N-acetyl Beta-endorphin Research Articles

Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio).

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.

Beta-endorphin inhibits the production of interleukin-8 by human chorio-decidual cells in culture.

Interleukin-8 (IL-8) is produced by human decidual cells in culture, and may play a role in the initiation of parturition. beta-endorphin is released in significant amounts into the maternal and fetal circulation during labour. The effect of beta-endorphin on IL-8 production by human chorio-decidual cells in culture was investigated. Mixed cells were obtained from the decidual surfaces of 35 term placentas. The cells were plated out at 10x10(6) cells per well in Roswell Park Memorial Institute 1640 culture medium. After 48 h the cells were washed and incubated with either plain culture medium (control), 1 micromol/l progesterone, 1-100 nmol/l beta-endorphin or 1 nmol/l N-acetyl beta-endorphin. After 48 h, IL-8 concentrations were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Experiments were repeated in the presence of naloxone (1 micromol/l) and using calcium-deficient culture medium. Progesterone (P < 0.0002) and beta-endorphin (P < 0. 0005) significantly inhibited the production of IL-8. The inhibitory effect of beta-endorphin was blocked by naloxone and by using calcium-deficient medium. N-acetyl beta-endorphin had no significant effect on IL-8 production. These findings suggest that beta-endorphin has an inhibitory effect on IL-8 production by decidual cells, and that the effect is mediated via opioid receptors and is calcium-dependent.

Peripheral plasma levels of beta-endorphin in alcoholics and highly trained athletes and the relationship to a measure of central opioid tone.

Human beta-endorphin-like immunoactivity was measured in highly trained athletes (n = 10), alcoholics in the early phase of abstinence (n=9) and normal controls (n=15) using the Nichols Allegro immunoradiometric assay. The assay was examined for cross reactivity against related peptides, beta-lipotropin and human N-acetyl beta-endorphin. Venous blood sampling was carried out in the morning at 0900 and 1100 hours in a fasting state. Using two-way analysis of variance there was a significant effect of subject group on beta-endorphin concentration (p=0.029). Post-hoc analysis using the Bonferroni t-test showed that the source of the difference was the alcoholic group having significantly lower beta-endorphin immunoreactivity (p < 0.05). There was no difference between the controls and the athletes. There was a positive correlation between plasma beta-endorphin level at 1100 hours and the subsequent ACTH incremental response to naloxone in the group as a whole (r=0.48, p=0.004). The assay showed 100% cross reactivity with beta-lipotropin and 73% cross reactivity with N-acetyl-beta-endorphin. We conclude that alcoholics have reduced levels of beta-endorphin-like immunoactivity. While beta-endorphin is known not to cross the blood-brain barrier, levels of plasma beta-endorphin-like immunoactivity may indirectly reflect central opioid activity.

Melanocortin receptors and δ‐opioid receptor mediate opposite signalling actions of POMC‐derived peptides in CATH.a cells

The locus coeruleus is innervated by proopiomelanocortin (POMC)-derived peptide immunoreactive fibres. The biological effects of ( melanocyte-stimulating hormone (aMSH) and [-endorphin on second messengers (cAMP, inositol phosphates) and gene transcription were studied in the locus cceruleus-derived cell line CATH.a. RT-PCR analysis revealed the presence of four MSH receptor subtypes (1, 3, 4 and 5). Activation of these receptors by diacetyl alphaMSH stimulated cAMP accumulation in a dose-dependent manner (EC50: 4 x 10(-9) M). Diacetyl alphaMSH stimulated transcription from reporter genes driven by the c-fos or tyrosine hydroxylase promoter. This effect was abolished when protein kinase A was inactivated with a dominant inhibitory mutant. RT-PCR analyses revealed the presence of delta-, but not mu-and kappa-opioid receptor. Pharmacological analysis showed that beta-endorphin (EC50: 2.5 x 10(-8)M), but not N-acetyl beta-endorphin, antagonized the biological effect of diacetyl alphaMSH on cAMP production and gene transcription. Since N-acetylation regulates the biological activity of alphaMSH and beta-endorphin in an opposite manner, we propose a model where the rate of secretion dictated by the bioelectric activity of the presynaptic neuron modulates POMC-derived peptide maturation and the resulting biological signal sensed by the postsynaptic plate.

POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: differential subcellular processing in the Golgi and secretory granules.

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.

Alpha-melanocyte-stimulating hormone and N-acetyl-beta-endorphin immunoreactivities are localized in the human pituitary but are not restricted to the zona intermedia.

The adult human pituitary lacks a well defined intermediate lobe, and it is uncertain whether the POMC cells that remain in the zona intermedia represent melanotropes or corticotropes. In the present study, we investigated whether the N-acetylated beta-endorphin- and alpha-MSH-related peptides that are characteristically produced by melanotropes in the rat and other species are localized in the human pituitary. Sequential gel filtration and ion exchange HPLC analysis revealed that small amounts of alpha-N-acetyl-beta-endorphin-(1-31), as well as beta-endorphin-(1-27) and beta-endorphin-(1-26), were detectable in human pituitary extracts, although beta-endorphin-(1-31) was clearly the major form. Consistent with this analysis, low levels of alpha-MSH, but not N,O-diacetyl-alpha-MSH, were identified by reverse-phase HPLC, although again, the desacetyl form of alpha-MSH predominated. Immunohistochemistry revealed that N-acetyl-beta-endorphin immunoreactivity was colocalized with ACTH and beta-endorphin in a subpopulation of zona intermedia cells. Unexpectedly, immunoreactive N-acetyl-beta-endorphin was also observed in a comparable proportion of corticotropes dispersed throughout the anterior lobe. alpha-MSH immunoreactivity was similarly distributed. These results indicate that N-acetylation is not restricted to the zona intermedia, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in the rat and other species does not extend to the human pituitary.

Alpha-melanocyte-stimulating hormone and N-acetyl-beta-endorphin immunoreactivities are localized in the human pituitary but are not restricted to the zona intermedia

The adult human pituitary lacks a well defined intermediate lobe, and it is uncertain whether the POMC cells that remain in the zona intermedia represent melanotropes or corticotropes. In the present study, we investigated whether the N-acetylated beta-endorphin- and alpha-MSH-related peptides that are characteristically produced by melanotropes in the rat and other species are localized in the human pituitary. Sequential gel filtration and ion exchange HPLC analysis revealed that small amounts of alpha-N-acetyl-beta-endorphin-(1-31), as well as beta-endorphin-(1-27) and beta-endorphin-(1-26), were detectable in human pituitary extracts, although beta-endorphin-(1-31) was clearly the major form. Consistent with this analysis, low levels of alpha-MSH, but not N,O-diacetyl-alpha-MSH, were identified by reverse-phase HPLC, although again, the desacetyl form of alpha-MSH predominated. Immunohistochemistry revealed that N-acetyl-beta-endorphin immunoreactivity was colocalized with ACTH and beta-endorphin in a subpopulation of zona intermedia cells. Unexpectedly, immunoreactive N-acetyl-beta-endorphin was also observed in a comparable proportion of corticotropes dispersed throughout the anterior lobe. alpha-MSH immunoreactivity was similarly distributed. These results indicate that N-acetylation is not restricted to the zona intermedia, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in the rat and other species does not extend to the human pituitary.

Des-acetylated variants of α-melanocyte-stimulating hormone and β-endorphin can antagonize the mammotrope-recruiting activity of their acetylated forms

We have previously reported that hypophysial neurointermediate lobe peptides, di-acetylated alpha-melanocyte-stimulating hormone (di-ac-alpha-MSH) and N-acetylated beta-endorphin (N-ac-beta-END), can acutely increase the relative number of prolactin-secreting cells in anterior pituitary cell cultures from ovariectomized rats. Inasmuch as the des-acetylated forms of these peptides (des-ac-alpha-MSH and beta-END) were not effective in this regard, we concluded that acetylation was an absolute requirement for manifestation of the recruitment response. The aim of the present study was to determine whether these des-acetylated variants could antagonize the mammotrope-recruiting activity of their acetylated congeners. Treatment of anterior pituitary cell cultures with di-ac-alpha-MSH and N-ac-beta-END increased the relative amount of prolactin secretors above control values. Interestingly, des-acetylated variants of alpha-MSH and beta-END blocked the mammotrope-recruitment activity of their respective acetylated forms. In addition, beta-END antagonized the mammotrope-recruitment activity of di-ac-alpha-MSH while des-ac-alpha-MSH did not attenuate the stimulatory effect of N-ac-beta-END. Given that mammotropes maintained in vivo are exposed to all these peptides, it is possible that these acetylated and non-acetylated congeners may act in an opposing manner to regulate dynamic prolactin release.

Beta-endorphin is a potent inhibitor of thymidine incorporation into DNA via mu- and kappa-opioid receptors in fetal rat brain cell aggregates in culture.

Thymidine incorporation into DNA was inhibited dose-dependently by beta-endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by mu (cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr- Pen-Thr amide) or kappa (norbinaltorphimine) antagonists. Complete blockade of the beta-endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie-Hofstee analysis revealed that beta-endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, beta-endorphin decreased thymidine incorporation with an IC50 of 0.7 nM. Truncated and N-acetylated beta-endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that beta-endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.

Effects of Pentobarbital on Proopiomelanocortin Opioid Products of Neonatal Piglets During Normoxia and Hypoxia

Abstract Endogenous opioids have been shown to suppress physiological functions in the neonate. It has been suggested that anesthesia with barbiturates might enhance this suppression by influencing opioid systems directly. To explore this possibility, naive piglets, 2.2+/-0.8 (X+/-SD) days old, underwent one of five protocols: 1) normoxia (control); 2) 10% 0(2)/90% N(2) (hypoxia); 3) saline injection ip during normoxia (sham anesthesia); 4) pentobarbital sodium, 25 mg/kg ip, during normoxia (barbiturate anesthesia); and 5) pentobarbital sodium, 25 mg/kg ip, during hypoxia (combined hypoxia and barbiturate anesthesia). Following the inhalation of either gas mixture for at least 30 min, and precisely 30 min after an injection, blood, cerebrospinal fluid and a dorsal medullary slice containing the nucleus tractus solitarii were collected and processed for measurement by radioimmunoassay of opioid proopiomelanocortin products. These comprised beta-lipotropin (the precursor), beta-endorphin-like immunoreactivity (containing the active peptide beta-endorphin) and N-acetyl beta-endorphin (a deactivated peptide). The most striking result was seen in the cerebrospinal fluid: As compared to barbiturate anesthesia, peptide levels with all other treatments, including combined hypoxia and barbiturate anesthesia, were consistently higher. In the plasma, peptide levels after either combined hypoxia and barbiturate anesthesia or hypoxia alone were generally higher than those of their respective controls (sham anesthesia, control). Plasma levels of beta-endorphin-like immunoreactivity and estimated beta-endorphin with combined hypoxia and barbiturate anesthesia were also higher than those with barbiturate anesthesia. The latter pattern was reversed in the nucleus tractus solitarii, in which beta-endorphin-like immunoreactivity and estimated beta-endorphin levels were lower with combined hypoxia and barbiturate anesthesia than with barbiturate anesthesia alone, although no significant differences were achieved. These results suggest that pentobarbital may decrease the central neuronal release of active endorphins, and thus decrease the quantity of these ligands available for interaction with opioid receptors. Hypoxia, on the other hand, appears to increase such release even in the presence of pentobarbital. Thus, during a hypoxic insult, the suppressive influence of opioids on physiological functions would be enhanced regardless of the presence of barbiturate anesthesia.

Enhancement of germ tube formation in Candida albicans by ß-endorphin

Candida albicans is a dimorphic yeast that causes vaginal infections after its transition from a budding yeast to a germinating hyphal form. We report here that physiologic concentrations of beta-endorphin, a neuropeptide with immunomodulating activity produced during stress or physical exercise, stimulates germ tube formation in C. albicans. The percent of germination was proportional to the endorphin concentration, over the 5 x 10(-12) to 5 x 10(-10) mol/L range tested. beta-Endorphin modified by removal of the 4-carboxy-terminal amino acids and (D-Ala2)-beta-endorphin, a peptide with a protease-resistant amino terminal end, were equally effective in stimulating germination. In contrast, N-acetylated beta-endorphin did not stimulate germination. Antisera to beta-endorphin also completely blocked beta-endorphin-stimulated germ tube formation. Two clinical isolates of C. albicans were also responsive to beta-endorphin. Stimulation of germ tube formation by beta-endorphin occurred only in sera from ovulating women. Germination in sera from women using oral contraceptives, in sera from men, or in glucose beef extract broth was not influenced by beta-endorphin. Thus C. albicans may be able to recognize and respond to neuroendocrine signals in ovulating women.

Differential mechanisms for the N-acetylation of alpha-melanocyte-stimulating hormone and beta-endorphin in the intermediate pituitary of the frog, Xenopus laevis.

Immunohistochemical analysis of the pituitary of Xenopus laevis revealed the colocalization of alpha-melanocyte-stimulating-hormone (MSH)-related immunoreactivity and N-acetyl-beta-endorphin-related immunoreactivity in the cells of the intermediate pituitary. In order to determine whether the immunoreactive N-acetylated beta-endorphin is released in parallel with the immunoreactive alpha-MSH, intermediate pituitaries were incubated in L-15 medium for 24 h. The medium and an acid extract of the intermediate pituitaries from each incubation were separately fractioned by a combination of gel filtration chromatography, reverse-phase high-performance liquid chromatography, and cation exchange chromatography. In the intermediate pituitary extract, the major form of alpha-MSH had chromatographic properties which corresponded to nonacetylated alpha-MSH (ACTH)(1-13)amide; whereas the major form of beta-endorphin had an apparent molecular weight of 1.2 kDa and was N-acetylated. The 1.2-kDa form of beta-endorphin and ACTH(1-13)amide were present in equimolar amounts. Analysis of the medium indicated that both end products were released in parallel. However, as reported in the literature, there was a significant increase in the N-acetylation of ACTH(1-13)amide during secretion. There was no further processing of beta-endorphin during secretion. Collectively, these observations indicate that in the intermediate pituitary of X. laevis there are separate mechanisms for the N-acetylation of alpha-MSH and beta-endorphin.

Induction of the Intermediate Lobe Pro-Opiomelanocortin System with Chronic Swim Stress and β-Adrenergic Modulation of this Induction

Swimming at 25-30 degrees C for 30 min stimulates release of beta-endorphin from both the anterior and intermediate lobe of the pituitary in rats. Measurement of N-acetyl beta-endorphin-immunoreactivity (IR), which is specific for intermediate lobe secretion, indicates a 2- to 3-fold increase in N-acetyl beta-endorphin IR in plasma following this challenge. When swim is repeated on a daily basis, there is an increase in the amount of N-acetyl beta-endorphin IR released with repeated swim over time. As well as increased response to the swim challenge, these animals demonstrate an increase in the resting plasma levels of N-acetyl beta-endorphin IR and an increase in the intermediate lobe content of N-acetyl beta-endorphin IR. Molecular sieving of plasma from rats which were swum repeatedly demonstrates that this N-acetyl beta-endorphin IR consists of both larger molecular weight N-acetyl beta-endorphin IR, e.g. N-acetyl beta-endorphin1-31 and C-terminally shortened forms, e.g. N-acetyl beta-endorphin1-27. Administration of propranolol (3 mg/kg), a beta-adrenergic antagonist, 30 min before the onset of swim is able to block the intermediate lobe release of N-acetyl beta-endorphin IR with acute swim challenge. However, repeated administration of propranolol in conjunction with repeated swim is not able to block the swim stress-induced increase in plasma N-acetyl beta-endorphin IR or the increase in N-acetyl beta-endorphin IR content of the intermediate lobe. This is not due to decreased sensitivity to propranolol with repeated administration since in rats given chronic propranolol treatment an acute dose of propranolol is still able to block swim stress-induced release of N-acetyl beta-endorphin IR. Similarly, it is not due to a decreased efficacy of this dose of propranolol in rats which were swum chronically.

Induction of the intermediate pituitary by stress: synthesis and release of a nonopioid form of beta-endorphin.

beta-Endorphin in the intermediate lobe of the pituitary gland is posttranslationally modified to produce opioid inactive peptides. Whether these are metabolites or biologically relevant products has not been known. It was found that repeated stress induces increased biosynthesis and release of beta-endorphin-like substances from the intermediate lobe of rats and that opioid-inactive N-acetylated beta-endorphin-(1-31) is selectively made and liberated. The possible role of this nonopioid product and the selective release of peptide forms are discussed.

Postnatal ontogeny of acetylated and non-acetylated B-endorphin in rat pituitary

Extracts of the anterior lobe and intermediate lobe of postnatal (P) (Day P1, P7, P14, P21, P28, P35, P42) and adult male Spraque-Dawley rats were analyzed by both a Beta-endorphin (B-END) radioimmunoassay and a radioimmunoassay for N-acetyl-B-END. In the anterior lobe, on P1, less than 2% of the adult level of B-END was present. By P42 this level had increased to 21% of adult levels. In the intermediate lobe, on P1, the B-END levels were less than 0.1% of the adult level, and by P42 this level approached approximately 45% of the adult levels. N-acetylated B-END was identified in both anterior lobe and intermediate lobe from P1 through adulthood. In the anterior lobe at P1, N-acetyl-B-END immunoreactivity contributes approximately 25% of the total B-END immunoreactivity. This level drops to less than 10% by P21, and to adult-like levels by P42 (less than 5%). On the other hand, in the intermediate lobe, the N-acetyl-B-END levels start at 70% of the total B-END immunoreactivity at P1 and by P14 reaches adult-like proportions of 90% or more of the total B-END immunoreactive material.

Biosynthesis and release of beta-endorphin-, N-acetyl beta-endorphin-, beta-endorphin-(1-27)-, and N-acetyl beta-endorphin-(1-27)-like peptides by rat pituitary neurointermediate lobe: beta-endorphin is not further processed by anterior lobe

Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive beta-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Cell and medium extract immunoreactive beta-endorphin (specific immunoprecipitation and radioimmunoassay) exhibiting a Kav similar to authentic beta-endorphin upon gel filtration was characterized further by nonequilibrium isoelectric focusing, cation exchange chromatography, reverse phase high pressure liquid chromatography, and partial tryptic and chymotryptic mapping. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive beta-endorphin. Four of these forms were physicochemically similar to authentic beta-endorphin, N-acetylated beta-endorphin, beta-endorphin-(1-27), and N-acetylated beta-endorphin-(1-27). Pulse-chase studies indicated that a beta-lipotropin-like molecule served as a metabolic intermediate for a beta-endorphin-like molecule. As beta-endorphin-like material accumulated in the cell, some of it was N-acetylated (approximately 18% at 2 hr chase and approximately 65% at 18 hr chase). At later chase times, beta-endorphin-(1-27)- and N-acetylated beta-endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (10(-8) M norepinephrine), or inhibitory (10(-7) M dopamine) incubation conditions. In all cases, beta-endorphin-(1-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive beta-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic beta-endorphin molecule. Continuous labeling (72 hr) revealed the beta-lipotropin/beta-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the beta-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a biosynthetic intermediate.