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.     

Immunohistochemical localization of neurotensin and β-endorphin in the rat anterior pituitary gland

Nakane's enzyme-labeled antibody technique revealed that cells containing neurotensin-like immunoreactivity were widely distributed in the anterior lobe of the pituitary body. Immunohistochemical studies on serial sections showed that a part of neurotensin positive anterior lobe cells contained β-endorphin-like peptide simultaneously. The results show that β-endorphin and neurotensin occur together in certain pituitary cells and this is an evidence of coexistence of more than one peptide within one anterior pituitary cell.     

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.     

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.     

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.     

Circadian variations in β-endorphin concentrations in pituitary and in some brain nuclei of the adult male rat

Using both the ‘punch’ microdissection and a radioimmunological technique, circadian variations in β-endorphin concentrations can be observed in the pituitary and in some discrete brain regions of the male rat (Wistar CFY). Animals were synchronized with light from 06.00 to 18.00 h, then darkness. Water and food were available ad libitum. Very well marked circadian rhythms were in evidence in the anterior lobe of the pituitary, the septum, the pons, the medulla obblongata and the cerebellum. There crest time locations were situated between 20.00 and 24.00 h. No significant circadian rhythms but biphasic variations were observed in the intermidiate lobe of the pituitary, the POA, the thalamus, the central gray and the caudatus. There crest time locations were synchronized around 08.00 and 20.00 h.The most striking finding was that, regardless of the brain area investigated so far, maximal vaalue were observed a short time after the beginning of the activity period of rats. This fact is identical with the which has been observed for substance P and LH-RH contents in brain areas where these peptides are mostly present in nerve terminals in high concentrations.     

Changes in the β-endorphin content of discrete hypothalamic nuclei during the estrous cycle of the rat

Concentrations of β-endorphin were measured in discrete brain nuclei of 4-day cycling rats on each day of the estrous cycle. On the afternoon of proestrus β-endorphin levels were significantly higher in median eminence and suprachiasmatic nucleus, and lower in arcuate nucleus, when compared to levels found on other days of the cycle. These changes coincided with the peak of plasma prolactin, which was blocked by prior administration of naloxone.     

Brain β-endorphin concentrations in experimental chronic liver disease

β-Endorphin, Met-enkephalin, substance P and somatostatin concentrations have been evaluated in brain areas of rats with severe liver disease obtained by chronic pretreatment with CCl₄ for 3 or 9 weeks. β-Endorphin, but not Met-enkephalin, somatostatin or substance P concentrations were significantly decreased in the hypothalamus of both the three and nine week-treatment groups. The β-endorphin decrease we observed might be tentatively attributed to a modification of GABA levels, but not serotonin, since the stimulation of the serotoninergic system induced a significant increase, while the potentiation of the GABAergic system induced a clear decrease of β-endorphin concentrations in the hypothalamus of treated rats.     

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.     

Brain β-endorphin-like immunoreactivity in adult rats given β-endorphin neonatally

β-endorphin-like immunoreactivity was measured by radioimmunoassay in the brains of adult rats treated neonatally with β-endorphin, naloxone, or vehicle. After treatment with β-endorphin, the decreases observed in β-endorphin-like immunoreactivity in the hypothalamus, pineal, midbrain, pons-medulla, hippocampus, striatum, frontal cortex, occipital cortex, and posterior cortex were highly significant but the 23% decrease in the thalamus was not significantly different from that of control rats. Neonatal administration of naloxone only resulted in a significant decrease in β-endorphin-like immunoreactivity in the hypothalamus. In contrast, no differences were discernible in content of either β-endorphin-like immunoreactivity or ACTH-like immunoreactivity in the pituitary of rats treated with β-endorphin, naloxone, or vehicle in the neonatal period. These same rats had shown an increased threshold to painful thermal stimulation by the tail-flick test after administration of either β-endorphin or naloxone at birth. The results suggest that neonatally injected β-endorphin may alter the levels of β-endorphin-like immunoreactivity in rat brain as well as the response to pain.     

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.     

Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, β-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area

The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or β-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEP) (5 μg) and progesterone (500 μg). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or β-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of β-endorphin, neither the opiate antagonist nor anti-β-endorphin-γ-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of β-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-β-endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-γ-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA. Conversely, naloxone, anti-β-endorphin-γ-globulin, anti-CRF-γ-globulin and ACTH infused into the MPOA produced high levels of lordosis in female rats normally showing low levels of lordosis by treatment with low doses of OEB (2 μg) and normal doses of progesterone (500 μg). In each case the facilitation could be blocked by pretreatment of the MPOA site with a potent antagonist analogue of LH-RH. The results indicate the key role of LH-RH in the action of endogenous CRF, β-endorphin and ACTH in the regulation of lordosis behaviour. Each of these substances may act directly on the LH-RH neurone. The postulated presynaptic relationship between CRF and β-endorphin neuronal systems that seem to exist in the mediobasal hypothalamus and the central gray may be less predominant in the MPOA. The results, however, provide clear further evidence for the potent effects of CRF and opiocortin peptides in the regulation of LH-RH and reproduction.     

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.     

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.