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.     

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.     

The stimulation of glycogenolysis in isolated hepatocytes by opioid peptides.

The opioid peptides [Leu]enkephalin and dynorphin-(1-13) were shown to enhance glycogen breakdown when added directly to hepatocytes. This was the result of a concerted effect on the enzymes of glycogen metabolism, with a stimulation of glycogen phosphorylase activity and a simultaneous decrease in glycogen synthase I activity. The latter only became significant when the enzyme was activated by incubating the cells in presence of 20 mM- or 40 mM-glucose. The effect of the opioid peptides was independent of an increase in cyclic AMP or any change in the activity ratio of the cyclic AMP-dependent protein kinase and was abolished by depleting the cells of Ca2+. Both [Leu]enkephalin and dynorphin-(1-13) produced a significant decrease in cyclic AMP formation, suggesting that in liver, as in neuronal tissue, they may act by inhibiting adenylate cyclase activity.     

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.     

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.     

Morphine- and opioid peptide-induced inhibition of the release of dopamine from tuberoinfundibular neurons

Dopamine concentrations in pituitary stalk plasma ovariectomized rats and ovariectomized, estrogen-treated rats were measured following the subcutaneous administration of morphine or the intraventricular administration of ß-endorphin or [D-Ala²]-methionine-enkephalinamide, a synthetic enkephalin analog. The administration of morphine or the opioid peptides led to an 85–89% reduction in the concentration of dopamine in pituitary stalk plasma when compared to the mean concentration of dopamine in stalk plasma of vehicle-treated animals. Pretreatment of rats with naloxene, an opiate antagonist, prevented the opioid peptide-induced reduction in the stalk plasma concentration of dopamine. These results are supportive of the hypothesis that endogenous opioid peptides modulate the release of dopamine by tuberoinfundibular neurons into hypophysial portal blood.     

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.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

Putative enkephalin precursors in bovine adrenal medulla.

Extracts from bovine adrenal medulla and adrenal medullary chromaffin granules were found to contain three proteins, 20,000, 10,000 and 5,000 approximate molecular weights which yield tryptic peptides with opioid activity. The opioid activity of these peptides was demonstrated with a radioreceptor assay and two radioimmunoassays. The three proteins yield the same active peptides all of which are chromatographically distinct from the tryptic opioid nonapeptide β-LPH 61–69, generated by trypsin digestion of pituitary endorphins and their precursors. Furthermore, these endorphins and their precursors do not appear to be present in the adrenal medulla. These findings further support the hypothesis that the enkephalin biosynthetic pathway is distinct from that leading to β-endorphin.     

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.     

Preparation and opioid activities of N-methylated analogs of [D-Ala2,Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala2,Leu5]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH3I-KHCO3 in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me3+Tyr1,D-Ala2,Leu5]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH3I-Ag2O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala2,Leu5]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.     

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.     

Comparison of the effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism.

The effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism are compared. Both peptides stimulated glycogenolysis as a result of an increase in phosphorylase a activity and enhanced glucose synthesis from [2-14C]pyruvate, although neither had any significant effect on pyruvate kinase activity. Although the magnitudes of the effects of both peptides on glycogenolysis were comparable and unaffected by the presence of insulin. [Leu]enkephalin proved to be more efficacious in enhancing gluconeogenesis, the response being comparable with that to glucagon. Both effectors decreased the intracellular concentration of cyclic AMP in hepatocytes when incubated under control conditions and after addition of sub-optimal concentrations of glucagon. This was correlated with the ability of the two peptides to inhibit both basal and hormone-stimulated adenylate cyclase activity in purified liver plasma membranes.     

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain.

Our observations that opioid peptides have direct effects on islet insulin secretion and liver glucose production prompted a search for endogenous opiates and their receptors in these peripheral tissues. Mu-, delta- and kappa-receptor-active opiates were demonstrated in brain, pancreas and liver extracts by displacement studies using selective ligands for the three opiate receptor subtypes [( 3H][D-Ala2,MePhe4,Gly5-ol]enkephalin, [3H][D-Ala2,D-Leu5]enkephalin and [3H]dynorphin respectively). Receptor-active opiates in brain extracts exhibited a stronger preference for delta-opiate-receptor sites than for mu and kappa sites. Pancreatic extract opiates demonstrated a similar activity at mu and delta sites, but substantially less at kappa sites. Liver extracts displayed similar selectivity for all three sites. The affinities of the receptor-active opiates for mu-, delta- and kappa-receptor subtypes displayed a rank order of potency: brain much greater than pancreas greater than liver. Total immunoreactive beta-endorphin and [Met5]enkephalin levels in liver and hepatocytes were greater than those in brain. Immunoreactive [Met5]enkephalin levels in pancreas were similar to, but beta-endorphin levels were substantially higher than, those in brain. Delta and kappa opiate-binding sites of high affinity were identified in crude membrane preparations of islets of Langerhans, but no specific opiate-binding sites could be demonstrated in liver membrane preparations. Immunoreactive dynorphin and beta-endorphin were demonstrated by immunogold labelling in rat pancreatic islet cells. No positive staining of liver sections for opioids was observed. These results suggest that the tissue content of opiate-receptor-active compounds in the pancreas and the liver is very significant and could contribute to the regulation of normal blood glucose levels.     

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.     

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.     

Potent side-chain to side-chain cyclized dermorphin analogues containing a carbonyl bridge.

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method and ring formation was achieved via a ureido group incorporating the side chain amino functions of D-Aaa2 (D-Lys, D-Orn) and Aaa4 (Lys, Orn, Dab, Dap). The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Most showed very high agonist potency in the GPI assay. The peptide containing D-Lys in position 2 and Dab in position 4 was 210 times more active than enkephalin, and that containing Orn and Dab, respectively, was 150 times more active than enkephalin. The latter peptide was also very active in the MVD assay, and showed an IC50 MVD/GPI ratio of 0.816. NMR spectra of selected peptides were recorded, and structural parameters were determined. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. With the help of the NMR spectra each peptide was described as an ensemble of conformations. The conformations have been interpreted with regard to the opioid activities, and comparisons have been made with a model proposed earlier for enkephalin analogues.     

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.     

Tissue Content of Opioid Peptides in the Myenteric Plexus-Longitudinal Muscle of Guinea-Pig Small Intestine

We have developed a method that is based on two HPLC systems and permits the separation of endogenous opioid peptides in tissue extracts. The individual peptides are bioassayed on the mouse isolated vas deferens; naloxone (100 nM) ensures opioid specificity. In the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine, the tissue content of prodynorphin-derived peptides is lower than those of proenkephalin-derived peptides. No beta-endorphin was detected. Of the prodynorphin fragments, alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), and dynorphin B are present in equimolar concentrations (12-15 pmol/g) whereas the tissue content of dynorphin A is only 0.8 pmol/g. Processing of proenkephalin leads to at least six opioid peptides. The tissue contents of [Leu5]enkephalin, [Met5]enkephalyl-Arg-Gly-Leu, and [Met5]enkephalyl-Arg-Phe are 90-100 pmol/g and the content of [Met5]enkephalin is 405 pmol/g. BAM-18 and [Met5]enkephalyl-Arg-Arg-Val-NH2 are present in much lower concentrations, 24 and 5 pmol/g, respectively. Although present in low amounts, BAM-18 and [Met5]-enkephalyl-Arg-Arg-Val-NH2 have high affinity for the mu-opioid binding site and to a lesser extent for the kappa-site; this binding profile differs from that of the other proenkephalin fragments all of which have high affinities for the mu- and delta-sites.     

Predominant involvement of mu-rather than delta- or kappa-opiate receptors in LH secretion

Opiate alkaloids and opioid peptides have been shown to suppress plasma LH and FSH levels via a naloxone sensitive mechanism in several species including man. Three subtypes of opiate receptors have been characterized: mu, delta and kappa. The present study was designed to investigate their role in gonadotropin release. Three highly selective opioid ligands, DAGO, MRZ and DTE12 (a dimeric tetrapeptide enkephalin), were injected intraventricularly into chronically ovariectomized rats. Injection of the mu-agonist at doses of 1 and 10 nmol produced a significant suppression of LH secretion, while the delta- and kappa-agonists had no significant effect. Thus, the mu-receptor seems to be the primary opiate receptor involved in the regulation of LH secretion. None of the opiate agonists employed had an effect on FSH secretion.