Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum.

β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [¹²⁵I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At different time intervals the release of [¹²⁵I]-tyrosine or the change in immunoreactivity of the endorphins was determined. The cSPM preparation displayed both high aminopeptidase and endopeptidase activities. In contrast, human serum mainly contained aminopeptidase activity. The data suggest that functional endorphin metabolism may occur at the synaptosomal plasma membrane. These membranes may potentially be involved in the formation of behaviorally active endorphin fragments.     

Generation of methionine and leucine-enkephalin from precursor molecules by cation-sensitive neutral endopeptidase of bovine pituitary

Highly purified preparations of cation-sensitive neutral endopeptidase, from bovine pituitary, and also rabbit brain, generate methionine-enkephalin, from α-endorphin, a peptide containing the amino acid sequence 61–76 of β-lipotropin (β-LPH), The enzyme also catalyzes the hydrolysis of the Leu-Thr bond in the synthetic peptide Tyr-Gly-Gly-Phe-Leu-Thr-2-naphthylamide with the release of leucine-enkephalin and Thr-2-naphthylamide. Neither Met- nor Leu-enkephalin are degraded. The data indicate that the presence of a free N-terminal group of tyrosine inhibits the further degradation of Leu- and Met-enkephalin by the endopeptidase. It is suggested that cation-sensitive neutral endopeptidase is one of the enzymes capable of generating Met- and Leu-enkephalin $\text{in}\text{,}\text{vivo}$.     

Conversion of Des-tyrosine-γ-endorphin by brain synaptic membrane associated peptidases: Identification of generated peptide fragments

Des-tyrosine-γ-endorphin, a β-endorphin fragment with neuroleptic-like properties, was digested with a cSPM fraction of rat brain. A profile of metabolites and a time course of conversion were obtained by HPLC analysis of the digests. Quantitative amino acid analysis and a second HPLC fractionation step which was designed to separate and to identify very similar des-tyrosine-γ-endorphin fragments, combined with dansyl end group determination allowed the characterization of β-LPH 65–77, β-LPH 66–77 and β-LPH 62–73 as main conversion products. In the digests the C-terminal leucyl peptides β-LPH 67–77 and β-LPH 68–77 as well as the N-terminal glycyl peptides β-LPH 62–74 and β-LPH 62–76 were minor components. The data indicate the involvement of several types of peptidase activities in the conversion process. It is suggested that these peptidases have a role in mediating in vivo des-tyrosine-γ-endorphin effects. In addition, this study points to the capacity of the brain to gene-rate small peptides with neuroleptic-like properties.     

The influence of beta-LPH fragments on alpha-MSH release: the involvement of a dopaminergic system

β-Endorphin was able to enhance plasma α-MSH levels in rats after intracerebroventricular injection. This effect could be inhibited by naloxone or by removing tyrosine from position 61 of the peptide. Neither α- and γ-endorphin nor their des-tyrosine analogs appeared to be able to modify plasma α-MSH levels. The stimulating effect of β-endorphin on plasma α-MSH levels could be completely blocked by a simultaneous injection of apomorphine, in an amount in which apomorphine itself had no effect on α-MSH levels in plasma. A single injection of haloperidol increased plasma α-MSH levels in a dose related manner. A dose of haloperidol, which caused an apomorphine antagonizable increase in plasma α-MSH, did not modify β-endorphin elevated α-MSH levels. A high concentration of haloperidol was able to stimulate the basal release of α-MSH from isolated pituitaries $\text{in bitro}$, whereas β-endorphin appeared to be inactive in this respect.These observations indicate a central opiate receptor-mediated influence of β-endorphin on α-MSH release and the possible involvement of a dopaminergic system, mediating the β-endorphin effect.     

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.     

Solid phase synthesis of porcine α-endorphin and γ-endorphin,two hypothalamic-pituitary peptides with opiate activity

The synthesis by solid phase methodology of α-endorphin (Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-OH) and γ-endorphin (Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH), two morphinomimetic peptides isolated from pig hypothalamus-pituitary extracts, is described. The sequences of these two peptides correspond to residues 61–76 and 61–77, respectively, of porcine β-lipotropin. The two synthetic compounds were shown to have the same physical, chemical and opiate activity as the respective native substances.     

Modulation of in vitro release of beta-endorphin from the separate lobes of the rat pituitary.

The rate of in vitro release of β-endorphin immunoreactivity from the anterior lobe of rat pituitary increased in response to hypothalamic extract and lys-vasopressin. Lys-vasopressin, at a low concentration, initiated a pronounced (5–6 fold) dose-dependent, parallel increase in the release of β-endorphin and ACTH from the anterior lobe. Corticosterone (5·10^?7 M) did not influence basal but could suppress such stimulated release. These stimulants did not, however, change the rate of release from the intermediate/posterior lobe.Chromatography of incubation media showed that β-endorphin and β-lipotropin were released in parallel from the anterior lobe but only β-endorphin from intermediate/posterior lobe tissue.These findings suggest that the β-endorphin pools in anterior and intermediate lobes differ both in their mechanism of release and in the regulation of this process.     

Presence of immunoassayable beta-lipotropin in bovine brain and spinal cord: lack of concordance with ACTH concentrations.

Discrete areas of freshly obtained adult bovine brain were assayed for their content of immunoreactive β-lipotropin (β-LPH), ACTH and β-endorphin. Highest concentrations (pg/100ug protein) of β-LPH were present in hypothalamus (517 ± 81), hippocampus (218 ± 60), central grey rostral mesencephalic level, pons, striatum, and spinal cord (163–258). Lesser concentrations (49–138) were present in other parts of the limbic system, brain stem, cortex and thalamus. Immunoreactive ACTH concentrations were highest in hypothalamus (1702 ± 487) and hippocampus (210 ± 40), with markedly lesser concentrations (5–24) being present in all the other aforementioned areas. Immunoreactive β-endorphin concentrations in hypothalamus were 1990 ± 510, in hippocampus 280 ± 50.     

Endorphins and the central inhibition of urinary bladder motility

The involvement of endogenous opioid mechanisms in the central neurogenic control of urinary bladder function has been examined in anesthetized rats. Intracerebroventricular (ICV) microinjection of β-endorphin (0.5–2.0 μg) produced powerful inhibition of rhythmic bladder contractions initiated by central reflex activity. The peptide fragments γ-endorphin and α-endorphin (4–16 μg), formed by the processing of β-endorphin by membrane homogenates of brain, were less active than the parent compound. The inhibitory effects of β-endorphin was reversed by ICV naloxone (1–2 μg) but higher doses were required to reverse γ- or α-endorphin effects. ICV naloxone administered alone increased intravesicular pressure and bladder contraction frequency. These observations support the hypothesis that the endorphins have a physiological role in the central regulation of urinary bladder activity.     

Beta-endorphin induced akinesia in rats: effect of apomorhine and alpha-methyl-p-tyrosine and related modifications of dopamine turnover in the basal ganglia

β-Endorphin (amino acid sequence 61–91 of β-lipotropin) administered intraventricularly at a dose of 13 n moles in rat induced akinesia and loss of corneal reflex. Apomorphine (20 mg/kg) which had been injected subcutaneously 20 minutes after the administration of β-endorphin fully reversed akinesia and elicited characteristic stereotyped behavior. During complete disappearance of akinesia, the corneal reflex was found to be still absent. Apomorphine (5 mg/kg) only partially reversed akinesia. Pretreatment with α-methyl-p-tyrosine (α-MT, 250 mg/kg) potentiated the effect of β-endorphin upon muscle rigidity. In a biochemical study, rats received β-endorphin (15 n moles) 60 minutes before sacrifice. Concentrations of dopamine (DA) and norepinephrine (NE) were not altered in any brain regions. A significant increase in concentrations of 5-hydroxytryptamine was obtained in the midbrain. In a DA and NE turnover study, rats received α-MT (250 mg/kg) 4 hours prior to β-endorphin and were sacrificed 60 minutes later. β-Endorphin partially corrected the decreased concentrations of DA induced by α-MT in the midbrain. A similar tendency toward correction of the decreased DA concentrations was observed in the striatum. The concentrations of NE decreased by α-MT in the midbrain, striatum and hypothalamus were not modified by β-endorphin     

Effect of bacitracin on the biodegradation of β-endorphin

β-endorphin was incubated with rat brain homogenate, and the amino acids released were measured by amino acid analysis. Phe, Leu, Tyr, and Lys were liberated in the greatest amount indicating that the cleavage of Leu⁷⁷-Phe⁷⁸ and some Lys-X peptide bonds with endopeptidases followed by the removal of the terminal residues by exopeptidases are the main routes of β-endorphin degradation in the brain. Bacitracin considerably reduced the amino acid release from β-endorphin incubated with rat brain homogenate, and its action is suggested to be due to the inhibition of brain amino- and carboxypeptidases. Bacitracin also potentiated and prolonged the $\text{in vivo}$ analgesic activity of β-endorphin.     

Low permeation of systemically administered human ß-endorphin into rabbit brain measured by radioimmunoassays differentiating human and rabbit ß-endorphin

Two antisera against human β-endorphin were generated in rabbits. They were found to differ largely in their specificities. One antiserum did not recognize rabbit β-endorphin. This antiserum was used to investigate the permeation of human β-endorphin into rabbit brain and cerebrospinal fluid after systemic injection of the synthetic peptide (50 μg/kg). Over a period of two hours, a low but significant permeation was found to occur only into the hypothalamus. All other brain areas remained below radioimmunoassay detection limits of 100 fmoles/g. Post-injectional cerebrospinal fluid concentrations of human β-endorphin showed very low values (90 fmoles/ml maximally). A regional distribution of rabbit brain β-endorphin, very similar to other species, was found using the antiserum which detected rabbit β-endorphin.     

β-Endorphin inhibits met-enkephalin breakdown by a brain aminopeptidase: Structure-activity relationships

An aminopeptidase solubilized and isolated from rat brain membranes selectively splits the Tyr¹-Gly² peptide bond of Met-enkephalin. β_h-Endorphin, which is apparently resistant to the aminopeptidase, inhibited the action of this peptidase on Met-enkephalin degradation competitively; the K_i value was 11.5 μM. Arg⁰-β_h-endorphin was found to be 10 times more potent than β_h-endorphin. From further structure-activity data it is concluded that the N-terminal amino group and some residues within region 18–31 of the β-endorphin structure are cooperatively involved in binding to the active site of the aminopeptidase.     

Comparison of the anticonvulsant effects of opioid peptides and etorphine in rats after icv administration

The effect of acute icv administration of β-endorphin (5–160 μg), D-ala²-D-leu⁵-enkephalin (DADL; 5–160 μg), D-ala²-met-enkephalinamide (DAME; 10–160 μg), and etorphine (0.05–1.6 μg) on brain excitability was studied by measuring flurothyl seizure thresholds in rats. Each test compound produced a behavioral stupor characterized by muscle rigidity, exophthalmos, and the absence of spontaneous movement. Wet-dog shakes occured only after injection of the opioid peptides. All four compounds produced a dose-related increase in seizure threshold. Naloxone antagonized the behavioral and anticonvulsant effects; the increase in seizure threshold induced by β-endorphin was the most resistant to naloxone. These results indicate that the opioid peptides, in addition to their known EEG epileptogenic potential, are also anticonvulsant in the rat, thus raising the possibility of a dual action for the opioid peptides on central nervous system excitability.     

Alpha-endorphin, gamma-endorphin and their des-tyrosine fragments in rat pituitary and brain tissue

Enkephalins, endorphins and related peptides were determined in pituitary and brain tissue of rats which were killed by decapitation or microwave irradiation. The tissues were heated in 1M acetic acid prior to homogenization and the levels of the various peptides were measured by means of a combination of HPLC and radioimmunoassays. Enkephalin levels in pituitary and brain of irradiation-killed rats were much higher as compared to those in tissue of rats sacrificed by decapitation. Similar data were obtained with respect to pituitary levels of γ-endorphin, des-Tyr-γ-endorphin and des- Tyr-α-endorphin. However, brain levels of α- and γ-endorphin and their respective des-Tyr-fragments were not different with the two methods of sacrifice used. The concentrations of β-endorphin in the pituitary gland were similar in rats killed by microwave irradiation and decapitation, but irradiation showed higher β-endorphin levels in the brain than decapitation. These results suggest that β-endorphin fragments like α- and γ-endorphin and des-Tyr-α- and des-Tyr-γ-endorphin are endogenous peptides in the rat pituitary gland and the 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.     

On the degradation of dermorphin and D-Arg2-dermorphin analogs by a soluble rat brain extract

Degradation of dermorphin, [D-Arg2]dermorphin and [D-Arg2, Gly3, Phe4]dermorphin in a soluble rat brain extract was examined. The former two heptapeptides were degraded in a similar fashion to produce corresponding N-terminal tetrapeptide as the main degradation product along with the parallel release of Tyr5, Pro6 and Ser7-NH2. Tyr-D-Arg-Phe-Gly showed a good enzymatic stability. When captopril, an angiotensin-converting enzyme inhibitor, was present in the incubation mixture, hydrolysis of the Gly4-Tyr5 bond was markedly suppressed and resulted in release of the corresponding N-terminal hexapeptide as the main degradation product. Combined use of captopril and amastatin, an aminopeptidase inhibitor, markedly suppressed the hydrolysis of these peptides. On the other hand, [D-Arg2, Gly3, Phe4]dermorphin was hydrolyzed easier than the other two heptapeptides and considerable amounts of Tyr1 and Phe4 were released after 20 hr incubation while the N-terminal tetrapeptide, Tyr-D-Arg-Gly-Phe, showed a good enzymatic stability. On the basis of these results, possible degradation pathways of these heptapeptides were discussed.     

beta-Endorphin concentration in the brain of intact and hypophysectomized rats.

The brain concentration and distribution of β-endorphin immunoreactivity in the brain have been studied in intact and hypophysectomized rats. The results obtained with different methods for killing the animals and extracting β-endorphin are compared. Different methodologies of killing the rat and extracting the brain yield concentrations of β-endorphin which vary ten fold. Consistently the highest concentrations of β-endorphin have been found in the hypothalamus, midbrain and hindbrain. After hypophysectomy major reduction of β-endorphin concentration in the brain was observed.     

beta-Endorphin: a pituitary peptide with potent morphine-like activity.

The morphine-like peptide, β-endorphin, from pituitary glands of various species including man, has been characterized chemically and biologically. It is a 31-amino acid peptide corresponding to the amino acid sequence of residues 61–91 of β-lipotropin. It possesses potent analgesic activity when injected intracerebroventricularly or intravenously. In addition to analgesic activity, β-endorphin causes behavioral changes in experimental animals. This paper presents current knowledge of this psychopharmacologically active peptide.     

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.