enkephalin--leucine-2-alanine and ketazocine

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

Previous studies using a variety of opiate ligands have suggested the existence of several subclasses of opiate receptors in crude membrane fractions of rat brain, and a similar diversity in bovine adrenal medulla. To examine the receptor profile of bovine adrenal medulla in detail we have studied the binding of classical ligands for mu (mu), delta (delta) and kappa (kappa) opiate receptors. [3H]naloxone ([3H]NAL), [3H]morphine ([3H]MOR), [3H]D-Ala2-D-Leu5-enkephalin ([3H]DAL) and [3H]ethyl-ketocyclazocine ([3H]EKCZ) were used as tracers; unlabeled competitors were NAL, MOR, DAL and ketocyclazocine (KCZ). In adrenal medulla [3H]NAL was specifically bound with a hierarchy of displacement NAL greater than MOR greater than KCZ much greater than DAL. No specific binding of [3H]DAL or [3H]EKCZ was found; for [3H]MOR very low levels of binding were seen, with no displacement by NAL or DAL, inconsistent displacement by KCZ and substantial displacement by MOR with an ED50 of 1.5 nM. In parallel studies rat brain membranes bound each labeled ligand with affinity and specificity consistent with previously published reports. Identical results were obtained in membranes from both tissues prepared with a preincubation step including 100 mM Na+, suggesting that the results were not influenced by occupation of binding sites by endogenous ligands. We interpret these data as supporting the existence of opiate receptors of the mu subtype in bovine adrenal medulla. We find, however, no evidence of delta or kappa sites in this tissue.

Topics: Adrenal Medulla; Animals; Binding Sites; Brain; Brain Chemistry; Cattle; Cyclazocine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; In Vitro Techniques; Ligands; Male; Morphine; Naloxone; Rats; Rats, Inbred Strains; Receptors, Opioid