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.     

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.     

The effect of α-melanocyte stimulating hormone (α-MSH) on the metabolism of blogenlc amines in the rat brain

The effect of the chronic administration of α-MSH on the incorporation of tritiated tyrosine into noradrenalin and dopamine and of tritiated tryptophan into serotonin was studied in different regions of the rat brain. α-MSH increased the incorporation of tritiated tryptophan into serotonin in the cortex and slightly decreased that of tyrosine into the dopamine in the hypothalamus. As the brain concentration of serotonin was unchanged in the animals treated with α-MSH, it is suggested that some of the changes in behavior, which other investigators have found following the administration of peptides containing the same sequence of amino acids to that found in MSH, could be associated with an increased turnover of cortical serotonin.     

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.     

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.     

Dopamine inhibits the release of immunoreactive β-endorphin from rat hypothalamus in vitro

Mediobasal hypothalamus tissue (MBH) from adult male rats was incubated in Krebs-Ringer bicarbonate medium (KRB). KRB was changed at 15 min intervals and the concentration of immunoreactive β-endorphin (β-ENDi) in the medium was measured by radioimmunoassay. Incubation of MBH tissue in normal KRB resulted in a constant release rate of β-ENDi of approximately 1% of the tissue content per h. KRB containing 45 mM K⁺ causes a two fold increase in the release rate of β-ENDi which was Ca²⁺ dependent. Dopamine (0.01–1.0 μM) inhibits both the spontaneous and the K⁺-stimulated release of β-ENDi in a dose related manner. The dopamine receptor blocking agent haloperidol prevents this inhibitory effect of dopamine. The selective D-1 receptor agonist SKF 38393 does not affect the release rate of β-ENDi; whereas the selective D-2 receptor agonist LY 141865 inhibits both the spontaneous and K⁺-stimulated release of β-ENDi. The effects of LY 141865 can be blocked by (−)-sulpiride, a selective D-2 receptor antagonist. Norepinephrine only weakly inhibits the K⁺-stimulated release of β-ENDi, and effect that can be blocked by haloperidol but not by the α-adrenoceptor blocker phentolamine. At concentrations tested (0.01–1.0 μM), isoproterenol, 5-hydroxytryptamine, carbachol and 8-Br-cAMP (1.0 μM) do not affect β-ENDi release. It is concluded that dopamine can inhibit the release of β-ENDi from hypothalamic neurons via a D-2 receptor mechanism.     

Age-related changes in cortico-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and β-endorphin in specific rat brain areas

We investigated the age-related changes in the tissular protein, cortico-releasing factor (CRF), somatostatin (SOM), neuropeptide Y(NPY), methionine enkephalin (M-ENK) and beta-endorphin (beta-END) levels in frontal cortex, hippocampus, striatum and hypothalamus of young (4-month-old), mature (18-month-old) and senescent (26-month-old) Wistar male rats, bred in a specific pathogen free environment. Between the age of 4 and 18 months, the tissular protein levels increased in all 4 structures studied. The CRF and SOM levels increased in the hippocampus, while the NPY levels decreased. During this time, the NPY content increased in the striatum, whereas the SOM and M-Enk striatal levels decreased. Concomitantly, the NPY and beta-End levels decreased in the hypothalamus. Interestingly, no significant variations were found to occur in the frontal cortex whatever the neuropeptide studied. Between the age of 18 and 26 months, no significant changes in the tissular protein levels were detected, except in the hippocampus. The changes in the neuropeptide concentrations observed during this period depended on the neuropeptide and the brain structure studied. The CRF and beta-End levels decreased in the frontal cortex and the hypothalamus, respectively. The NPY peptidergic systems seem to be preferentially affected by aging processes since 3 out of the 4 structures studied--the frontal cortex, the striatum and the hypothalamus--showed a decrease in their tissular NPY content. During the same period, none of the 5 neuropeptides studied were affected in the hippocampus.     

Subcellular localization of immunoreactive α-melanocyte stimulating hormone in human brain

The regional and subcellular distribution of immunoreactive α-melanocyte stimulating hormone (α-MSH₁) in the post mortem adult human brain was investigated. α-MSH_i was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of α-MSH_i were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of α-MSH_i in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, α-MSH_i was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of α-MSH_i were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that α-MSH is a neuronal peptide in the human brain.     

Lack of effect of interleukins 1α and 1β, during in vitro perifusion,on anterior pituitary release of adrenocorticotropic hormone and β endorphin in the male rat

It has been demonstrated that interleukin 1 (IL1) injection provokes a great variety of biological effects, notably an activation of the corticotropic axis, increasing plasma adrenocorticotropic hormone (ACTH) and corticosterone. However, the primary site of action of IL1 is still controversial. In the present study, we first verified the in vivo capability of human interleukins 1α (hIL1α) and 1β (hIL1β) to release ACTH and β endorphin (β EP) in the normal male rat, before investigating, through an anterior pituitary (AP) perifusion system, the hIL1α and hIL1β effects on basal and corticotropin-releasing factor (CRF)-induced ACTH and β EP secretions. This system enabled the examination of a dynamic profile of hormones secretion, avoiding the possibility of feedback mechanisms, as is the case with the use of regular but very often longtime incubations. The results showed that in a perifusion system, with a short duration treatment (below 2 hr) compatible with the kinetics of action observed in vivo, basal and CRF-induced ACTH and β EP release were not modified in the presence of a broad range of concentrations (from 10^?12 to 10^?9 M) of hIL1α or hIL1β. Taken together, these results clearly show that in an in vitro situation close to physiological conditions, the primary site of action of hIL1α and hIL1β on ACTH and β EP release is not located at the AP level in the male rat. © 1993 Wiley-Liss, Inc.     

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.     

Effect of opioid antagonism on β-endorphin processing and proopiomelanocortin-peptide release in the hypothalamus

Previous studies have shown that chronic opioid receptor blockade has significant effects on POMC gene expression and peptide levels in the hypothalamus. We have now examined the effects of the opioid antagonist naltrexone on β-EP processing in the hypothalamus and on the release of 2 POMC-derived peptides, β-EP andγ₃-MSH, from the perifused hypothalamus in vitro. The β-EP immunoactivity in the medial basal hypothalamus (MBH) of 7 rats infused for 1 week with naltrexone by osmotic minipump, was individually analyzed by HPLC and compared to 7 control rats. The mean ratio of β-EP_1–31 compared to β-EP_1–27 plus β-EP_1–26 was 2.34 ± 0.41 in the naltrexone treated rats, significantly higher than the ratio of 1.26 ± 0.09 in the control rats (P < 0.02). Thus in the setting of chronic opioid antagonism although β-EP content decreases, there is relatively more β-EP_1–31, the biologically active opioid form of the peptide, compared to the C-terminally cleaved forms of β-EP which have reduced biological activity. To study the effects of naltrexone on β-EP andγ₃-MSH release, hypothalami were perfused in vitro with 10⁻⁶M naltrexone. Basal release ofγ₃-MSH was significantly higher from the naltrexone treated brains compared to the controls (221 ± 20pg/60min vs.161 ± 6.7pg/60 min) (P < 0.01); KCl stimulatedγ₃-MSH was also significantly higher in the naltrexone group (951 ± 94 vs.543 ± 85pg/60 min) (P < 0.005). Basal release of β-EP was136 ± 45pg/60 min in the naltrexone treated brains compared to 93 ± 15pg in the controls, but this difference was not significant; KCl stimulated release of β-EP, however was significantly higher in the naltrexone group (558 ± 103 vs. 275 ± 49pg/60 min (P < 0.02). To study the acute and chronic effects of naltrexone in vivo on β-EP andγ₃-MSH release, rats were either injected with naltrexone and sacrificed 40–60 min later or were infused with naltrexone for 7 days. Baselineγ₃-MSH release was significantly higher in rats treated with naltrexone 40–60 min prior to the perifusion (P < 0.01). Baseline β-EP release was below the limit of assay detection. No differences were noted in the responses ofγ₃-MSH or β-EP to KCl in either group. In contrast after chronic treatment with naltrexone for 1 week, baseline peptide release was not different from the control animals despite a more than 50% fall in peptide content. Theγ₃-MSH and β-EP responses to KCl stimulation, however, were significantly less in the naltrexone treated animals. Thus there is an increase in POMC peptide release acutely after treatment with naltrexone in vitro and in vivo. After 1 week of naltrexone, baseline POMC peptide release continues unchanged despite the fall in peptide content, however, the response to KCl is blunted possibly reflecting the decrease in peptide content after chronic stimulation with naltrexone. We conclude that naltrexone has significant effects on POMC peptide release and on β-EP processing in the hypothalamus. These results further demonstrate that the brain POMC system can respond to opioid blockade at several levels and are consistent with inhibitory feedback mechanisms for the autoregulation of the POMC system by endogenous β-EP.     

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.     

α-Melanocyte-stimulating hormone structure-activity studies: Comparative analysis of melanotropic and CNS bioactivities

The structure-activity relationships in vitro of α-MSH (α-melanocyte-stimulating hormone, α-melanotropin) analogs as determined on normal and transformed (melanoma cell) melanocyte bioassays are summarized. Specifically, the characterization of potent and metabolically stable melanotropic agonist analogs and a newly discovered antagonist of α-MSH are highlighted. Comparison of these data versus the known structure-activity relationships of α-MSH related to CNS bioactivities suggests the existence of nonclassical α-MSH receptor-mediated pathways or, perhaps, a yet undefined endogenous neuropeptidergic pathway(s) having different selectivities for α-MSH analogs. In summary, several of the α-MSH analogs reported here may be useful molecular probes in future strategies aimed at the identification and systematic characterization of both peripheral and central α-MSH receptors.     

Plasma and cerebrospinal fluid α-MSH levels in the rat after hypophysectomy and stimulation of pituitary α-MSH secretion

Immunoreactive α-MSH was measured in cerebrospinal fluid (CSF) and plasma of rats. While treatment with haloperidol increased α-MSH levels in the plasma concentration of α-MSH in the CSF showed little change. Hypophysectomy also had little effect on the concentration of α-MSH in the CSF despite the fall in plasma α-MSH levels. This lack of correlation between α-MSH levels in the CSF and plasma suggests that the systemic circulation does not deliver α-MSH to the CSF. The apparently normal levels of α-MSH in the hypothalamus after hypophysectomy suggests that this tissue is able to synthesize α-MSH and it is possible that the hypothalamus is a source of the α-MSH in the CSF.     

Localization of neurons containing pro-opiomelanocortin-related peptides in the hypothalamus and midbrain of the lizard, Anolis carolinensis evidence for region-specific processing of β-endorphin

Immunohistochemical analyses of the lizard brain, following colchicine pretreatment, revealed two populations of POMC-producing cell bodies located in medial-basal hypothalamus and the mesencephalic tegmentum. Analyses of extracts of lizard brain regions by radioimmunoassay and gel filtration chromatography indicate that β-endorphin-sized and α-MSH-sized peptides are the major POMC-related end products. Evidence is presented for region-specific processing of β-endorphin in the lizard brain.     

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.