-MSH and -ACTH significantly and concomitantly in hypothalamus, thalamus, and brainstem. (2) PCPA, repeatedly injected intraperitoneally 8 times every 3 days, decreased the levels of these peptides in some of these brain regions. (3) There was no significant change of IR-β-EP, -α-MSH, -ACTH in the anterior and the inter-mediate-posterior pituitaries after the treatment of 5,7-DHT, or PCPA. (4) A single injection of the same dose of PCPA induced no significant effects on these peptide levels in both brain and pituitary. These data suggest that central serotoninergic neurons might affect β-EP-α-MSH-ACTH containing neurons in rat brain.     

Expression of the conditioned NK cell activity is β-endorphin dependent

We are interested in identifying the pathways which are responsible for triggering the conditioned enhancement of natural killer (NK) cell activity. Earlier studies have suggested that central opioid(s) are involved in eliciting the expression of the conditioned NK cell activity. The purpose of this study was to identify the central opioid peptides that allow the central nervous system (CNS) to communicate with the immune system. Mediators that activate the efferent pathway of communication between the CNS and immune system was examined by injection of the mediator via the cisterna magna (CM). Conditioning was used as a tool to show that the bi-directional communication between the CNS and the immune system does take place. We found that β-endorphin but not dynorphin could stimulate NK cell activity, when β-endorphin or dynorphin was injected into the CM. In addition, when anti-β-endorphin or anti-dynorphin antibody was injected into the conditioned animals via CM the conditioned response was blocked by anti-β-endorphin but not by anti-dynorphin antibody. These observations suggest that β-endorphin appears to be one of the signals that is induced in the brain at the CS recall step of the conditioned response to trigger the elevation of NK cell activity.     

Effects of sex steroids on brain β-endorphin

Brain β-endorphin was measured by radioimmunoassay in female rats during different stages of the estrous cycle, during pregnancy, 3 weeks after ovariectomy, and 3 weeks after ovariectomy plus estradiol or estradiol and progesterone replacement. No change in hypothalamic β-endorphin content was noted on the afternoon of diestrus, proestrus, or estrus. However, in 9 rats studied between days 8–20 of pregnancy the mean hypothalamic β-endorphin concentration of41.6 ± 2.24ng/mg protein was significantly higher than the concentration of32.7 ± 1.01 in 21 non-pregnant animals (P < 0.001). Although hypothalamic β-endorphin content did not change 3 weeks after ovariectomy, when ovariectomized rats were treated iwth silastic estradiol capsules for 3 weeks, hypothalami β-endorphin decreased significantly from25.5 ± 1.2to18.3 ± 1.3and15.5 ± 0.94ng/mg protein after low and high dose estradiol treatment respectively (P < 0.001). In a second experiment hypothalamic β-endorphin in ovariectomized rats decreased from27.1 ± 1.5to20.7 ± 1.9ng/mg protein after 3 weeks of estradiol treatment (P < 0.02); the β-endorphin content of the thalamus and midbrain also decreased from8.95 ± 1.5and4.11 ± 0.70to5.24 ± 0.39and2.42 ± 0.25ng/mg protein, respectively (P < 0.025). When progesterone was administered together with estradiol, the decrease in β-endorphin induced in the hypothalamus, thalamus and midbrain by estradiol treatment was partially blocked and β-endorphin concentrations in the latter two regions were no longer significantly different from controls.We conclude that physiologic concentrations of estradiol and progesterone can alter the content of brain β-endorphin and suggest that ovarian steroids may be important regulators of this brain peptide.     

Changes of immunoreactive neuropeptide Y, somatostatin and corticotropin-releasing factor (CRF) in the brain of a novel epileptic mutant rat, Ihara's genetically epileptic rat (IGER)

Ihara's genetically epileptic rat (IGER) is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures. In the present study, the possible changes of three neuropeptides, neuropeptide Y (NPY), somatostatin (SRIF) and corticotropin-releasing factor (CRF), in the brains of IGER were investigated. Increased contents of immunoreactive (IR) NPY were found only in the hippocampus of 2-month IGERs before developing convulsive seizures, while similar increases of IR-NPY were discovered in the striatum and pyriform and entorhinal cortex as well as hippocampus in 8-month IGERs with repetitive seizures. There were no significant differences in the brain contents of IR-SRIF and IR-CRF between IGERs and the controls at both ages. These findings indicate an enhanced rate of NPY synthesis in this experimental model of epilepsy which may play a critical role in the development of epileptogenesis.     

Clonidine releases immunoreactive β-endorphin from rat pars distalis

Clonidine (10⁻⁶, 10⁻⁷ M) evokes the release of β endorphin-like immunoreactivity (β-END-LI) from cell cultures of anterior (pars distalis) but not neurointermediate (pars nervosa plus pars intermedia) lobe of the rat pituitary. This drug-induced secretion is blocked by α-adrenergic (phenoxybenzamine, yohimbine; 10⁻⁵ M) but not β-adrenergic (propranolol, 10⁻⁵ M) antagonism. Gel filtration (Sephadex G-50) reveals that β-END-LI released from anterior lobe cells consists of 2 major forms of immunoreactivity which coelute with β-lipotropin or β-endorphin standards. Conversely, β-END-LI released spontaneously from neurointermediate lobe cells almost entirely corresponds to β-endorphin. The data show that α-adrenergic stimulation by clonidine releases β-END-LI selectively from cells of anterior but not neurointermediate lobe in vitro and suggests that the clonidine-induced release of pituitary β-END-LI we have observed in vivo occurs in part by direct action on the corticotrophs of the pars distalis.     

Effects of hypophysectomy and dexamethasone treatment on plasma β-endorphin and pain threshold during pregnancy

During pregnancy, rats and humans show an increase in pain threshold that is mediated by an endorphin system. In order to determine whether plasma β-endorphin and/or other factors of pituitary origin are involved in pregnancy-induced analgesia in the rat, the effects of hypophysectomy (day 12 of pregnancy) or pharmacological suppresson of pituitary function via dexamethasone administration (day 14–21 of pregnancy) were investigated. Hypophysectomy did not affect either the magnitude of the increase or the pattern of change in pain threshold despite the resulting decrease in stress-induced plasma β-endorphin concentrations. However, the observed effect of the surgical and/or postsurgical procedure on pain threshold confounded unequivocal interpretation of these results. Pharmacological suppression of pituitary function with dexamethasone (2 μg/ml), a non-invasive procedure, also produced a significant decrease in resting plasma β-endorphin levels. As was observed for surgical removal of the pituitary gland, this treatment did not produce a significant alteration in the magnitude of the increase in jump threshold. Furthermore, no correlation was found between plasma β-endorphin concentrations and jump threshold values on day 21 of pregnancy. These results indicate that the pituitary gland does not play an essential role in the maintenance of opioid analgesia during pregnancy. It is suggested that pregnancy-induced analgesia depends on central rather than peripheral opioid systems.     

Regional distribution of β-lipotropin converting enzymes in rat pituitary and brain

Among the brain areas studied only pars distalis and pars intermedia are found to contain β-lipotropin activating enzyme indicating that these may be the exclusive organs for a physiologically significant conversion of β-lipotropin into β-endorphin. β-Endorphin inactivating enzyme is found to be rather uniformly distributed in all the pituitary and brain regions, α- and γ-endorphins are presumably formed by the action of this enzyme on β-endorphin.     

Immunocytochemical demonstration of β-endorphin and β-lipotropin in cultured human spinal ganglion neurons

Human fetal spinal ganglion neurons isolated and cultured in vitro were found to contain immunoreactive β-endorphin and β-lipotropin as demonstrated by immunoperoxidase and immunofluorescence techniques. The specificity of the immunoreactions was confirmed by the negative staining by prior absorption of the specific antisera with added peptides. The culture system described may provide a valuable model system in which cellular mechanisms underlying the functions of opioid peptides can be investigated.