enkephalin-leu--ala(2)-cys(6)- and naltrindole-5--isothiocyanate

A number of recent investigations have proposed the existence of two pharmacologically distinct delta opioid receptor subtypes, named delta 1 and delta 2. In the present study, we have investigated the involvement of the two delta receptors in the opioid stimulation of adenylyl cyclase activity in rat olfactory bulb. In addition, we have conducted a similar investigation in rat striatum, where delta agonists are known to inhibit cyclic AMP formation. Both (D-Ala2, Glu4) deltorphin (DELT), a delta 2 agonist, and [D-Pen2, D-Pen5] enkephalin (DPDPE), a delta 1 agonist, stimulated adenylyl cyclase activity in rat olfactory bulb in a concentration-dependent manner, DELT being 25-fold more potent than DPDPE. The selective delta 2 antagonist naltriben counteracted the stimulatory effects of both agonists with a potency about 10-fold higher than that of the selective delta 1 antagonist 7-benzylidenenaltrexone. Moreover, pretreatment of olfactory bulb membranes with the nonequilibrium antagonist naltrindole 5'-isothiocyanate, which irreversibly blocks the delta 2 subtype, reduced the stimulatory effects of both DELT and DPDPE, whereas pretreatment with [D-Ala2, Leu5, Cys6]enkephalin, which binds covalently to delta 1 receptors, failed to affect the response to the agonists. Similar results were obtained in rat striatum. These data indicate that delta opioid receptors coupled to either stimulation or inhibition of adenylyl cyclase in two different brain areas predominantly belong to the delta 2 subtype.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Corpus Striatum; Enkephalin, Leucine-2-Alanine; Enzyme Activation; In Vitro Techniques; Isothiocyanates; Male; Molecular Sequence Data; Naltrexone; Olfactory Bulb; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta

In this study, the delta receptor-selective nonequilibrium affinity ligands, 5'-NTII and DALCE, and the nonspecific sulfhydryl reagent NEM were evaluated over a range of concentrations and treatment conditions for their ability to selectively alter the binding properties of delta 1- or delta 2-preferring opioid radioligands in brain homogenate. Treatment of tissue preparations with DALCE (0-10,000 nM) or NTII (0-10,000 nM) resulted in an equivalent concentration-dependent loss of binding capacity for the delta 1 agonist 3H-DPDPE and the mu/delta 2 agonist 3H-DSLET. In contrast, treatment of tissue with NEM (0-8000 microM) resulted in greater loss of 3H-DPDPE binding. Scatchard analysis of the binding of 3H-DPDPE, 3H-DSLET, and 3H-NTI in 3 mM NEM-treated rat brain P2 preparation revealed an equivalent decrease in affinity for the agonist ligands, but a significantly greater decrease in Bmax for 3H-DPDPE compared with control tissue values. Comparison of the K(i) values for a series of delta-selective compounds against 3H-DSLET binding in control vs 3 mM NEM treated P2 fraction showed differential effects of NEM on affinity within the series that were consistent with a selective depletion of delta 1 sites. Overall, these results indicate that NEM treatment selectively reduced delta 1 receptor binding, resulting in a preparation that is enriched in delta 2 sites.

Topics: Affinity Labels; Animals; Brain; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylmaleimide; Isothiocyanates; Male; Mice; Naltrexone; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta

Previously, we have shown that the development of physical dependence on morphine in mice is inhibited substantially by treatment of mice with the highly selective, nonequilibrium delta-2 opioid receptor antagonist, naltrindole-5'-isothiocyanate. With the availability of the highly selective, nonequilibrium delta-1 opioid receptor antagonist, [D-Ala2,Leu5,Cys6]enkephalin, it was possible, in the present report, to examine the possible involvement of delta-1 opioid receptors in the development of opiate dependence. Mice were made physically dependent on morphine by s.c. implantation of morphine pellets (75-mg free base) for 3 days. The degree of dependence was quantified by determining the ED50 values of naloxone to precipitate withdrawal jumping and diarrhea. Neither sign of opiate withdrawal was affected by chronic treatment of animals with [D-Ala2,Leu5,Cys6]enkephalin during the morphine implant period. The data suggest that delta-1, as opposed to delta-2, opioid receptors are not involved in the development of physical dependence on morphine. This fact takes on added significance because the recently cloned delta opioid receptors appear to be the delta-2 subtype and the present data together with previous findings suggest that the cloned receptors may be proper models for the study of opiate dependence.

Topics: Affinity Labels; Animals; Diarrhea; Enkephalin, Leucine-2-Alanine; Injections, Intraventricular; Injections, Spinal; Isothiocyanates; Male; Mice; Morphine; Naloxone; Naltrexone; Receptors, Opioid, delta; Substance-Related Disorders

The interaction of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2,Glu4]deltorphin with delta-opioid receptor subtypes was investigated. Pretreatment of mice with the delta 1-opioid receptor antagonist, [D-Ala2,Leu5,Cys6]enkephalin (DALCE), produced a virtually complete antagonism of the antinociceptive actions of DPDPE, but had no effect on those of [D-Ala2,Glu4]deltorphin. In DALCE pretreated mice (i.e., delta 1-opioid receptors blocked), DPDPE was able to significantly antagonize the antinociceptive effects of [D-Ala2,Glu4]deltorphin. Pretreatment of mice with the delta 2-opioid receptor antagonist, naltrindole-5'-isothiocyanate (5'-NTII) produced a virtually complete antagonism of the antinociceptive effects of [D-Ala2,Glu4]deltorphin, but had no effect on the antinociception produced by DPDPE. In 5'-NTII pretreated mice (i.e., delta 2-opioid receptors blocked), [D-Ala2,Glu4]deltorphin had no effect on the antinociception produced by DPDPE. These data suggest that [D-Ala2,Glu4]deltorphin is highly selective for the delta 2-opioid receptor in vivo, and that neither agonist nor antagonist actions can be demonstrated at delta 1-opioid receptors for this peptide. In contrast, under appropriate conditions, DPDPE can be shown to interact with both delta 1- and delta 2-opioid receptor subtypes; DPDPE may have limited efficacy (i.e., is a partial agonist) at the delta 2-opioid receptor.

Topics: Amino Acid Sequence; Analgesics; Animals; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Injections, Intraventricular; Isothiocyanates; Male; Mice; Mice, Inbred ICR; Molecular Sequence Data; Naltrexone; Oligopeptides; Pain Measurement; Reaction Time; Receptors, Opioid, delta

The identification of opioid delta receptor subtypes in mouse brain led to the investigation of the nature of the opioid delta receptors in the mouse isolated vas deferens in vitro. Noncumulative concentration-effect curves were constructed for DPDPE (delta 1 agonist) and [D-Ala2, Glu4]deltorphin (delta 2 agonist) in control tissues, or in tissues which had been incubated with either [D-Ala2, Leu5, Cys6] enkephalin (DALCE) (noncompetitive delta 1 antagonist) or 5'-naltrindole isothiocyanate (5'-NTII) (noncompetitive delta 2 antagonist). Incubation of the tissues with DALCE, under either oxygenated or nonoxygenated conditions, did not alter the concentration-effect curves for either agonist. In contrast, incubation of the tissues with 5'-NTII resulted in a significant rightward displacement of the concentration-effect curves of both DPDPE and [D-Ala2, Glu4] deltorphin. Additionally, naltriben, a selective and competitive delta 2 antagonist, showed no significant difference in its ability to antagonize a fixed, submaximal concentration of either DPDPE or [D-Ala2, Glu4]deltorphin. Furthermore, there was no significant difference in the affinity of naloxone (i.e., pA2) at the receptor(s) acted upon by either DPDPE or [D-Ala2, Glu4]deltorphin. Tolerance to DPDPE or [D-Ala2, Glu4]deltorphin was produced by incubation of the tissues with these agonists; construction of the [D-Ala2, Glu4]deltorphin concentration-effect curve in DPDPE-tolerant tissues demonstrated cross-tolerance between these agonists and, conversely, construction of DPDPE concentration-effect curves in [D-Ala2, Glu4]deltorphin-tolerant tissues revealed cross-tolerance between these agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Benzylidene Compounds; Drug Tolerance; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; In Vitro Techniques; Isothiocyanates; Male; Mice; Mice, Inbred ICR; Morphinans; Naltrexone; Oligopeptides; Receptors, Opioid, delta; Thiocyanates; Vas Deferens

Previous studies from our laboratory have indicated that i.c.v. pretreatment of mice with the novel, selective opioid delta receptor antagonists, [D-Ala2,Leu5,Cys6]enkephalin (DALCE) and naltrindole-5'-isothiocyanate (5'-NTII), differentially antagonized the direct antinociceptive effects of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2]deltorphin II (DELT). These findings, and others, suggested the existence of subtypes of opioid delta receptors which could be classified as activated by DPDPE and DALCE sensitive (delta 1 receptor), or selectively activated by DELT and 5'-NTII sensitive (delta 2 receptor). The present study has extended these observations to the characterization of delta-mediated antinociception effects of DPDPE and DELT after i.t. administration in mice using pretreatment with DALCE and 5'-NTII in order to selectively antagonize the delta subtypes. Additionally, the acute antinociceptive actions of DALCE itself were studied to ensure activity of this compound at the spinal level. The respective antinociceptive A50 value (95% CL) for i.t. DPDPE, DELT and DALCE were 19.0 (12.9-28.1), 19.3 (16.1-23.1) and 2.0 (1.4-3.0) nmol. The delta antagonist, N,N-diallyl-Try-Aib-Aib-Phe-Leu-OH (ICI 174,864) (where Aib is alpha-aminoisobutyric acid) blocked the antinociceptive effects of DPDPE and DELT, but not those of i.t. morphine or [D-Ala2,NMPhe4,Gly-ol5]enkephalin (DAMGO), indicating that the observed antinociceptive effects of DPDPE and DELT were delta mediated. Pretreatment 24 hr before testing with graded doses of i.t. 5'-NTII blocked the i.t. antinociceptive effects of DPDPE and DELT, although at least a 10-fold higher dose of 5'-NTII was needed to produce equivalent antagonism of DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Affinity Labels; Analgesics; Animals; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Isothiocyanates; Male; Mice; Mice, Inbred ICR; Morphinans; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Receptors, Opioid, delta; Spinal Cord; Thiocyanates

The present study has investigated the direct opioid delta receptor-mediated antinociception produced by i.c.v. administration of the highly selective delta agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, as well as that of the less delta-selective [D-Ser2,Leu5,Thr6]enkephalin (DSLET), by using two novel nonequilibrium opioid antagonists, [D-Ala2,Leu5,Cys6] enkephalin (DALCE) and naltrindole 5'-isothiocyanate (5'-NTII). At times ranging from 8 to 48 hr after a single i.c.v. pretreatment of mice with 5'-NTII, the antinociceptive effects of [D-Ala2] deltorphin II were significantly antagonized. In contrast, 5'-NTII pretreatment at times between 10 min and 24 hr failed to antagonize the antinociceptive effects of DPDPE. Previous studies have shown that pretreatment with i.c.v. DALCE produces a dose- and time-related antagonism of DPDPE, but not morphine, antinociception. However, pretreatment with i.c.v. DALCE failed to antagonize the antinociceptive effects of [D-Ala2]deltorphin II. Similarly, i.c.v. administration of DSLET produced time- and dose-related antinociception which was partially antagonized by either beta-funaltrexamine (beta-FNA) or by ICI 174,864 (N,N-dialyl-Tyr-Aib-Aib-Phe-Leu-OH), suggesting mixed activity at mu and delta receptors. ICI 174,864 produced essentially complete antagonism of DSLET antinociception in beta-FNA-pretreated mice. Pretreatment with 5'-NTII (at -8 to -48 hr), blocked the antinociception produced by DSLET in control or in beta-FNA-pretreated mice. In contrast, pretreatment with DALCE failed to antagonize the antinociception produced by i.c.v. DSLET in either control or in beta-FNA-pretreated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Dose-Response Relationship, Drug; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Injections, Intraventricular; Isothiocyanates; Male; Mice; Mice, Inbred ICR; Morphinans; Naltrexone; Narcotic Antagonists; Oligopeptides; Receptors, Opioid; Receptors, Opioid, delta; Thiocyanates