chlornaltrexamine and norbinaltorphimine

Kappa opioid receptor (KOR) desensitization was previously shown to follow agonist-dependent phosphorylation of serine 369 by G-protein receptor kinase (GRK) and beta-arrestin binding in transfected cells. To study the in vivo effects induced by phosphorylation of KOR(S369), C57Bl/6 mice were administered single or repeated doses of the KOR agonist, U50,488, and isolated brain glycoprotein was probed with an antibody, KOR-P, that specifically recognized phosphoserine 369 KOR. Western blot analysis using KOR-P antibody showed that labeling intensity increased after either single or repeated treatment of mice with U50,488 by 59 +/- 22% and 101 +/- 29%, respectively. In contrast, there was no change in labeling intensity by nonphosphoselective KOR antibodies following acute or chronic in vivo treatment with kappa agonist. Moreover, mice lacking GRK3 showed no increase in KOR-P labeling and developed significantly less analgesic tolerance following treatment with kappa agonist. The result suggests that tolerance to kappa agonists includes phosphorylation of serine 369 within KOR by GRK3. Recovery of analgesic potency and reduction of elevated KOR-P labeling in wild-type mice both required 2 weeks to return to base line. Consistent with these results, in vitro phosphorylation by GRK3 of KOR isolated from tolerant mice resulted in 46 +/- 7% less (32)P incorporation than in KOR isolated from untreated mice. In addition, in vitro (32)P incorporation returned to base line levels only in KOR isolated from tolerant mice allowed to recover for 2 weeks. The coincident reversal of analgesic tolerance and slow return to a basal phosphorylation state matched the regeneration rate of functional kappa receptors following irreversible antagonism and suggested that receptor replacement rather than dephosphorylation was required to restore sensitivity.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Animals; Cell Line; Drug Tolerance; G-Protein-Coupled Receptor Kinase 3; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, Opioid, kappa

GI 87084B (3-[4-methoxycarbonyl-4-[(1-oxopropyl) phenylamino]1-piperidine]propanoic acid, methyl ester, hydrochloride) was found to be a potent opioid agonist in the guinea pig ileum (EC50 = 2.4 +/- 0.6 nM), the rat vas deferens (EC50 = 387 +/- 44 nM) and the mouse vas deferens (EC50 = 39.5 +/- 7.4 nM). In the guinea pig ileum, GI 87084B, was roughly equivalent in potency to fentanyl (EC50 = 1.8 +/- 0.4 nM). GI 87084B was more potent in this tissue than alfentanil (EC50 = 20.1 +/- 1.2 nM) and less potent than sufentanil (EC50 = 0.3 +/- 0.09 nM). Schild analyses of antagonism of GI 87084B by naloxone yielded pKB values of 8.2 and slopes indistinguishable from unity in the guinea pig ileum and the mouse vas deferens. Insurmountable antagonism of GI 87084B by naloxone was observed in the rat vas deferens. However, an empirical measure of antagonist potency could be made: apparent pA2 = 8.1. The agonist dissociation constant (KA) for GI 87084B (220 +/- 90 nM) was determined by receptor alkylation with beta-chlornaltrexamine in the guinea pig ileum. Calculation of receptor occupancy suggested poor receptor-effector coupling and limited receptor reserve in the rat vas deferens, which could explain the insurmountable antagonism seen with higher concentrations of naloxone. These data suggest that GI 87084B acted through the mu class of opioid receptors to inhibit contraction induced by field stimulation in these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkylation; Animals; Enkephalin, Leucine; Guinea Pigs; Ileum; Kinetics; Male; Mice; Mice, Inbred Strains; Muscle Contraction; Muscle, Smooth; Naloxone; Naltrexone; Narcotic Antagonists; Piperidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Remifentanil; Vas Deferens