enkephalin--leucine-2-alanine and chlornaltrexamine

The affinity of morphine, normorphine, methadone, Tyr-D-Ala-Gly-MePhe-NH(CH2)2(N-O)(CH3)2 (RX 783030), [D-Ala2,D-Leu5]enkephalin (DADLE), ketazocine and ethylketocyclazocine (EKC) were determined for their pharmacological receptors in two bioassay tissues, the guinea-pig ileum and the mouse vas deferens (MVD). The method involved the use of the irreversible antagonist, beta-chlornaltrexamine (beta-CNA), and the method of partial receptor blockade. The agonist concentration-effect curves were displaced to the right with decreasing maximum effect, a pattern typical of partial, irreversible blockade of receptors. The concentrations of beta-CNA required to produce a rightward displacement in the concentration-effect curves for different agonists, ranged between 2 and 3000 nM. No similarity was found between the IC50 and the dissociation constant (KA), values predicted to be equivalent only if a linear relationship exists between receptor occupation and observed effect; the dissociation constant for the agonists were between 3 and 218 times larger than the IC50 values. When methadone was used as the agonist in the guinea-pig ileum, beta-CNA produced parallel displacement of the concentration-effect curve, regardless of the blocking concentration chosen, preventing the determination of KA for this agonist, in this tissue; this problem was not encountered in the mouse vas deferens. The KA of morphine, RX 783030 and ketazocine were found not to differ in the guinea-pig ileum and mouse vas deferens. As expected, DADLE had significantly different affinity in the two tissues, showing 117-fold lower affinity in the guinea-pig ileum. Surprisingly, the normorphine affinity was found to be 7-fold higher in the guinea-pig ileum. While the difference in affinity of DADLE may be due to the suggested lack of functional delta receptors in the guinea-pig ileum, the difference in affinity seen with normorphine, but not morphine, in the two tissues is difficult to explain. Taken together with the insensitivity of methadone to beta-CNA blockade in the guinea-pig ileum, but not mouse vas deferens, the difference in the affinity of normorphine in these tissues may suggest the possibility of differences in local milieu of mu receptors or of mu receptor subtypes in the two tissues. The results provide fundamental information regarding opioid agonist affinity in two standard bioassays in vitro, and support the view of (1) a difference in receptors activated by DADLE in the guinea-pig

Topics: Animals; Cyclazocine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Guinea Pigs; Ileum; Male; Methadone; Mice; Morphine; Morphine Derivatives; Naltrexone; Receptors, Opioid; Vas Deferens

Opioid receptors in intact NG 108-15 cells were irreversibly inactivated with increasing concentrations of the alkylating antagonist beta-chlornaltrexamine (CNA). The consequence of the reduction in density of opioid binding sites (quantified by saturation analysis of opioid binding in membranes) was studied at two steps of opioid receptor-mediated responses, (a) stimulation of high affinity GTPase and (b) inhibition of basal adenylate cyclase. Both agonist-mediated stimulation of GTPase and inhibition of adenylate cyclase activities were progressively reduced as the concentration of CNA in the pretreatment was increased. However, the loss of responsiveness for the two enzymes differed in two aspects. First, the diminution of GTPase responsiveness was in agreement with the loss of binding sites and took place at concentrations of CNA that were lower than those necessary to reduce responsiveness of adenylate cyclase. Second, the loss of responsiveness of GTPase occurred simply as reduction of maximal stimulation, whereas that of adenylate cyclase involved an initial reduction of apparent agonist affinity (10-fold) that was followed by a decrease in maximal effect. We next examined the loss of responsiveness of both GTPase and adenylate cyclase in membranes prepared from cells that had been exposed to increasing concentrations of pertussis toxin (PTX) to inactivate PTX-sensitive G proteins in vivo. Also in this case, the extent of reduction in responsiveness was more pronounced for GTPase than for adenylate cyclase, especially in membranes treated with high concentrations of PTX. However, the pattern of loss was identical for the two enzymes and involved a main reduction in maximal effect of the agonist that was followed only after a large degree of inactivation (greater than 60%) by a diminished apparent affinity for the agonist. Opioid receptor-mediated inhibition of cAMP accumulation in intact cells exhibits an IC50 for the agonist that is 30-10 times lower than that measured in membranes for stimulation of GTPase or inhibition of cyclase, respectively. Treatment of cells with either CNA (1 microM) or various concentrations of PTX altered the concentration-response curves for agonist-mediated inhibition of cAMP accumulation in a manner similar to that observed for adenylate cyclase in membranes, inasmuch as both maximal inhibition and apparent affinities for the agonist were decreased. However, this decrease in affinity (5-fold) was not sufficient to eli

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Cyclic AMP; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Naltrexone; Pertussis Toxin; Protein Binding; Receptors, Opioid; Tumor Cells, Cultured; Virulence Factors, Bordetella

beta-Chlornaltrexamine and beta-funaltrexamine analogues 4-7 with different length "arms" to which an electrophilic moiety is attached were synthesized in an effort to obtain affinity labels that would selectively and irreversibly block specific opioid receptor types and subtypes. One of the compounds, 4, was a potent, irreversible blocker of opioid receptors in the guinea pig ileum and mouse vas deferens preparations. The results of this study suggest that nucleophiles that are remote from the recognition locus are capable of alkylation by reactive electrophiles.

Topics: Naltrexone; Narcotic Antagonists; Receptors, Opioid; Structure-Activity Relationship

Cryptic receptor sites in the guinea-pig ileum preparation have been uncovered by the treatment of the preparation with the highly selective, irreversible mu opioid receptor antagonist, beta-funaltrexamine. These beta-funaltrexamine-insensitive sites appear to interact only with opioid peptides ([D-Ala2, D-Leu5]enkephalin, [D-Ala2, Met5]enkephalinamide, Tyr-D-Ser-Gly-Phe-Leu-Thr and [D-Ala2, MePhe4, Gly-ol5]enkephalin) but not with nonpeptide agonists. These new sites could not be protected by either mu-selective (morphiceptin and [D-Ala2, MePhe4, Gly-ol5]enkephalin) or delta-selective ([D-Ala2, D-Leu5]enkephalin, Tyr-D-Ser-Gly-Phe-Leu-Thr, (Allyl)2-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu, and (Allyl)2-Tyr-Aib-Aib-Phe-Leu) peptides against beta-chlornaltrexamine alkylation. However, naloxone afforded full protection of these sites against beta-chlornaltrexamine alkylation. The delta-selective antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu, had no activity at these cryptic sites at concentrations that effectively blocked delta receptors in the mouse vas deferens. The cryptic sites do not appear to be typical mu or delta receptors. The new receptors were termed mu', a mu subtype, and a receptor model that is consonant with our data is presented.

Topics: Alkylation; Animals; Dose-Response Relationship, Drug; Electric Stimulation; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Guinea Pigs; Ileum; Male; Mice; Naloxone; Naltrexone; Oligopeptides; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Vas Deferens

Topics: Animals; Dose-Response Relationship, Drug; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Guinea Pigs; Ileum; Male; Mice; Muscle Contraction; Muscle, Smooth; Naloxone; Naltrexone; Receptors, Opioid; Receptors, Opioid, kappa; Structure-Activity Relationship; Vas Deferens