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A purified opioid‐binding protein has been characterized by cDNA cloning. The cDNA sequence predicts an extracellularly located glycoprotein of 345 amino acids. This protein does not possess a membrane‐spanning domain but contains a C‐terminal hydrophobic sequence characteristic of membrane attachment by a phosphatidylinositol linkage. It displays homology to the immunoglobulin protein superfamily, featuring three domains that resemble disulfide‐bonded constant regions. More specifically, the protein is most homologous to a subfamily of proteins which includes the neural cell adhesion molecule (NCAM) and myelin‐associated glycoprotein (MAG) and one subgroup of the tyrosine kinase growth factor receptors comprising the platelet‐derived growth factor receptor (PDGF R), the colony‐stimulating factor 1 receptor (CSF‐1 R) and the c‐kit protooncogene. These sequence homologies suggest that the protein could be involved in either cell recognition and adhesion, peptidergic ligand binding or both.

A single dose of dynorphin A-(1-13) [dyn A(1-13)] is effective in suppressing the expression of opioid withdrawal and tolerance in morphine-dependent mice. In addition, this modulatory activity is retained by the corresponding non-opioid [des-Tyr1]-dynorphin A peptide [dynA(2-17)]. We have further investigated the non-opioid nature of this activity by comparing the efficacies of dyn A(1-13) and (2-17) under different experimental protocols with a variety of dosing regimens. The effect of dyn A(1-13) on withdrawal and tolerance expression was dose-dependent and could be enhanced by repeated dosing. Thus, the ED50 of naloxone to precipitate withdrawal jumping was increased 1.8-fold when morphine-dependent mice were treated with 4.2 mumol/kg dyn A(1-13) on the fourth day after pellet implantation and 2.4-fold on the sixth day with continued daily dyn A(1-13) treatment. The maximal effect was observed on day 6 when the ED50 of mice treated with 8.4 mumol/kg of dyn A(1-13) was increased nearly 6-fold over that of saline controls. Dyn A(2-17) proved to be nearly as effective as dyn A(1-13).

Nanomolar concentrations of neurotensin caused a dose-dependent contraction of the longitudinal muscle layer of the guinea-pig ileum. The contractile activity of neurotensin was partially blocked by tetrodotoxin or atropine, indicating that a component of the neurotensin-mediated contraction is indirect in nature and likely involves the release of endogenous acetylcholine from nervous terminals in the myenteric plexus. Dynorphin and related peptide fragments also blocked in part the neurotensin contraction; the potency of this opioid peptide was about the same as that of atropine. Other peptides and alkaloids tested for ability to block the neurotensin contractures included the enkephalins, beta-endorphin, normorphine and the ketocyclazocines; all these opioids inhibited in a dose-dependent fashion the neuronal component of the excitatory effect of neurotensin. The potency of these compounds to reduce the contractions of neurotensin showed good correlation with the potency of these agents to depress by 50% the electrically evoked neuromuscular twitches in the same tissue (r = 0.99); in these tests dynorphin was found to be the most potent of the endogenous opioid-like peptides. The dynorphin blockade was selective to the excitatory effect of neurotensin because the opioid peptide did not antagonize the contractile action of acetylcholine, histamine, substance P, angiotensin II, bradykinin, Ba++ or K+ ions. In addition, somatostatin, vasointestinal peptide, gastrin or adenosine did not modify the potency of neurotensin whereas thyrotropin releasing hormone and epinephrine caused a modest doubling of the neurotensin EC50. The inhibitory action of dynorphin was reduced in the presence of naloxone, suggesting that the interaction involved opiate receptors. Morphine tolerance was not extended to the inhibitory action of dynorphin as evidenced by the finding that the potency of dynorphin-(1-13) to block the neurotensin responses was increased after chronic morphine exposure. In contrast, the potency of dynorphin-(1-13) was significantly reduced in tissues rendered tolerant to the action of ketocyclazocine or ethylketocyclazocine, suggesting that the action of dynorphin could be partially mediated via occupation of K-opiate receptors. Thus, a cholinergic-neuronal component activated by neurotensin on the myenteric plexus appears to be under the inhibitory influence of opiate receptors, suggesting that dynorphin may play a role in the modulation of cholinergic synapses on the enteric nervous system.

The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM). This population of receptors, to which opioate alkaloids and opiod peptides display very low affinity, is probably the sigma receptor. Another class of binding sites was identified by the simultaneous addition of the selective agonists Sandoz FK-33824 and D-Ala2-D-Leu5-enkephalin, which blocked the access of EKC to mu and delta opioid receptors, respectively, leaving a portion of naloxone-displaceable benzomorphan binding still detectable. Analysis of this remaining binding revealed a small population of receptors of high affinity, the kappa receptor. Therefore, EKC binds to the mu, delta, kappa and sigma receptors in the mouse brain, with similar affinities for the mu and kappa (0.22 and 0.15 nM). These results confirm the existence of a kappa opioid receptor type in the mouse brain.

We have previously reported that interleukin-1-induced proliferation of thymocytes is accompanied by the appearance of [3H]morphine binding sites on these cells. In the present study, we have characterized these binding sites. They differ from classical opioid receptors in the brain in several ways, including: 1) lack of stereoselectivity; 2) relatively low affinity (Kd = 50 nM) and high capacity (Bmax = 3 pmol/mg of protein); 3) binding is strongly inhibited by Ca++, Mg++, Mn++ and Cl- ions and 4) binding is inhibited by proteinase K or E and by phospholipase A2 but not trypsin treatment of thymocyte membranes. The binding sites, which were found largely on the CD4+ subset of T-cells, also showed a preference for opioid alkaloids over peptides. These [3H]morphine binding sites may mediate a negative feedback effect on interleukin-1-induced proliferation of thymocytes in vivo.

The role of dynorphin-(1-13) and dynorphin-(1-10)-amide in the neuroendocrine control of primate anterior pituitary hormones was studied in nonrestrained, ovariectomized rhesus monkeys. The effects of these opioids on plasma concentrations of prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyrotropin (TSH), and interactions with naloxone are reported here. Intravenous administration of dynorphin-(1-13), 30 to 120 micrograms/kg, significantly increased plasma PRL levels 3- to 4-fold. These PRL increases occurred within 5 min and levels remained elevated for at least 60 min. Administration of naloxone (1.0 mg/kg i.v.) antagonized the rise in PRL levels. Dynorphin-(1-13) had no significant effect on plasma LH, FSH or TSH levels. Dynorphin-(1-10)-amide (30-120 micrograms/kg) increased plasma PRL levels 2- to 4-fold at 5 to 40 min after administration. Plasma LH levels were significantly depressed 100 to 120 min postdrug. Dynorphin-(1-10)-amide produced no change in plasma FSH or TSH levels. These results indicate that dynorphin is involved in the modulation of PRL and perhaps LH secretion, although not affecting TSH or FSH release.

Mu, delta and kappa opiate receptors have been implicated in the production of analgesia. In order to study the relative contributions of these receptor types to supraspinally mediated analgesia, apparent pA2 values and rank order potencies were determined for i.c.v. injected highly selective opioid agonists in the mouse using a thermal nociceptive test. Drugs used included the prototypical mu agonist morphine, putative mu agonists [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin and BAM 22P, putative delta agonists [D-Pen2, D-Pen5] enkephalin, [D-Thr2, Thr6, Leu5] enkephalin and [D-Ser2, Leu5, Thr6] enkephalin and the putative kappa agonists [trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methane sulfonate hydrate] and Dynorphin A (1-13). We were unable to demonstrate significant analgesic potencies for i.c.v. injected Dynorphin A (1-13) or BAM 22P in the absence of marked behavioral abnormalities. The rank order potency of the remaining compounds studied was found to be: [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin greater than [D-Thr2, Thr6, Leu5] enkephalin greater than [D-Ser2, Leu5, Thr6] enkephalin greater than Morphine greater than [D-Pen2, D-Pen5] enkephalin greater than [trans-3, 4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methane sulfonate hydrate]. Apparent pA2 values of morphine, [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin and [D-Pen2, D-Pen5] enkephalin in naloxone antagonism trials did not differ significantly. These results indicate that although both mu- and delta-selective ligands produce potent analgesia, a single receptor (mu) is sufficient to explain the supraspinal effects of opiate drugs.

The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.

A monoclonal antibody generated against the tertiary structure of a partially purified opioid binding protein was used to probe the structure of the dynorphin and beta-endorphin receptors. The Fab fragment 3B4F11 inhibited completely the binding of 125I-beta-endorphin and [3H]dynorphin to rat brain P2 membranes with IC50 values of 26 ng/ml and 40 ng/ml, respectively. To explore further the interaction of 3B4F11 with the beta-endorphin receptor, the effect of the Fab fragment on 125I-beta-endorphin cross-linking to rat brain membranes was examined. 125I-beta-endorphin was covalently bound to three major species of approximate molecular weights 108,000, 73,000, and 49,000. The delta-selective ligand D-Pen2, D-pen5enkephalin was least effective at inhibiting the cross-linking of beta-endorphin, whereas the micro-selective ligand Tyr-D-Ala-Gly-NMe-Phe-Gly-ol and kappa-selective ligand U50488 inhibited beta-endorphin cross-linking to the 108,000 and 73,000 Da species. Both 3B4F11 and beta-endorphin prevented the covalent binding of 125I-beta-endorphin to all three labeled species. These findings suggest that micro and kappa receptor types might have some structural similarities, whereas the delta receptor type might differ in molecular size. In addition, the micro, kappa, and delta ligands might have different primary sequences, whereas their tertiary structures might share regions of molecular homology with all three receptor constituents labeled by 125I-beta-endorphin. 3B4F11 will be a valuable tool for the purification and isolation of the several components of the beta-endorphin receptor complex.

Human beta-endorphin adopts a partial helical conformation in aqueous solutions of cerebroside sulfate, ganglioside GM1, phosphatidylserine, and phosphatidic acid, but not of cerebroside and phosphatidylcholine, as evidenced by circular dichroic spectra. Addition of Ca2+ to the peptide in cerebroside sulfate solution can break up the helix; at 10 mM Ca2+ the peptide (12 microM) essentially exists in an unordered form. For comparison, sheep beta-lipotropin in acidic cerebroside sulfate solution (pH less than 4) also has a partial helical conformation of the complex between human beta-endorphin and lipids may be related to the opiatelike function of this peptide hormone.

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).

The dynorphin family of peptides stands out among the opioids in that its members are not antinociceptive after central administration in the common antinociceptive assays. In addition, reports of spinal antinociception have been conflicting. We have tested the antinociceptive activity of i.v. dynorphin A-(1-13) in the writhing assay and have found it to be very potent, with an ED50 of 1.0 (0.99-1.02) mumol/kg. Remarkably, [des-tyr1]dyn A-(2-17) was equally active with an ED50 of 1.1 (0.99-1.20). This activity was also retained by several smaller, non-opioid dynorphin A fragments and was not affected by the presence of either 50 mumol/kg naloxone or 20 mumol/kg Nor-BNI. Further, ED50 values were not different in morphine-dependent mice. The peak effect of dyn A-(1-13) and A-(2-17) was observed 5 min after administration and the effect of dyn A-(1-13) or dyn A-(2-17) was still measurable 1 hr after i.v. administration with a 5- to 6-fold increase in ED50 at this time. The ED50 values after i.c.v. and i.t. administration of dyn A-(1-13) were similar to those reported previously. Dyn A(2-17) was also effective by these routes with ED50 values not significantly different from those of dyn A-(1-13). Both dyn A-(1-13) and A-(2-17) were also active when injected i.p., whereas ED50 values increased substantially after s.c. administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitory activity of a variety of opioid peptides was tested on the electrically stimulated rat vas deferens. Human beta-endorphin (beta h-EP) was the most potent of the opoioid-like peptides; it produced half-maximal inhibition of the neuromuscular twitching at a concentration of about 100 nM. The potency of beta h-EP was greater than that of camel, porcine, ovine or leucine5 beta h-EP, alpha-N-acetyl beta-ovine-EP was inactive. In contrast to beta-endorphin (beta-EP), methionine and leucine-enkephalin, dynorphin-(1-13), morphine and other narcotic analgesics were devoid of opioid-like activity. Fragments of beta-EP with amino acid deletions at the carboxy end of the molecule were considerably less potent than the parent compound. The fragment beta h-EP 1-21 was 70 times less potent than beta h-EP, whereas the segments beta h-EP 1-19 and beta h-EP 1-16 were both completely inactive. Deletions at the amino terminal of beta-EP yielded inactive compounds. The potency of beta h-EP was reduced in a dose-related fashion by applications of nanomolar concentrations of naloxone or N-allylnormetazocine and by micromolar concentrations of levorphanol or morphine. In decided contrast, opioid peptides such as methionine enkephalin, dynorphin-(1-13), beta-casomorphan derivatives or short chain beta h-EP segments such as beta-EP 1-16, beta-EP 1-19, beta-EP 6-31, beta-EP (1-5)-(16-31), methionine enkephalin plus beta-EP 6-31 or the N-acetyl beta-bovine-EP did not antagonize the inhibitory action of beta h-EP. The present results demonstrate that the rat vas deferens contains opioid receptors with considerable selectivity for beta-EP. It is concluded from this structure activity relationship study that the activation of the beta-EP receptor involves at least two sites of recognition on the beta-EP structure.

Previously, we demonstrated that the expression of opiate withdrawal and antinociceptive tolerance can be suppressed by dynorphin (dyn) A-(1-13) in morphine-dependent mice. In this study, it was shown that the normal, endogenous dyn, dyn A-(1-17) also possessed this suppressive property. While using the nonopioid dyn analog, [des-Tyr1]dyn A [dyn A-(2-17)] as a negative control, we discovered unexpectedly that this peptide fragment also suppressed naloxone-induced withdrawal and the expression of morphine tolerance in morphine-dependent mice. Thus, an extensive structure activity relationship was studied using 11 peptide fragments. It was determined that the amino acid sequence of dyn A was required for the suppressive activity because dyn B and alpha-neoendorphin both failed to suppress naloxone-precipitated withdrawal jumping. Of the [des-Tyr1]dyn fragments, the minimal amino acid sequence required to suppress naloxone-induced withdrawal was determined to be dyn A-(2-8), containing the sequence G-G-F-L-R-R-I.

Treatment of the isolated mouse was deferens with the enzyme arylsulfatase (E.C. 3.1.6.1) had an effect on the ability of this tissue to respond to various opiates. It increased the IC50 values and slopes of their dose-response curve for enkephalins and their analogs, and shifted to the right the curves for FK33824, levorphanol and normorphine. There was no effect on the action of etorphine, beta-endorphin or dynorphin. With morphine there was a biphasic effect, IC50 values increasing at low enzyme concentrations and decreasing at high enzyme concentrations. A further comparison of arylsulfatase effects on morphine and on D-Ala-2-D-Leu5-enkephalin indicated that the morphine effect, unlike the D-Ala-2-D-Leu5-enkephalin effect, could not be reversed by washing and that morphine, unlike D-Ala2-D-Leu5-enkephalin, became much less sensitive to naloxone antagonism. The observed modifications in the shape of the dose-response curves indicate that the effect of the opiates in the mouse vas deferens is more complex than that expected through the occupation of a simple receptor. There is either more than one type of functional receptor for each agonist or only one receptor, which can interact with every drug in different ways; this complexity is discussed in terms of various possibilities, including fractional occupancy, positive cooperativity and multiple sites.