Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10^?11?10^?5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10^?4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10^?9?10^?7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.     

Corticotropin/beta-endorphin precursor: concomitant storage of its fragments in the secretory granules of anterior pituitary corticotropin/endorphin cells.

Serial semithin und ultrathin sections of Epoxy resin embedded rat anterior pituitaries were sequentially immunostained for corticotropin, β-lipotropin, β-endorphin and 16k-fragment. The 16k-fragment corresponds to the non-β-lipotropin-non-corticotropin part of the corticotropin/β-endorphin precursor. Immunostaining was performed by the peroxidase-antiperoxidase method. Corticotropin, β-lipotropin, β-endorphin and the 16k-fragment were stored in the same cells of the anterior pituitary. Cross absorption experiments confirmed the specificity of the immunocytochemical reactions. At the ultrastructural level immunoreactivities to all these parts of the corticotropin/β-endorphin precursor were found to be stored together in the same secretory granules. It is concluded that under all stimulatory or inhibitory conditions all fragments will be released together during secretion by granule extrusion.     

Affinity purification of β-endorphin-like material from NG108CC15 hybrid cells by means of the monoclonal β-endorphin antibody 3-E7

Neuroblastoma × glioma hybrid cells (NG108CC15) were examined for the presence of β-endorphin-like material. In order to differentiate this β-endorphin-like material from crude cell extract, a procedure for immunoaffinity chromatography was developed. The monoclonal antibody 3-E7 employed possesses the unique property of recognizing the N-terminal sequence of virtually all endogenous opioid peptides, but not their precursors. By means of this immunoaffinity procedure about 90% of exogenous β-endorphin was recovered from 10 ml phosphate buffered saline samples. Affinity chromatography served as first-step purification of crude NG108CC15 cell extract for the separation and concentration of β-endorphin-like material. The eluate of the immunoaffinity gel was subjected either to Sephadex gel filtration or to high pressure liquid chromatography. Under either condition, immunoreactive β-endorphin which eluted with synthetic β-endorphin was detected. The concentration in six different batches varied from 4 to 17 fmol/10⁸ cells. This would be 10–200-fold lower than that observed for the enkephalins or dynorphin A/α-neo-endorphin. It is concluded that the utilization of the monoclonal antibody 3-E7 for a first-step purification of cell extracts was an essential pre-requisite for the separation of β-endorphin-like material from the hybrid cells. The presence of enkephalin-like material, of dynorphin A/α-neo-endorphin-like material and of β-endorphin immunoreactive material suggests that NG108CC15 cells are able to generate opioid peptides related to the precursors pre-proenkephalin A, pre-proenkephalin B and pro-opiomelanocortin.     

Biosynthesis of beta-endorphin, beta-lipotropin and the putative ACTH-LPH precursor in the frog pars intermedia.

When frog pars intermedia are incubated for 3 h with radioactive methionine, the predominant labeled peptide is one with an apparent molecular weight of 33, 100. This peptide can be immunoprecipitated with antisera against β-melanotropin (β-MSH), adrenocorticotrophin (ACTH), and β-endorphin and is believed to be the common precursor of ACTH and β-lipotropin (β-LPH). Immunoprecipitation experiments have also demonstrated the presence of labeled β-LPH and β-endorphin. The labeled β-endorphin has been shown to behave identically to sheep β-endorphin on both carboxymethyl-cellulose chromatography and polyacrylamide gel electrophoresis. Frog β-endorphin has methionine as the fifth residue, as do all other β-endorphins that have been sequenced.     

Isolation of a new low molecular weight beta 2-globulin from urine of a worker with chronic cadmium poisoning

The in vitro biosynthesis of β-endorphin and its subsequent N-acetylation was studied in the pars intermedia/nervosa (PIN) of the rat pituitary. Freshly isolated PINs were incubated with ³H-tyrosine for various periods of time. Tissue extracts were subjected to double-immunoprecipitation with an antiserum raised against β-endorphin. The immunoprecipitated compounds of a molecular weight corresponding to that of β-endorphin were separated from β-endorphin precursor molecules by gel-chromatography and subjected to chymotryptic cleavage. ³H-tyrosine- or N-acetyl-³H-tyrosine-containing peptide fragments were analysed by thin-layer chromatography. It was found that N-acetylation of β-endorphin occurred immediately after its formation from its precursor molecules as a modification step closely linked with the post-translational formation of β-endorphin. Up to 70% of newly synthesized radiolabelled β-endorphin was found to be N-acetylated during the course of prolonged in vitro incorporation.     

Antibodies Specific for α-N-Acetyl-β-Endorphins: Radioimmunoassays and Detection of Acetylated β-Endorphins in Pituitary Extracts

Abstract: Antibodies specific for α-N-acetyl-β-endorphins have been prepared by injecting into rabbits either α-N-acetyl-β-endorphin(1-31) or [α-N-acetyl, ε-acetyl-Lys⁹]-β-endorphin(1-9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α-N-acetyl-β-endorphins. The radioimmunoassays were used to measure α-N-acetylated β-endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β-endorphin and α-N-acetyl-β-endorphin immunoreactivity, a relative ratio of β-endorphin acetylation was obtained. The relative acetylation of β-endorphin was highest in rat posterior-intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β-endorphin. Human pituitary extracts did not contain acetylated β-endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β-endorphin were resolved in extracts of rat posterior-intermediate pituitaries. Two of these peptides corresponded to α-N-acetyl-β-endorphin(1-31) and -(1-27). One acetylated β-endorphin fragment had the same size as α-N-acetyl-β-endorphin(1-27) but was eluted earlier from the cation exchange column. This peptide had full cross-reactivity with antibodies directed against the middle and amino-terminal parts of β-endorphin. Compared with α-N-acetyl-β-endorphin(1-27), it had much less cross-reactivity with antibodies directed against the COOH-terminal part of β-endorphin, suggesting that it was a COOH-terminally modified derivative of β-endorphin(1-27). The remaining N-acetylated β-endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y-endorphin, i.e., β-endorphin(1-17), but smaller than β-endorphin(1-27). They had full cross-reactivity in an amino-terminally directed β-endorphin radioimmunoassay and a greatly diminished cross-reactivity with antibodies to the middle region of β-endorphin.     

Sex Differences in the Content of β-Endorphin and Enkephalin-Like Peptides in the Pituitary of Obese (ob/ob) Mice

Abstract: The β-endorphin content in pituitary extracts of male and female obese (ob/ob) and lean (+/?) mice was determined by radioimmunoassay. The amount of β-endorphin-like material contained in the pituitary of 3-month-old ob/ob male mice is similar to that of lean male mice. In contrast, the pituitary glands of female ob/ob mice have a greater amount of β-endorphin-like material than lean female mice. To determine with greater precision the molecular nature of the polypeptide that accounts for the increase in β-endorphin immunoreactivity, the various molecular forms of β-endorphin immunoreactivity were resolved by Biogel P-30 column chromatography. At least four peaks of immunoreactive material were detected. The first peak elutes in the void volume, and the second and the third peaks appear in the elution volumes of β-lipotropin and β-endorphin, respectively. That the material present in the void volume might be proopiocortin is supported by adrenocorticotropic hormone radioimmunoassay. The increased total β-endorphin immunoreactivity in pituitary glands of ob/ob mice is accounted for mainly by β-endorphin. The β-endorphin content of various brain structures of ob/ob mice is similar to that of lean littermates.     

β-endorphin proteolysis by guinea-pig ileum myenteric plexus membranes: Increased γ-endorphin turnover after chronic exposure to morphine

The influence of chronic morphine exposure $\text{in vitro}$ on the biotransformation of β-endorphin (βE) was investigated using the myenteric plexus-longitudinal muscle of guinea-pig ileum. A membrane preparation was incubated with βE and the degradation of βE as well as the accumulation of several βE fragments in the incubation medium were followed with time. The levels of peptides were determined by specific radioimmunoassays after separation by high-pressure liquid chromatography. It was found that exposure to morphine did not affect the disappearance of βE, but altered the time course of accumulation of βE fragments. In fact, the accumulation of γ-endorphin, α-endorphin and des-tyrosine¹-α-endorphin was enhanced, while that of des-tyrosine¹-γ-endorphin was not changed. Additionally, the disappearance of γ-endorphin appeared to be stimulated by morphine exposure. These data provide evidence that the fragmentation of βE is changed by chronic morphine exposure in such a way that the turn-over of γ-endorphin is increased.     

Effects of peptides of pituitary origin on the formation of C21 steroid by fetal calf adrenal cells in culture.

Corticotrophic activity of opiate-like peptides was assessed by their ability to stimulate the formation of C₂₁ steroids from [³H] progesterone by three-day old cultures of fetal calf adrenal cells. ACTH_1–39, ACTH_α1–24 and a purified preparation of pituitary ovine β-endorphin caused a marked increase in 17α and 21-hydroxylation while a preparation of pure synthetic porcine β-endorphin gave a minimal stimulation. The activity of the purified ovine β-endorphin preparation could not be accounted for by contamination by ACTH or by a synergistic action between the two peptides. The novel pituitary factor described here may be due to a contaminant of the β-endorphin peak which is different from ACTH_1–39.     

Effects of β-endorphin on brain serotonin metabolism

Human β-endorphin (15 μg) administered intracisternally increased concentrations of serotonin (5HT) and its metabolite, 5-hydroxyindoleacetic. acid (5-HIAA), in brain stem and hypothalamus and decreased 5-HIAA concentrations in hippocampus. These data are compatible with the hypothesis that β-endorphin increases 5HT turnover in brain stem and hypothalamus and decreases 5HT turnover in hippocampus. β-endorphin increased in brain stem and hypothalamus and decreased in hippocampus the rate of pargyline-induced decline of 5-HIAA. β-endorphin decreased the rate of pargyline-induced accumulation of 5HT in all these brain regions. The probenecid-induced accumulation of 5-HIAA in brain stem was decreased by β-endorphin. These data are compatible with the hypothesis that β-endorphin increases release of 5HT from neurons in brain stem and hypothalamus and decreases release of 5HT from neurons in hippocampus. The data require further a hypothesis that β-endorphin either decreases 5HT reuptake in these three brain regions or increases 5-HIAA egress from brain.     

Radioimmunoassay of brain peptides: Evaluation of a methodology for the assay of β-endorphin and enkephalin

The brain levels of β-endorphin, α-endorphin and enkephalin were measured by radioimmunoassay after different methods of sacrifice. Microwave irradiation proved not to be better than decapitation followed by boiling of the intact tissue, the latter procedure giving values of β-endorphin 10 fold higher than decapitation alone. Concurrently when decapitation was followed by boiling, α-endorphin was no longer detected. Evaluation in brain tissue of several extraction media--phosphate buffered saline, 5% TCA, HCl methanol, and 1N HOAc--showed the last to be the most satisfactory for both β-endorphin and enkephalin. Since β-endorphin was found to be readily hydrolized by brain homogenates with consequent appearance of α-endorphin, these results indicate that disruption of tissue modifies the content of opioid peptides in brain.     

Content of beta-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Radioimmunoassays (RIAs) specific for β-LPH_1–47, β-endorphin, α-MSH and β-MSH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50. When components in extracts of both lobes, eluting at the same position, were measured with the β-endorphin and β-LPH_1–47 RIA systems, marked quantitative differences were seen. The main components reacting with the β-LPH_1–47 system in anterior pituitary extract co-migrated with β-LPH and γ-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than β-endorphin. When the β-endorphin RIA system was used, relatively low amounts of immunoreactive material co-migrating with β-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than β-endorphin was observed in intermediate lobe extract. Some β-MSH was seen in the intermediate lobe. These date indicate that the processing of β-LPH is markedly different in the anterior and intermediate bovine pituitary lobes: β-endorphin immunoreactive material predominates in the intermediate lobe whereas β-LPH and γ-LPH predominate in the anterior lobe.     

Localization of multiple forms of ACTH- and β-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Molecular interpretation of ACTH-β-endorphin coaggregation: relevance to secretory granule biogenesis

Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.     

Endotoxin-induced formation of specific β-endorphin binding sites in human serum

Specific binding of human β-endorphin is demonstrated to sites that are present in endotoxin-treated, but not in native, human serum. Binding is saturable and reversible and is mediated by a nonopioid segment of β_H-endorphin to sites that are located on the terminal SC5b-9 complement complex. Since endotoxemia causes both the secretion of β-endorphin into blood (1) and the formation of specific β-endorphin binding sites therein, both effects might be elements of a common physiological process.     

Isoelectric point determination of human and camel beta-endorphin, alpha-endorphin and enkephalins

The isoelectric point of the camel and the human β-endorphin, of the α-endorphin and the enkephalins were determined by analytical isoelectric focusing on 1 mm thin polyacrylamide gel slab. The difficulty of staining peptides as short as β-endorphin or smaller was overcomed using a modification of Bibring and Baxandall's or Faupel and Von Arx's staining method. The camel β-endorphin gives two bands having isoelectric point of 10.3 and 10.4, the human β-endorphin focus at pH 9.9, while α-endorphin, leu and met-enkephalin at pH 5.9, 5.5 and 5.45 respectively. The staining method described coupled with the isoelectric focusing seems to be fit for discriminating β-endorphin in a crude rat pituitary extract.     

Beta-Endorphin: lack of correlation between opiate activity and immunoreactivity by radioimmunoassay.

A sensitive radioimmunoassay for human β-endorphin has been developed. When natural human β-lipotropin and various synthetic analogs of β-endorphin were assayed for their immunoreactivity, lack of correlation was found between opiate activity and immunoreactivity. These data suggest that residues 6–15 of β-endorphin are the antigenic determinant.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.