Binding characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides

The present paper describes the isolation and characterization of a clone of hybrid myelomas (3-E7) secreting a mouse monoclonal antibody to beta-endorphin. An examination of its specificity against a series of human beta-lipotropin fragments and other opioid peptides revealed that the N-terminus portion of beta-endorphin is the determinant. Complete or almost complete cross-reactivity was obtained to methionine- and leucine-enkephalin, beta-lipotropin 60-65, and BAM 22; partial cross-reactivity was seen to dynorphin1-13 and alpha-neo-endorphin, whereas beta-lipotropin, alpha-N-acetyl-beta-endorphin, Des-Tyr1-beta-endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross-reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for beta-endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of beta-endorphin iodinated with its N-terminal tyrosine protected by coupling to an antibody. A solid-phase RIA was developed involving the internally 3H-labeled monoclonal antibody, which resulted in a 10-fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.     

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

Opioid peptide effects on insulin release and c-AMP in islets of Langerhans

The time course and specificity of the effect of opioid peptides on c-AMP production in the islets of Langerhans was examined. An enkephalin analogue, d-Ala2Me Phe4 Met(O)-ol enkephalin (DAMME, Sandoz) produced a significant stimulation of basal c-AMP levels, with a peak of stimulation at 5 minutes and a decline thereafter. These changes in intracellular c-AMP levels were of the same order of magnitude as those induced by other secretagogues, but did not coincide in time with the more rapid peak of enkephalin-induced insulin release. The rise in islet c-AMP and insulin secretion induced by DAMME and alpha-endorphin but not leu enkephalin was antagonised by naloxone. The effects of high and low concentrations of a variety of opioid peptides and naloxone on insulin release and islet c-AMP levels were determined, alpha-endorphin, dynorphin, leu enkephalin and met enkephalin all stimulated insulin secretion significantly, though not to the same extent. Higher concentrations of alpha-endorphin, dynorphin and met enkephalin inhibited insulin release relative to effects at low opiate concentrations. However, higher concentrations of leu enkephalin stimulated insulin release further. We conclude from these results that the mode of action of opioid peptides in stimulating insulin release is not via increased islet c-AMP exclusively. Furthermore, the results obtained with different classes of opioid suggest the presence of distinctive types of opiate receptor in islets of Langerhans.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Structural-functional relations of short analogs of enkephalin

The similarity of action of narcotic analgesics and opioid peptides is due to activation of a common opiate receptor as the primary step in initiating biochemical chains responsible for diverse morphine-like effects. The most widely used assays for opioid and analgesic activities are presented and evaluated. Approximately 180 short enkephalin analogues (di-, tri- and tetrapeptides), described in the literature, are systematized and their opioid and systemic analgesic activities compared with methionine-enkephalin and morphine as the reference compounds, respectively. The analysis of structure-opioid activity relationships among these enkephalin analogues substantiates the hypothesis that only a limited N-terminal region of the peptide molecule is essential for the binding of opioid peptides to the subclass of opiate receptors interacting with narcotic alkaloids (mu-receptors). An attempt has been made to identify minimal structural elements responsible for the mu-receptor activation. Shortening of the molecule and modification of its elements are examined with regard to the mu- and delta-receptor selectivity. It is emphasized that the aromatic structure of the C-terminal region of the peptide is not obligatory for the mu-receptor binding. Modifications of short enkephalin analogues which might confer them antagonistic properties are reviewed. The correlation between the ability of short enkephalin analogues to interact with mu-receptors and their antinociceptive properties is discussed along with some structural features pertinent to the analgesic effect after systemic administration of peptides. On the basis of this analysis, peptides containing no more than four amino acids are considered as the most probable morphine-like analgesics.     

Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors.     

Expression of opioid genes in bovine seminal vesicles

In seminal vesicles, the organ producing most of seminal plasma in the bovine species, the pro-opiomelanocortin and the proenkephalin genes are transcribed and translated, and their translation products processed into opioid peptides, which are secreted into the seminal plasma. By using a micro-organ preparation of seminal vesicles we found that, after 20 h of incubation with labelled methionine, a multiplicity of opioids was produced. Among these, [Met]enkephalin and beta-endorphin were positively identified, whereas in the newly formed secretion only [Met]enkephalin was detected. This may be correlated to the finding that the concentration of beta-endorphin in an extract of seminal plasma was one order of magnitude lower than that of [Leu]enkephalin and [Met]enkephalin. These findings expand the picture of the presence of opioid peptides in the male reproductive tract, indicating that they should have a role(s) in the physiology of reproduction, not only in the hypothalamus-pituitary-gonadal axis, determining the reproductive potential, but also in the so-termed sex accessory glands, determining the actual events leading to reproduction. To our knowledge this is also the first case studied of opioid peptides produced as exocrine hormones.     

Pertussis toxin substrate is a guanosine 5''-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.

Addition of the opioid peptides, [Leu]enkephalin and [Met]enkephalin, to isolated hepatocytes was shown to produce a stimulation of glycogenolysis comparable with that observed in the presence of maximal concentrations of glucagon, adrenaline or angiotensin. This stimulation was demonstrated to be the result of an activation of phosphorylase by a rapid Ca2+-dependent mechanism and was not decreased by the presence or either alpha- or beta-adrenergic antagonists, although it was dependent on the presence of the N-terminal tyrosine residue in the enkephalin molecule. It is suggested that this may be further evidence for specific opioid receptors in the liver. Addition of [Leu]enkephalin also inhibited lactate formation, indicating that the opioid peptides exert a concerted effect on hepatic carbohydrate metabolism to enhance glucose output. The transient nature of the effect of the enkephalins was shown to be the result of a rapid breakdown of the peptides in the incubation as a result of aminopeptidase activity, the initial product being the inactive des-tyrosine derivative.     

Isolation and Characterization of Opioid Peptides from Rabbit Cerebellum

The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.     

Stereospecific synthesis of (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent delta opioid antagonist and a somewhat less potent mu antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to delta and mu receptors but may be required for signal transduction.     

Types of opioid receptors: relation to antinociception

The endogenous opioid peptides are derived from three large precursors. Pro-opiocortin and proenkephalin yield [Met]enkephalin, carboxy-extended [Met]enkephalins and [Leu]enkephalin. The fragments of prodynorphin are all carboxy-extended [Leu]enkephalins. Three approaches are of importance for an analysis of the physiological functions of the different endogenous opioid peptides. First, since these peptides interact with more than one of the mu-, delta- and kappa-binding sites and thus with their receptors, it is necessary to synthesize peptides or non-peptides, which bind to only one of the sites. As far as narcotic analgesics are concerned, morphine fulfils these conditions since it interacts almost exclusively with the mu-receptor. Secondly, antagonists are required that are selective for only one of the opioid receptors, even when used in high concentrations. Finally, it is important to find circumscribed areas in the nervous system that possess only one type of opioid receptor. It is now known that in the rabbit cerebellum the opioid receptors are almost exclusively of the mu-type whereas in the guinea-pig cerebellum they are almost exclusively of the kappa-type.     

The stimulation of glycogenolysis and gluconeogenesis in isolated hepatocytes by opioid peptides.

The opioid agonists [leucine]enkephalin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13)-peptide, but not morphine, stimulated the conversion of [2-14C]pyruvate into glucose and glycogenolysis when added directly to isolated hepatocytes. Naloxone produced a small but significant inhibition of both the basal and stimulated rate of incorporation of label into glucose but had no effect on the total glucose output by the cells. The effects of the opioid peptides were mediated by a cyclic AMP-independent mechanism.     

Target tissue distribution of the proenkephalin peptides F, E, and B

The adrenal medulla is a rich source of endogenous opioid peptides. These peptides exist predominantly in the form of larger (25-34 amino acid long) enkephalin containing peptides, whose biological roles have yet to be elucidated. We report here the tissue binding distribution of three iodinated enkephalin containing peptides, Peptides F, E, and B, in rats, rabbits, and guinea pigs following intravenous injection. Each [125I]peptide has a unique distribution profile but all three are found to bind to the pituitary in each species of animal examined. A number of lines of evidence point toward the enkephalin containing peptides, Peptides F, E, and B, having physiological roles distinct from the enkephalins. The distribution profile of these [125I]peptides reported here gives insight into their potential effector sites.     

Opioid peptides and primary biliary cirrhosis.

Patients with liver disease have increased plasma concentrations of the endogenous opioid peptides methionine enkephalin and leucine enkephalin. As an initial investigation to determine whether opioid peptides contribute to any of the clinical manifestations of hepatic disease nalmefene, a specific opioid antagonist devoid of agonist activity, was given to 11 patients with cirrhosis. They all experienced a severe opioid withdrawal reaction on starting the drug. In the nine patients with primary biliary cirrhosis pruritus was greatly alleviated, fatigue seemed to improve, and plasma bilirubin concentration, which had been rising, showed a modest fall in all except one patient. These results indicate that blocking opioid receptors has an effect on some of the metabolic abnormalities of liver disease.     

Properties and functions of human placental opioid system.

Human placental villus tissue contains opioid receptors and peptides. Kappa opioid receptors (the only type present in this tissue) were purified with retention of their binding properties. The purified kappa receptor is a glycoprotein with an apparent molecular weight of 63,000. Two opioid receptor mediated functions were identified in trophoblast tissue, namely regulation of acetylcholine and hormonal (human chorionic gonadotrophin and human placental lactogen) release. Placental content of kappa receptors increases with gestational age. Term placental content of kappa receptors correlates with route of delivery (higher in those abdominally obtained). Opioid use and/or abuse during pregnancy affects placental receptor content at delivery, as well as its mediated functions. Opioid peptides identified in placental extracts were beta-endorphin, methionine enkephalin, leucine enkephalin and dynorphins 1-8 and 1-13. Dynorphin 1-8 seem to be the predominant opioid peptide present in placental villus tissue.     

Nicotine-induced alteration in Tyr-Gly-Gly and Met-enkephalin in discrete brain nuclei reflects altered enkephalin neuron activity

Nicotine acts in CNS, but the pathways and mechanisms of its actions are poorly understood. Recent studies suggest an interaction between brain nicotinic receptors and endogenous opioid peptides. Acute administration of nicotine may alter enkephalin release without affecting brain enkephalin level. Tyr-Gly-Gly has been shown previously to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A. Concentrations of Tyr-Gly-Gly in brain were used to provide an index of enkephalin release in vivo. Thus we examined the thesis that nicotine alters brain neuronal enkephalin release, by measuring Tyr-Gly-Gly levels in specific brain nuclei from rats treated with nicotine 0.3 mg/kg SC 10 min before decapitation. Of 30 brain regions investigated, acute nicotine increased Tyr-Gly-Gly immunoreactivity in nucleus accumbens and in lower brain stem areas including dorsal raphe, pontine reticular formation, gigantocellular reticular formation, locus coeruleus, sensory trigeminal nucleus and the caudal part of ventrolateral medulla oblongata. Concomitantly, nicotine produced a significant decrease in native Met-enkephalin in central amygdala, flocculo-nodular lobe of cerebellum, caudal part of the ventrolateral medulla and intermediolateral cell column of the spinal cord. It is probable that the effects of nicotine to increase Tyr-Gly-Gly and alter Met-enkephalin concentration are mediated by nicotine-induced release of enkephalin at these brain sites. Furthermore, some of the physiologic and pharmacologic effects of nicotine may be mediated by such enkephalin release.     

Brain endopeptidase generates enkephalin from striatal precursors

An enzyme capable of converting putative opioid peptide intermediates to free enkephalin has been purified 300-fold from washed rat brain membranes. The action of this enzyme, an enkephalin-generating endopeptidase (EGE), was compared with the action of carboxypeptidase B after trypsin treatment on enkephalin precursor peptides present in rat striata. After Sephadex G-100 gel filtration of striatal material, fractions were radioimmunoassayed for enkephalin content using an antiserum specific for the carboxyl terminal of enkephalin. Additionally, aliquots of the column fractions were treated with either trypsin and carboxypeptidase B, trypsin and EGE, or EGE alone. The peak of enkephalin immunoreactivity increased with the enzymes' treatment indicating the conversion of the low molecular weight proenkephalin precursor peptides to enkephalin. Trypsin and EGE generated almost as much enkephalin as trypsin and carboxypeptidase B in the conditions of the experiment. Thus EGE is capable of processing precursors to enkephalin after the action of trypsin-like enzyme(s) in the brain. The gel filtration fractions containing enkephalin and its low molecular weight precursors were pooled and one-half treated with EGE. The contents were analyzed by HPLC and the increase in immunoreactivity co-eluted with enkephalin and Leu-enkephalin. Small peptides found to be the most potent competitive inhibitors of this enzyme are Met-Arg-Phe-Ala, and Met-Arg-Phe.     

Pharmacology of amphibian opiate peptides

In 1980 the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective mu-opioid agonists, and named dermorphins. Since 1987 deltorphins, a family of highly selective delta-opioid peptides were identified either by cloning of the cDNA from frog skins or isolation of the peptides. The distinctive feature of opioid peptides is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe. The discovery of the amphibian opiate peptides, provided new insights into the functional role of the mu- and delta-opiate systems. It also provided models for novel analgesics with enhanced therapeutic benefits and reduced toxicity.     

Hemorphins derived from hemoglobin have an inhibitory action on angiotensin converting enzyme activity.

The hemorphins are opioid active peptides, which are enzymatically released from the beta-chain of hemoglobin. In this paper we report an inhibitory effect of these peptides on angiotensin converting enzyme (ACE) activity, known to be involved in blood pressure regulation. The hemorphins were found to be quite stable in tissue extracts containing ACE, and their importance as naturally occurring ACE inhibitors is discussed.