Beta-endorphin enhances interleukin-2 (IL-2) production in murine lymphocytes

Beta-endorphin has been reported to enhance T lymphocyte proliferation and cytolytic activity. In this report, it is demonstrated that beta-endorphin enhances the production of the T cell lymphokine, interleukin-2, from mitogen-stimulated, unfractionated murine splenocytes, as well from a cloned T cell line. The enhancement is naloxone irreversible and dependent on the integrity of the C-terminal amino acids, though the N-terminal amino acids appear to contribute to the potency of the enhancement. The data suggest that beta-endorphin interacts with a nonopioid receptor that has specificity characteristics similar to a nonopioid beta-endorphin receptor described in the central nervous system.     

Enhancement of the generation of cytotoxic T cells by endogenous opiates

Previous reports have shown that endogenous opiates (β-endorphin and met-enkephalin) have effects on cells of the immune system at physiologic concentrations. Using murine spleen cells, we herein report that β-endorphin and met-enkephalin can enhance the generation of cytotoxic T lymphocytes (CTLs) at suboptimal concentrations of alloantigen in a one-way mixed lymphocyte culture (MLC). This enhancement is seen at levels ranging from 2.9 × 10⁻⁸ M to 2.9 × 10⁻¹⁴ M and 1.45 × 10⁻⁶ M to 1.45 × 10⁻¹² M for β-endorphin and met-enkephalin, respectively. This enhancement can be partially blocked by naloxone at 10⁻⁷ M. Furthermore, the simultaneous addition of met-enkephalin and β-endorphin does not increase the enhancement of CTL generation by either opiate peptide alone suggesting that they are acting through the same receptor. This report gives added proof for the regulatory-loop theory between the neuroendocrine and immune systems.     

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.     

Transferred T lymphocytes are compromised when the donor is pretreated with β-endorphin

A single intracerebroventricular (i.c.v.) injection of 14 pmol of β-endorphin into 6–7-week-old BALB/c (+/+) donor mice, 24 h prior to isolation of their T lymphocytes for use for reconstitution of athymic BALB/c (nu/nu) nude mice, altered the immuno-protective effect of adoptive transfer against an intracerebral (i.c.) infection with a temperature-sensitive mutant of vesicular stomatitis virus (tsG31 KS5 VSV). Simultaneous injection of β-endorphin and naloxone into door animals negated the opiate effects on splenic lymphocytes. T lymphocytes, isolated from β-endorphin-treated donors, and then depleted with anti-asialo GM1 antiserum and complement failed to demonstrate the detrimental effects of β-endorphin.     

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.     

β-Endorphin concentrations in serum, hypothalamus and central gray of hypophysectomized and mediobasal hypothalamus lesioned rats

The serum and brain concentrations of β-endorphin immunoreactivity have been studied in intact, mediobasal hypothalamus (MBH) lesioned and hypophysectomized male rats. After hypophysectomy there is a major reduction (90%) of β-endorphin concentration in the serum but only a partial reduction (20%) in the mediobasal hypothalamus. However, MBH lesions enhance β-endorphin serum values in previously hypophysectomized rats. Long-term MBH lesions alone lead to an almost complete disappearance of β-endorphin in the central gray matter with a slight decrease in the serum.These data clearly show that: (1) the pituitary is the major source of β-endorphin in the serum; (2) the hypothalamus is the major source of β-endorphin in the central gray matter; and (3) there is clear influence of the pituitary on hypothalamic β-endorphin.     

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.     

The 21-aminosteroid antioxidant, U74389F, prevents estradiol-induced depletion of hypothalamic β-endorphin in adult female rats

A single intramuscular injection of 2 mg estradiol valerate (EV) results in neuronal degeneration and β-endorphin depletion in the hypothalamic arcuate nucleus of adult female rats. We have hypothesized that peroxidase-positive astrocytes in this brain region oxidize estrogens and catecholestrogens to semiquinone radicals which mediate oxidative neuronal injury. In the present study, dietary administration of the potent antioxidant 21-aminosteroid, U-74389F, completely blocked EV-induced β-endorphin depletion in the hypothalami of adult female rats. Neither EV nor 21-aminosteroid treatment had any effect on hypothalamic concentrations of neuropeptide Y and Met-enkephalin, confirming that the estradiol lesion is fairly selective for the β-endorphin cell population. The present findings support the hypothesis that the toxic effect of estradiol on hypothalamic β-endorphin neurons is mediated by free radicals.     

Autoradiographic Localization of β-Endorphin Binding in the Pancreas

Autoradiographic localization of ¹²⁵I-labeled β-endorphin binding in the rabbit pancreas demonstrated specific binding in the pancreatic islet cells. Binding was inhibited by (1) nonradioactive β-endorphin, (2) the opioid antagonist naloxone, (3) the μ receptor agonists morphine and [ -Ala², (Me)Phe⁴, Gly(ol)⁵]enkephalin, (4) the δ receptor agonist [ -penicillamine², -penicillamine⁵]-enkephalin, (5) the μ and δ agonist met-enkephalin and (6) the δ and κ agonist dynorphin. Specific binding was not clearly demonstrable in the acinar portion of the rabbit pancreas. The binding characteristics of ¹²⁵ I-β-endorphin in the pancreatic islets were comparable with those of μ and δ opioid receptors in the rabbit brain. In the pancreas, β-endorphin binding appeared to be concentrated in discrete areas in the islets. Combined immunohistochemistry and autoradiography demonstrated that β-endorphin binding was primarily concentrated in the glucagon-containing alpha and somatostatin-containing delta cells, but was also found in the insulin-containing beta cells to a lesser extent, Given the intraislet location of the opioid binding sites, and our previous finding of immunoreactive β-endorphin in the pancreatic beta cells and the inhibitory effect of β-endorphin on insulin secretion, it appears that β-endorphin may serve a paracrine or autocrine function in the regulation of pancreatic hormone secretion.     

An antibody to a peptide specified by an RNA that is complementary to γ-endorphin mRNA recognizes an opiate receptor

The putative δ-opiate receptor complex has been identified by a new approach which employed an antibody that is directed against a peptide which binds γ-endorphin and is specified by RNA that is complementary to that of γ-endorphin mRNA. This antibody competes with β-endorphin and naloxone for binding sites on the surface of neuroblastoma × glioma NG108-15 hybrid cells. The opiate receptor complex has an apparent molecular weight of 210 000 and is composed of four noncovalently associated subunits with apparent molecular weights of 68 000, 58 000, 45 000 and 30 000.     

Inhibition of interleukin-1β and prostaglandin E2 thermogenesis by glycyl-glutamine, a pro-opiomelanocortin-derived peptide

Interleukin-1β (IL-1β) and other cytokines produce fever by stimulating prostaglandin E₂ (PGE₂) synthesis in thermoregulatory regions of the preoptic area and anterior hypothalamus (POA/AH). Prostaglandin E₂ is thought to raise body temperature, at least in part, by stimulating β-endorphin release from pro-opiomelanocortin neurons that innervate the POA/AH. In this study, we investigated whether glycyl-glutamine (β-endorphin_30–31), an inhibitory dipeptide synthesized from β-endorphin post-translationally, inhibits IL-1β and PGE₂-induced hyperthermia. Hyperthermic sites were identified by microinjecting PGE₂ (3 fmol/1 μl) into the medial preoptic area (mPOA) of conscious, unrestrained rats. Interleukin-1β (1 U) injection into the same PGE₂ responsive thermogenic sites in the mPOA elicited a prolonged rise in colonic temperature (T_c) (+1.02±0.06°C) that persisted for at least 2 h. Glycyl-glutamine (3 nmol) co-injection into the mPOA inhibited IL-1β thermogenesis completely (T_c=−0.18±0.22°C). Glycyl-glutamine had no effect on body temperature when given alone to normothermic rats. Co-injection of individual amino acids, glycine and glutamine (3 nmol each amino acid), failed to influence IL-1β-induced thermogenesis, which indicates that Gly-Gln hydrolysis does not explain its inhibitory activity. Glycyl-glutamine (3 nmol) also prevented the rise in body temperature produced by PGE₂ (PGE₂=0.89±0.05°C; PGE₂ plus Gly-Gln=−0.16±0.14°C), consistent with evidence that PGE₂ mediates IL-1β-induced fever. These findings demonstrate that Gly-Gln inhibits the thermogenic response to endogenous pyrogens.     

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.     

Age-Dependent Muscarinic Stimulation of β-Endorphin Secretion From Rat Neurointermediate Lobe In Vitro

The effect of acetylcholine on the neurointermediate lobe β-endorphin secretion was studied in the neonatal and in the adult rat in vitro. Acetylcholine stimulated β-endorphin secretion from the 2-day- and 5-day-old neurointermediate lobe, the effect was dose dependent and more pronounced in the presence of the cholinesterase inhibitor eserine. The 10-day-, the 21-day-old and the adult rat neurointermediate lobes did not respond to acetylcholine, even in the presence of eserine. Basal β-endorphin secretion was elevated by the D₂ receptor antagonist sulpiride, but acetylcholine was without effect in the 10-day-old and in the adult neurointermediate lobe even after dopamine receptor blockade. The β-endorphin stimulatory response to acetylcholine was diminished by the M₁ muscarinic receptor antagonist pirenzepine and blocked by the M₃ > M₁ antagonist 4-diamino-phenyl-piperidine (4-DAMP). The selective M₂ antagonist methoctramine and nicotine had no effect. These data indicate that the neurointermediate lobe β-endorphin secretion is under special muscarinic cholinergic regulation for a relatively short time after birth. The disappearance of this stimulatory cholinergic effect in later life might be due to changes in the intracellular secretory machinery in the IL and/or to the uncoupling of the cholinergic receptors from the intracellular signal transduction system(s) responsible for the stimulated secretion in the rat melanotrope cells.     

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.     

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.     

Brain cholecystokinin and β-endorphin systems may antagonistically interact to regulate tissue DNA synthesis in rat pups

Previously, we have shown that intracisternal (i.c.) administration of β-endorphin suppresses brain and liver DNA synthesis in rat pups. This finding is consistent with the view that endogeneous CNS β-endorphin plays an important role in controlling postnatal growth. Recent evidence suggests that brain CCK₈, the sulfated carboxyterminal octapeptide fragment of cholecystokinin, may function physiologically as an endogenous opioid antagonist. We now report that CCK₈ injected i.c. together with β-endorphin effectively prevented β-endorphin from inhibiting brain and liver DNA synthesis in 10-day-old rats. CCK₈ blocked the liver DNA effect of β-endorphin via actions within the brain, as subcutaneous administration of CCK₈ was ineffective. In contrast to CCK₈, i.c. administration of CCK_8U (the unsulfated form of CCK₈) together with β-endorphin did not prevent β-endorphin from inhibiting liver DNA synthesis, and only slightly reversed the brain DNA effect.The results obtained support a role for endogenous brain CCK₈ in the modulation of tissue DNA responses to CNS β-endorphin and possibly to other endogenous opioids. If so, interference with brain CCK function could disrupt tissue growth. Thus, normal mammalian development may require a close functional interaction between the cholecystokinin and β-endorphin systems in the brain.     

α-Melanotropin and β-endorphin-like immunoreactivities are contained within neurons and nerve fibers of the rat duodenum

Both α-melanotropin and β-endorphin were revealed by immunofluorescence microscopy studies within neurons and nerve fibers of the rat duodenum. An immunohistochemical staining for α-melanotropin was seen within neuronal cell bodies and nerve fibers bundles of the myenteric and submucous plexus. A β-endorphin immunofluorescence was visualized within perikarya and nerve fibers of both the myenteric and submucous plexus. α-Melanotropin as well as β-endorphin immunoreactivities were strictly localized to structures of the enteric nervous system. In crypts and epithelial cells only a non-specific staining was observed.     

Release of immunoreactive met-enkephalin from the spinal cord by intraventricular β-endorphin but not morphine in anesthetized rats

Effect of β-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin_1–13 from the spinal cord was studied in anesthetized rats. Intraventricular β-endorphin, 16 μg, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 μg, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin_1–13 was not released by either β-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by β-endorphin had a retention time identical to [³H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for β-endorphin and morphine.     

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.