Activity of mu- and delta-opioid agonists in vas deferens from mice deficient in MOR gene.

1. Mice lacking the mu-opioid receptor have been recently generated. Centrally mediated responses of mu-opioid agonists are suppressed whereas some of the delta-opioid responses are preserved in these mutant mice. 2. The vas deferens bioassay has been used in this study to investigate the functional activity at a peripheral level of mu- and delta-opioid agonists in mice lacking mu-opioid receptors. 3. The different mu-opioid agonists evaluated, morphine, DAMGO, dermorphin and [Lys(7)]-dermorphin produced an inhibitory response in vas deferens from wild-type mice but had no relevant activity on vas deferens from mutant mice. 4. The selective delta-opioid agonists DPDPE, BUBU, deltorphin I, deltorphin II and [D-Met(2)]-deltorphin induced inhibitory effects in vas deferens from both wild-type and mutant mice. However, the biological activities of these ligands were slightly reduced in preparations from mutant mice. The inhibitory responses of all these delta-opioid agonists were prevented by the administration of the selective delta-opioid antagonist naltrindole. 5. These data indicate that delta-opioid agonists, but not mu-opioid agonists, are biologically active in vas deferens from mice lacking mu-opioid receptors. The decreased response of delta-agonists in mutant mice suggests that some cooperativity may exist between mu- and delta-opioid receptors in these vas deferens preparations.

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.

Effects of antisense oligonucleotides on brain delta-opioid receptor density and on SNC80-induced locomotor stimulation and colonic transit inhibition in rats.

1. To reduce the density of delta-opioid receptor protein, five antisense phosphorothioate oligodeoxynucleotides (aODN), targeting the three exons of rat delta-opioid receptor mRNA (DOR), were injected twice daily for 4 days or continuously infused for 7 days into brain lateral ventricles (i.c.v.) of Sprague-Dawley rats. Rats acting as controls were infused or injected with a mismatch sequence (mODN) of each aODN. The density of opioid receptors in rat brain membranes was measured by saturation binding experiments using selective ligands for delta, mu and kappa opioid receptors. 2. aODNs injected twice a day for 4 days left rat brain delta-opioid receptor density unchanged. The ODN targeting the DOR nucleotide sequence 280 - 299 (aODN280 - 299, exon 2), decreased brain delta-opioid receptor density significantly more than aODNs targeting exon 1 (aODN239 - 258), exon 2 (aODN361 - 380), or exon 3 (aODN741 - 760) (to 52% vs 79, 72, and 68%). None of the aODNs to the DOR changed the brain density of mu- or k-opioid receptors. 3. When in a novel environment (but not when kept in their home cages), the locomotor activity of aODN280 - 299 treated rats was significantly lower than that of saline or mODN treated rats. The delta-opioid agonist SNC80 (5 mg kg-1, s.c.) significantly and potently stimulated locomotion and delayed colonic propulsion in saline- and mODN-infused rats, but left motor behaviour and colonic transit of delta-knockdown rats unchanged. 4. The baseline nociceptive threshold and the antinociceptive response to morphine were unchanged in delta-knockdown rats.

Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats.

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

Respiratory and cardiovascular effects of the mu-opioid receptor agonist [Lys7]dermorphin in awake rats.

1. Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys7]dermorphin and its main metabolites, [1-5]dermorphin and [1-4]dermorphin. 2. Fifteen minutes after injection, doses of [Lys7]dermorphin producing antinociception (i.c.v., 36-120 nmol; s.c., 0.12-4.7 micromol kg(-1)) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5-HT receptor antagonist ritanserin (2 mg kg(-1), s.c.), was blocked by naloxone (0.1 mg kg(-1), s.c.), significantly reduced by the mu1 opioid receptor antagonist naloxonazine (10 mg kg(-1), s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg(-1), s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3-14.2 micromol kg(-1), i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO2. I.c.v. administration of [1-5]dermorphin and [1-4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO2-stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone. 3. In awake rats, [Lys7]dermorphin (0.1-1 mg kg(-1), s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine. 4. In conclusion, the present study indicates that analgesic doses of [Lys7]dermorphin stimulate respiration by activating central mu1 opioid receptors and this respiratory stimulation involves a forebrain 5-hydroxytryptaminergic excitatory pathway.

Postnatal development of delta-opioid receptor subtypes in mice.

1. The density and affinity of binding sites for the delta-selective opioid ligands [3H]-[D-Ala2, Asp4]deltorphin (DELT-I), [3H]-[D-Ala2Glu4]-deltorphin (DELT-II), [3H]-[D-Pen2,D-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. 2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. 3. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34%, high affinity site, Kd = 1.08 nM and 66% low affinity sites, Kd = 39.9 nM). 4. DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. 5. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. 6. These data suggest that a sub-population of delta-sites (probably the delta 2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. 7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three delta-agonists was: DELT-I > DELT-II > DPDPE in 10 day-old mice: and DELT-I- DELT-II > DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the delta 2-subtype.

The dermorphin peptide family.

1. 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. Given the name dermorphins, these were the first members of a peptide family that in the past 15 years has grown to reach a total of seven naturally occurring peptides and nearly 30 synthetic analogs. 2. Dermorphin peptides are potent analgesics in rodents and primates, including man. Some dermorphins can enter the blood-brain barrier and produce central antinociception after peripheral administration. 3. The dermorphin family also includes mu 1-opioid receptor selective agonists that produce intense opioid analgesia, but stimulate pulmonary ventilation. 4. Experiments in rats and mice chronically exposed to dermorphins have shown that not only do they have higher antinociceptive efficacy and potency than morphine, but they are also less likely than morphine to produce tolerance, dependence and opiate side effects.

Opioid receptor affinity and analgesic activity of O- and C-glycosylated opioid peptides.

O- and C-glycosylation of the mu-agonist dermorphin reduced neither its mu receptor affinity in binding assay nor its agonist potency in guinea-pig ileum assay (GPI). O- and C-glycosylation of the delta-agonist deltorphin reduced its delta-receptor affinity and its agonist potency in mouse vas deferens assay (MVD). O- and C-glycosylated dermorphin, administered i.c.v. and s.c., produced long-lasting antinociception in mice and rats. The ratio between i.c.v. and s.c. antinociceptive ED50 demonstrates facilitated transport into the CNS only for the galactosil peptide. Acetylation significantly reduced penetration of glycopeptides into the CNS indicating that facilitated transport into the CNS exists, but does not depend on the glucose transporter (GLUT-1).

Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding.

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.

Development of delta-opioid receptor subtypes and the regulatory role of weaning: radioligand binding, autoradiography and in situ hybridization studies.

Evidence from behavioral studies suggests that the process of weaning activates the development of a delta-opioid receptor subtype. We now report the influence of weaning on the development of delta receptors in the central nervous system assessed by membrane homogenate binding and autoradiography with selective delta radioligands and by in situ hybridization using a cRNA probe for the delta receptor. Binding was carried out by using [3H][D-Ala2]deltorphin I (DELT I), [3H]IIe5,6-deltorphin II (IIe5,6-DELT II) and [3H]naltrindole (NTI). [3H]IIe5,6-DELT II and [3H]NTI labeled an equivalent number of sites in brain and spinal cord from both weaned and nonweaned 25-day-old rats. The number of sites labeled by [3H]DELT I was similar in nonweaned rats but significantly higher in the brain and cord from weaned animals. Furthermore, the ontogenetic profile of these three ligands was distinct. Quantitative autoradiography showed identical levels of [3H]IIe5,6-DELT II binding in all brain regions in weaned and nonweaned rats. In contrast, levels of [3H]DELT I binding were significantly higher in weaned rats and this difference was localized to the deep layers of the frontal-parietal cortex and to the pontine nucleus. In situ hybridization experiments showed no differences in delta-opioid receptor mRNA density between weaned and nonweaned groups in the regions in which binding differences were observed. Weaning stimulates the development of a subpopulation of delta receptors recognized by [3H]DELT I but not by [3H]IIe5,6-DELT II or NTI. This effect is localized to specific brain regions and does not appear to reflect increased synthesis of mRNA coding for the delta receptor.

Production of antinociception by peripheral administration of [Lys7]dermorphin, a naturally occurring peptide with high affinity for mu-opioid receptors.

1. The opioid activity of the amphibian peptide, [Lys7]dermorphin, was studied in rats and mice. When administered intracerebroventricularly (i.c.v.), intravenously (i.v.) or subcutaneously (s.c.) it produced a long lasting analgesia. Its antinociceptive potency exceeded that of morphine 290 times by i.c.v. injection, and 25-30 times by peripheral administration. 2. The dose-response curves of [Lys7]dermorphin antinociception were shifted to the right by the pretreatment with naloxone (0.1 mg kg-1, s.c.) or with the mu 1-selective antagonist, naloxonazine (10 mg kg-1, i.v. 24 h before peptide injection). 3. The peptide also displayed potent antinociceptive effects in a chronic inflammatory pain model (rat Freund's adjuvant arthritis). In this pain model, systemic administration of the peptide raised the nociceptive threshold more in inflamed than in healthy paw. 4. High central and peripheral doses of [Lys7]dermorphin in rats produced catalepsy. The cataleptic response was antagonized by naloxone but left unchanged by naloxonazine pretreatment. 5. In rats and mice, central or peripheral administration of [Lys7]dermorphin induced a significantly slower development of tolerance to the antinociceptive effect than did morphine. 6. Upon naloxone precipitation of the withdrawal syndrome, [Lys7]dermorphin-dependent mice made fewer jumps and lost less weight than the morphine-dependent animals. Withdrawal hyperalgesia did not develop in [Lys7]dermorphin-dependent mice. 7. In conclusion, [Lys7]dermorphin seems to be a unique opioid peptide having a high penetration into the blood-brain barrier despite its low lipid solubility. This peptide causes fewer side-effects than other opioids and appears less likely than morphine to cause physical dependence in rats and mice.

A crystal-state, solution and theoretical study of the preferred conformation of linear C alpha, alpha-diphenylglycine derivatives and dipeptides with potential anticonvulsant activity.

Several linear molecules containing the C alpha, alpha-diphenylglycine residue were prepared as potential anticonvulsants. The conformational preferences of the C alpha, alpha-diphenylglycine residue were assessed in these synthetic derivatives and dipeptides by X-ray diffraction, FTIR absorption and 1H NMR techniques, and by conformational energy computations. Five (out of six) derivatives adopt the fully extended C5 conformation in the crystal state. This intramolecularly H-bonded form is largely populated in chloroform solution in all the derivatives investigated. Conformational energy computations in vacuo support the view that the intramolecularly H-bonded C7-ring form is the most stable structure for these compounds. Only one linear derivative exhibits a (modest) anticonvulsant activity.

Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain.

Previous biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a 3H-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.

[D-Ala2]deltorphin I binding and pharmacological evidence for a special subtype of delta opioid receptor on human and invertebrate immune cells.

The effects of the opioid neuropeptide [D-Ala2]deltorphin I, isolated from amphibian skin, on immunoregulatory activities were studied in representatives of vertebrates and invertebrates. The high potency of this compound parallels that of [Met]enkephalin, which was previously demonstrated in vertebrate plasma and invertebrate hemolymph. The addition of [D-Ala2]deltorphin I at 10(-11) M to human granulocytes or immunocytes of the mollusc Mytilus edulis resulted in cellular adherence and conformational changes indicative of cellular activation. This value is in line with the concentrations obtained with [Met]enkephalin, tested in the presence of the specific neutral endopeptidase 24.11 inhibitor phosphoramidon, and this opioid's synthetic analog [D-Ala2, Met5]enkephalin which, like [D-Ala2]deltorphin I, is resistant to proteolytic degradation. Both ligands appear to be acting on the same population of immunocytes. The same relationship was estimated to exist in the insect Leucophaea maderae, in which the high viscosity of the hemolymph makes the quantification of reactive cells more difficult than in Mytilus. In addition, [D-Ala2]deltorphin I is as potent as beta-endorphin in affecting the proliferation of lymphocytes in response to mitogen. Saturation experiments with unlabeled ligands and the radioligands [3H][D-Ala2]deltorphin I and [3H][D-Ala2,Met5]enkephalinamide revealed the presence of two high-affinity binding sites on human granulocytes, one sensitive to the nonequilibrium delta opioid antagonist [D-Ala2,Leu5,Cys6]enkephalinamide and the other relatively insensitive. The results obtained with [D-Ala2]deltorphin I support the view that the special role played by endogenous [Met]enkephalin in immunobiological activities of vertebrates and invertebrates is mediated by a special subtype of delta opioid receptor.

Dermorphin-related peptides from the skin of Phyllomedusa bicolor and their amidated analogs activate two mu opioid receptor subtypes that modulate antinociception and catalepsy in the rat.

Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.

Long-term sensitization to the activation of cerebral delta-opioid receptors by the deltorphin Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 in rats exposed to morphine.

In experiments to evaluate responses to the activation of cerebral delta-opioid receptors, repeated daily injection of the selective delta-opioid agonist Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 ([D-Ala2]deltorphin II) into rat brain resulted in the development of tolerance, whereas repeated daily injection or continuous infusion of morphine resulted in sensitization to the behavioral activating effects of the delta-opioid agonist. Although the rats did not modify their spontaneous locomotor activity after morphine withdrawal, they became markedly hyperresponsive to the locomotor and stereotypy-producing effects of a challenge dose of the delta-opioid agonist. Sensitization to activation of delta-opioid receptors persisted for at least 60 days after discontinuing morphine treatment. These results show that the development of tolerance and long-term sensitization to opioids involves delta-opioid as well as mu-opioid receptors.

A family of bombinin-related peptides from the skin of Bombina variegata.

A peptide fraction was isolated from the skin of Bombina variegata that showed antimicrobial activity. This fraction contained several molecular species, all of them consisting of 27 amino acid residues, with a constant C-terminal region (from residues 14-27), including an amidated carboxyl end and a variable N-terminal segment. These peptides are related but not identical to bombinin [Csordas, A. & Michl, H. (1970) Monatsh. Chem. 101, 182-189]. By using synthetic oligonucleotides corresponding to the C-terminal region of the peptides, a cDNA library from the skin of B. variegata was screened and several positive clones coding for the corresponding peptide precursors were isolated and sequenced. Each clone contained the genetic information for a different bombinin-like peptide. The antimicrobial activity towards different bacterial species of a synthetic peptide corresponding to one of the variants deduced from cDNA sequences was tested. This peptide was found to be mainly active against different isolates of Staphylococci and Escherichia coli.

(D-Ala2)deltorphin II: D1-dependent stereotypies and stimulation of dopamine release in the nucleus accumbens.

In order to investigate the relative role of central delta- and mu-opioid receptors in behavior, the effects of (D-Ala2)deltorphin II, a natural delta-opioid peptide, and PL017, a beta-casomorphin derivative specific for mu receptors, were compared after local intracerebral and intraventricular administration. Intracerebral infusion of the two peptides was done bilaterally in the limbic nucleus accumbens and in the ventral and dorsal caudate putamen of freely moving rats through chronic intracerebral cannulas. After intra-accumbens infusion, the two peptides elicited marked but opposite behavioral effects: while (D-Ala2)deltorphin II evoked dose-dependent motor stimulation characterized by locomotion, sniffing, and oral stereotypies, PL017 elicited motor inhibition with rigidity and catalepsy. These effects were site specific because they could not be evoked from the ventral or from the dorsal caudate. Low doses of naloxone (0.1 mg/kg, s.c.) blocked the effects of PL017 but not those of (D-Ala2)deltorphin II, which instead were reduced by high doses of naloxone (1.0 mg/kg) and by the putative delta-antagonist naltrindole; this drug failed to affect the catalepsy induced by PL017. Therefore, while (D-Ala2)deltorphin II effects were delta-mediated, PL017 effects were mu-mediated. Blockade of dopamine D1 receptors by SCH 23390 abolished (D-Ala2)deltorphin II effects, while blockade of dopamine D2 receptors by raclopride or by haloperidol was without effect. Local application by reverse dialysis of (D-Ala2)deltorphin II (5 microM) to the accumbens resulted in a naloxone-sensitive increase of extracellular dopamine concentrations; these effects could not be evoked from the caudate, nor by PL017 in the accumbens. Intracerebroventricular administration of (D-Ala2)deltorphin II or of PL017 elicited behavioral effects qualitatively similar to those obtained from the accumbens.

Structure-activity relationships of the delta-opioid-selective agonists, deltorphins.

Deltorphins are naturally occurring peptides with high affinity and selectivity for delta-opioid receptors. They share with dermorphin, another mu-selective opioid agonist, the same N-terminal tripeptide Tyr-D-Xaa-Phe, where D-Xaa is a D-Ala or a D-Met residue. This common sequence appears to be essential for the best fitting of the peptides to both mu- or delta-opioid sites. We studied the changes in receptor affinity and selectivity and in biological potency of deltorphins due to shortening of the sequence, C-terminal deamidation or single amino acid substitutions. The results support the view that a code addressing the molecule towards delta-opioid sites is expressed in the C-terminal region of these peptides. This addressing domain confers high delta-selectivity to the ligand in the following two ways: (i) increased affinity for delta-sites; (ii) decreased affinity for mu-sites. The sequence of the C-terminal tripeptide appears to be responsible for the high delta-affinity of the molecules. Negatively charged side chains inhibit mu-binding and enhance delta-selectivity.