Extracellular biotransformation of beta-endorphin in rat striatum and cerebrospinal fluid.

Numerous studies have investigated the behavioural effects of beta-endorphin, both endogenous and exogenously applied. However, the potential for biotransformation of beta-endorphin in the extracellular space of the brain has not been previously directly addressed in vivo. Utilising microinfusion/microdialysis and matrix-assisted laser desorption/ionisation mass spectrometry, we investigated beta-endorphin biotransformation in the striatum of rats. We infused 1.0 nmol beta-endorphin into the striatum of adult male Fischer rats and observed rapid cleavage resulting in beta-endorphin 1-18, as well as several fragments resulting from further N-terminal degradation. In vitro studies with incubation of full-length beta-endorphin, with and without protease inhibitors, in the incubation fluid of isolated striatal slices indicate that beta-endorphin is initially cleaved predominantly at the Phe(18)-Lys(19), position, as well as at the Leu(17)-Phe(18) position. Investigations of cerebrospinal fluid revealed similar enzymatic cleavage of beta-endorphin. The observed pattern of cleavage sites (Phe(18)-Lys(19) and Leu(17)-Phe(18)) is consistent with published in vitro studies of purified insulin-degrading enzyme cleavage of beta-endorphin. The binding affinities of full-length beta-endorphin, as well as previously identified beta-endorphin fragments alpha-endorphin (beta-endorphin 1-16) and gamma-endorphin (beta-endorphin 1-17), and the fragment identified in the present study, beta-endorphin 1-18, at heterologously expressed mu, delta and kappa-opioid receptors, respectively, were determined; the affinity of the truncation fragments is reduced at each of the receptors compared to the affinity of full length beta-endorphin.

Limited effects of beta-endorphin compared to loperamide or fentanyl in a neuroendocrine biomarker assay in non-human primates.

The in vivo pharmacodynamics of the opioid neuropeptide beta-endorphin (a major endogenous agonist at the mu-opioid receptor) is difficult to determine in non-human primate models with translational value, or in humans. The present studies therefore employed a neuroendocrine biomarker assay, prolactin release, to systematically compare the in vivo profile of i.v. beta-endorphin (0.01-0.32 mg/kg; i.v.) in gonadally intact male rhesus monkeys (n=4) to that of the peripherally selective mu-agonist loperamide (0.01-0.32 mg/kg; i.v.) and the centrally penetrating mu-agonist fentanyl (0.0056-0.018 mg/kg; i.v.). Studies utilized a standardized time course design (measuring prolactin levels 5-120 min after agonist administration). Beta-endorphin displayed only limited effectiveness in causing prolactin release when tested over this 30-fold dose range, compared to loperamide or fentanyl. Furthermore, two of the four subjects were only minimally responsive to beta-endorphin. This differential responsiveness was not due to the presence of a previously described single nucleotide polymorphism at the OPRM1 gene (C77G), known to affect beta-endorphin pharmacodynamics in vitro. In vivo biotransformation studies with MALDI-mass spectrometry determined that full-length beta-endorphin was detectable in all subjects up to at least 5 min after i.v. administration. Thus, the relative ineffectiveness of i.v. beta-endorphin in this assay does not appear to be principally due to rapid generation of non-opioid fragments of this neuropeptide.

Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers.

A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K(D)) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).

Detection of beta-endorphin in the cerebrospinal fluid after intrastriatal microinjection into the rat brain.

We have investigated to what extent microinjected beta-endorphin could migrate from the rat brain parenchyma into the CSF compartment. Exogenous rat beta-endorphin (0.1 nmol) was microinjected into the left striatum 1 mm from the lateral ventricle in anesthetized male rats. CSF samples were collected at different time points up to 2 h post-injection from a catheter affixed to the atlanto-occipital membrane of the cisterna magna. Radioimmunoassay and mass spectrometry were performed on the CSF samples, and brain sections were immunostained for beta-endorphin and mu-opioid receptors. The beta-endorphin injected rats showed a marked increase in beta-endorphin immunoreactive (IR) material in the CSF, with a peak at 30-45 min post-injection, and this beta-endorphin-IR material existed mainly as the intact beta-endorphin peptide. The immunohistochemistry results revealed the appearance of distinct beta-endorphin-IR cell bodies in the globus pallidus and the bed nucleus of stria terminalis supracapsular part, regions distant from the injection site, at 2 h post-injection of exogenous beta-endorphin. The beta-endorphin-IR in several of the globus pallidus cell bodies colocalized with the mu-opioid receptor-IR at the cell surface. These findings show that upon delivery of synthetic beta-endorphin, there is a significant intracerebral spread of the injected peptide, reaching regions far from the site of injection via diffusion in the extracellular space and flow in the cerebrospinal fluid. This may be of relevance when interpreting studies based on intracerebral injections of peptides, and advances our knowledge regarding the migration of compounds within the brain.

Opioid receptors in fast and slow skeletal muscles of normal and dystrophic mice.

The density of beta-endorphin receptors and the proportions of fibres that expressed the receptors was assessed in fast extensor digitorum longus muscle and slow soleus muscles of normal and dystrophic mice using [125I]beta-endorphin and autoradiography. In the EDL the density was approximately 3.5 times higher and the proportion of labelled fibres approximately 2.6 times higher in dystrophic mice than normal mice. In the soleus the density was approximately 6.4 times higher and the proportion of labelled fibres approximately 1.5 times higher in the dystrophic mice than the normal mice. The receptors were of the delta-opioid subtype.

Beta-endorphin disrupts seasonal and FSH-induced ovarian recrudescence in the lizard Mabuya carinata.

Administration (ip) of an opioid peptide, beta-endorphin (beta-EP) (0.1, 0.5, or 1 microg beta-EP/day/lizard for 30 days) during seasonal recrudescence phase of the ovarian cycle inhibited ovarian recrudescence as shown by the absence of vitellogenic follicles in the ovary in contrast to their presence in treatment controls in the lizard Mabuya carinata. In the germinal bed, treatment of 0.1 microg beta-EP did not affect primordial follicles, whereas their mean number was significantly lower in lizards treated with 0.5 or 1 microg beta-EP compared to those of treatment controls. There was also suppression of oviductal development as shown by a significantly lower relative weight of the oviduct and regressed oviductal glands in lizards treated with all the dosages of beta-EP compared to treatment controls. In another experiment, administration of FSH (10 IU FSH/alternate day/lizard for 30 days) during the regression phase of the ovarian cycle induced development of vitellogenic follicles, whereas the treatment controls showed only previtellogenic follicles. In addition, there was a significant increase in the ovarian and oviductal weights compared to initial and treatment controls. However, simultaneous administration of similar dosage of FSH and beta-EP (0.5 microg/day/lizard) did not induce ovarian recrudescence as shown by the absence of vitellogenic follicles in the ovary and significantly lower weight of the ovary and the oviduct and the mean number of oogonia, oocytes, and primordial follicles compared to those of FSH-treated lizards. The results indicate that beta-EP inhibits seasonal as well as FSH-induced ovarian recrudescence. Inhibitory effect of beta-EP on follicular development despite FSH administration implies its effect at the ovarian level in M. carinata. While adversely affecting the ovarian follicular development, beta-EP did not affect the adrenal gland as there was no significant variation in the mean nuclear diameter of the adrenocortical cells of treatment controls and beta-EP-treated lizards. Furthermore, administration of beta-EP caused a significant decrease in the mean number of islands of white pulp of the spleen indicating its adverse effect on immunity.

Transport of opioids from the brain to the periphery by P-glycoprotein: peripheral actions of central drugs.

Many peptides and transmitters found within the brain also have peripheral sites of action. We now demonstrate that the brain releases functionally active neurotransmitters/neuromodulators directly from the brain into the blood through a saturable P-glycoprotein (Pgp) transport system. Downregulating Pgp1 expression with antisense reduced the brain-to-blood transport of morphine, beta-endorphin and other opioids. Lowering Pgp expression significantly enhanced systemic morphine analgesia and prevented tolerance, but diminished the analgesic activity of centrally administered morphine, implying that supraspinal analgesia resulted from a combination of central and peripheral mechanisms activated by morphine transported from the brain to the blood. Similarly, mice with a disruption of the Mdr1a gene were more sensitive to systemic morphine and less sensitive to morphine given centrally. This ability of the Pgp transport system to pump functionally active compounds from the brain to periphery defines a potentially important mechanism for the central nervous system to modulate peripheral systems.

Inflammation of the rat paw enhances axonal transport of opioid receptors in the sciatic nerve and increases their density in the inflamed tissue.

The effect of inflammation, induced by unilateral intraplantar injection of Freund's adjuvant, on opioid receptors transported in the sciatic nerve and on opioid receptors present in the paw of the rat was studied by means of in vitro receptor autoradiography using [125I]beta-endorphin (human) as ligand. In the absence of inflammation, human beta-endorphin binding sites accumulated proximally and distally to a ligature placed on the sciatic nerve in a time-dependent manner, indicating bidirectional axonal transport. Some human beta-endorphin binding was also visible in non-inflamed paw tissue. Inflammation of the paw tissue massively increased human beta-endorphin binding on both sides of the sciatic nerve ligature and in the ipsilateral paw tissue. In inflamed paw tissue, beta-endorphin binding accumulated in the cutaneous nerve fibers as well as in the immune cells infiltrating the surrounding tissue. In the sciatic nerve and paw tissue, beta-endorphin binding was displaced by (D-Ala2, N-methyl-Phe4, Gly-ol5)enkephalin and (D-Pen2, D-Pen5)enkephalin, selective mu- and delta-opioid receptor agonists, respectively, and by the universal opioid antagonist naloxone, but not by U-50,488H, a k-selective receptor agonist. Taken together, these data provide neuroanatomical evidence for local inflammation-induced enhanced axonal transport of opioid receptors in rat sciatic nerve and accumulation in paw tissue.

Sympathoadrenal control by paraventricular hypothalamic beta-endorphin in hypertension.

The paraventricular hypothalamus regulates autonomic nerve outflow and is innervated with beta-endorphin-immunoreactive nerve terminals. This study examined the effects of beta-endorphin microinjected into the paraventricular hypothalamus on blood pressure, heart rate, and plasma catecholamine and glucose concentrations in conscious, unrestrained spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at the age of about 9 weeks. Thirty minutes after paraventricular hypothalamic injection of [125I] beta-endorphin (3.5 micrograms), most of the recovered radioactivity was detectable within +/- 0.5 mm from the injection site in the coronal, sagittal, and horizontal planes. Unilateral paraventricular hypothalamic injections of beta-endorphin (1 and 0.1 microgram/0.1 microliter) increased blood pressure and heart rate in both strains in a dose-independent manner with significantly greater increases in SHR. Plasma catecholamine and glucose concentrations were measured 15, 30, and 60 minutes after beta-endorphin injection. Norepinephrine concentrations were not significantly altered in WKY rats but increased in SHR. Epinephrine concentrations increased in both strains with significantly greater increases in SHR. Increases in catecholamine concentrations were not dose-related. Glucose concentrations also increased in both strains with significantly greater increases in SHR only at the lower dose. Ganglionic blockade with pentolinium significantly reduced beta-endorphin-induced pressor and tachycardiac responses in SHR. Pretreatment of the paraventricular hypothalamus with naltrexone (1.1 micrograms) in SHR blocked the initial pressor and tachycardiac responses to beta-endorphin (0.1 microgram) and blunted increases in epinephrine and glucose levels. When the animals were anesthetized with alpha-chloralose 2-5 days after the study in conscious animals, there were no differences in blood pressure or heart rate between strains after beta-endorphin (0.1 microgram) injection. The results indicate that conscious SHR show enhanced cardiovascular and sympathoadrenal responses to beta-endorphin injected into the paraventricular hypothalamus, suggesting that alterations in the activity of the paraventricular hypothalamic beta-endorphin system can modulate the development of hypertension in SHR.

Infusion of beta-endorphin has no suppressive effect on the releasing hormone-stimulated pituitary-adrenal-axis of normal human subjects.

The existence of a short-loop feedback inhibition of pituitary ACTH release by administration of beta-endorphin was postulated. However, data on the effect of peripherally administered beta-endorphin in humans are highly controversial. We infused human synthetic beta-endorphin at a constant rate of 1 microgram.kg-1.min-1 or normal saline to 7 normal volunteers for 90 min. Thirty min after starting the beta-endorphin or placebo infusion, releasing hormones were injected as a bolus iv (oCRH and GHRH 1 microgram/kg, GnRH 100 micrograms, TRH 200 micrograms) and blood was drawn for measurements of beta-endorphin immunoreactivity, all other pituitary hormones, and cortisol. Infusion of beta-endorphin resulted in high beta-endorphin plasma levels with a rapid decrease after the infusion was stopped. During the control infusion, beta-endorphin plasma levels rose in response to CRH. Plasma ACTH and serum cortisol levels in response to the releasing hormone were not different in subjects infused with beta-endorphin or placebo. The PRL response to TRH was significantly higher after beta-endorphin than after placebo (area under the stimulation curve 1209 +/- 183 vs 834 +/- 104 micrograms.l-1.h). There was no difference in the response of all other hormones measured. Our data on ACTH and cortisol secretion do not support the concept of a short-loop negative feedback of beta-endorphin acting at the site of the pituitary.

Chronic haloperidol and chlorpromazine treatment alters in vitro beta-endorphin metabolism in rat brain.

To determine if chronic haloperidol (3.0 mg/kg per day) or chlorpromazine (4.2 mg/kg per day) treatment alters central beta-endorphin metabolism, haloperidol and chlorpromazine were perfused via Alzet minipumps into male Sprague-Dawley rats for 8 days. Crude twice-washed membranes, purified synaptic plasma membranes and Golgi-enriched membranes, respectively, were isolated from rat brains and time course incubated with beta-endorphin. All samples were analyzed by high resolution, reversed-phase high performance liquid chromatography. The half-lives of beta-endorphin for animals treated with haloperidol or chlorpromazine were not statistically different from control animals at the crude washed membranes. At the purified synaptic plasma membranes, however, the half-lives of beta-endorphin from haloperidol (t 1/2 = 45.1 min)- and chlorpromazine (t1/2 = 47.0 min)-treated animals were significantly decreased as compared to the control animals (t1/2 = 78.0 min). The half-life of beta-endorphin at the Golgi-enriched membranes was increased for haloperidol (t1/2 = 112.3 min) and chlorpromazine (t1/2 = 103.0 min)-treated animals when compared to control animals (t1/2 = 80.2 min). The findings indicate a differential effect of the dopamine receptor antagonists haloperidol and chlorpromazine on the extracellular fate at the synaptic plasma membranes of beta-endorphin and the intracellular processing at the Golgi-enriched membranes in vitro.

Neuropeptide processing in regional brain slices: effect of conformation and sequence.

The central enzymatic stability of des-enkephalin-gamma-endorphin and its synthetic analogs [cycloN alpha 6, C delta 11]beta-endorphin-[6-17] and [Pro7, Lys(Ac)9]-beta-endorphin[6-17] was studied in vitro using a newly developed, regionally dissected rat brain slice, time course incubation procedure. Tissue slice viability was estimated as the ability of the brain slice to take up or release gamma-[3H]aminobutyric acid after high K+ stimulation. Results demonstrated stability of uptake/release up to 5 hr of incubation, suggesting tissue viability over this period. The estimated half-life of peptides based on the results obtained in our incubation protocol suggest that the peptides studied are metabolized at different rates in the individual brain regions tested. A good correlation exists between the high enzyme activity of neutral endopeptidase (EC 3.4.24.11) and the rapid degradation of des-enkephalin-gamma-endorphin and [cycloN alpha 6, C delata 11]beta-endorphin-[6-17] in caudate putamen. Proline substitution combined with lysine acetylation appears to improve resistance to enzymatic metabolism in caudate putamen and hypothalamus. However, cyclization of des-enkephalin-gamma-endorphin forming an amide bond between the alpha-NH2 of the N-terminal threonine and the gamma-COOH of glutamic acid did not improve peptide stability in any brain region tested. The present study has shown that the brain slice technique is a valid and unique approach to study neuropeptide metabolism in small, discrete regions of rat brain where peptides, peptidases and receptors are colocalized and that specific structural modifications can improve peptide stability.

Des-enkephalin-gamma-endorphin (DE gamma E): pharmacokinetics in dogs after intravenous and subcutaneous administration.

After intravenous dosing in dogs [3H-Lys9]-DE gamma E (Org 5878) was very rapidly eliminated from the circulation. Disappearance of the neuropeptide from blood followed a biphasic decay with half-lives of 0.6 +/- 0.1 min (+/- S.D.; alpha-phase) and 2.4 +/- 1.0 min (beta-phase). The central volume of distribution ranged between 0.05 and 0.23 l.kg-1. The mean blood clearance rate amounted to 0.15 l.min-1.kg-1, which is indicative of extensive hepatic and extrahepatic metabolism of DE gamma E. In contrast to intravenous dosing, subcutaneous injection of [3H]-DE gamma E in dogs resulted in low but relatively long-lasting peptide levels in blood. Peak values were found at 5-10 min, whereafter they declined to the limit of detection at 1.5-2 h. The bioavailability of DE gamma E for this route of administration was shown to be 20-23%.

Rectal absorption enhancement of des-enkephalin-gamma-endorphin (DE gamma E) by medium-chain glycerides and EDTA in conscious rats.

The stability of the neuroleptic peptide des-enkephalin-gamma-endorphin (DE gamma E; Org 5878) in the rectal lumen and the rectal bioavailability of DE gamma E were investigated in conscious rats. Furthermore, the influence of peptidase inhibition, peptidase saturation, and absorption enhancement on DE gamma E bioavailability were evaluated. Na2EDTA (0.25%, w/v) prolonged the degradation half-life of DE gamma E in the ligated colon from 33 +/- 7 to 93 +/- 45 min. Without adjuvant, tritium-labeled DE gamma E was absorbed from the rat rectum to a very low extent (0-4%). After administration of an excess of unlabeled DE gamma E or with Na2EDTA, comparable results were obtained. The medium-chain glyceride preparation MGK markedly enhanced the rectal DE gamma E bioavailability, up to 8-20%, which was further increased to 10-44% by coadministration of Na2EDTA. No substantial influence of varying the rectal delivery rate was observed. The results suggest that absorption enhancement and enzyme inhibition both are essential for effective increase of rectal peptide bioavailability.

Physiologically based pharmacokinetics of radioiodinated human beta-endorphin in rats. An application of the capillary membrane-limited model.

In order to simulate the distribution and elimination of radioiodinated human beta-endorphin (125I-beta-EP) after iv bolus injection in rats, we proposed a physiologically based pharmacokinetic model incorporating diffusional transport of 125I-beta-EP across the capillary membrane. This model assumes that the distribution of 125I-beta-EP is restricted only within the blood and the tissue interstitial fluid, and that a diffusional barrier across the capillary membrane exists in each tissue except the liver. The tissue-to-blood partition coefficients were estimated from the ratios of the concentration in tissues to that in arterial plasma at the terminal (pseudoequilibrium) phase. The total body plasma clearance (9.0 ml/min/kg) was appropriately assigned to the liver and kidney. The transcapillary diffusion clearances of 125I-beta-EP were also estimated and shown to correlate linearly with that of inulin in several tissues. Numerically solving the mass-balance differential equations as to plasma and each tissue simultaneously, simulated concentration curves of 125I-beta-EP corresponded well with the observed data. It was suggested by the simulation that the initial rapid disappearance of 125I-beta-EP from plasma after iv injection could be attributed in part to the transcapillary diffusion of the peptide.

Beta-endorphin infusion fails to modulate the hormonal and metabolic response to surgery.

The effects of an i.v. infusion of synthetic human beta-endorphin on the hormonal, metabolic and cardiovascular responses to surgery were investigated in female patients undergoing pelvic surgery. A beta-endorphin infusion (2 micrograms/kg as a bolus at induction of anaesthesia + 10 micrograms/kg/h for the first hour of surgery) increased plasma beta-endorphin immunoreactivity to values at least 100-fold greater than those seen during surgery in a control group of patients. In spite of this massive increase the only significant findings were a transient augmentation of the expected hyperglycaemic response and increased plasma glucagon values. There were no significant changes in ACTH, GH, insulin and cortisol secretion, in blood concentrations of lactate or glycerol, or in cardiovascular variables. Complete dissociation between plasma and cerebrospinal fluid concentrations of beta-endorphin was found even when plasma values exceeded 10,000 pmol/l in the presence of anaesthesia and surgery. These results show that the increase in circulating beta-endorphin immunoreactivity associated with clinical stress states are unlikely to modulate the associated hormonal, metabolic and cardiovascular changes.