Victor Bykov

Interaction of endogenous opioid peptides and other drugs with four kappa opioid binding sites in guinea pig brain

Guinea pig brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents, 2-(4-ethoxybenzyl)-1- diethylaminoethyl-5-isothiocyanatobenzimidazole.HCl (BIT) and N-phenyl-N-[1-(2-(4-isothiocyanato)phenethyl)-4- piperidinyl]-propanamide.HCl (FIT), were used in this study to test the hypothesis that guinea pig brain possesses subtypes of kappa receptors. Pretreatment of membranes with either (-)-(1S,2S)-U50,488 or the kappa selective acylating agent, (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, caused a wash-resistant inhibition of kappa 1 binding sites labeled by [3H]U69,593 binding, but not kappa 2 binding sites labeled by [3H]bremazocine. Binding surface analysis of [3H]bremazocine binding resolved two binding sites, termed kappa 2 and kappa 2b, present at densities of 212 and 225 fmol/mg protein, which had low affinity for (-)-(1S,2S)-U50,488 and U69,593. The kappa 2b site had high affinity for beta-endorphin(1-31) (Kd = 5.5 nM) and [D-Ala2,D-Leu5]enkephalin (Kd = 14 nM), and lower affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (Kd = 147 nM) and [Leu5]enkephalin (Kd = 46.0 nM). Binding surface analysis of [3H]U69,593 binding also resolved two binding sites, termed kappa 1a and kappa 1b, present at densities of 6.0 and 40.0 fmol/mg protein. The kappa 1a binding site was characterized by very high affinity for alpha-neoendorphin. Quantitative autoradiographic studies demonstrated that kappa 2a and kappa 2b binding sites are heterogeneously distributed in guinea pig brain, and that the anatomical distribution of kappa 1 binding sites reported in the literature is different from that observed in this study for the kappa 2 binding sites. Viewed collectively, these data provide evidence for four kappa receptor subtypes in guinea pig brain.

Preparation of rat brain membranes greatly enriched with either type-I-delta or type-II-delta opiate binding sites using site directed alkylating agents: Evidence for a two-site allosteric model

Although it is widely accepted that radiolabeled prototypic delta receptor agonists label two binding sites in vitro, the mechanism by which mu ligands inhibit peptide binding as well as the identity of the binding sites remains unsettled (Rothman and Westfall, Mol. Pharmacol. 21:538-547, 1982 ; Bowen et al., Proc. Natl. Acad. Sci. U.S.A. 78:4818-4822, 1981). Using the site directed, receptor selective alkylating agents, BIT and FIT (Rice et al., Science 220:314-316, 1983), we describe the preparation of membranes devoid of high affinity binding sites and demonstrate that the mu agonist oxymorphone noncompetitively inhibits the binding of [3H]DADL to the residual lower affinity binding sites.

Interaction of enantiomeric pairs of opiates with phencyclidine binding sites in rat brain: Identification of (+) pentazocine as a ligand potentially suitable for imaging sigma binding sites using positron emission tomography

Some unnatural opiates, which do not interact with classical opiate receptors, interact with phencyclidine (PCP) receptors. Among their many pharmacological actions, drugs which bind to the PCP receptor antagonize the actions of glutamic acid mediated via the NMDA excitatory amino acid receptor, leading to their potential use as anti-ischemic and anticonvulsant agents. Despite an enormous effort, identification of a PCP receptor antagonist, which would be useful for research and therapeutics, has not yet been reported. Chemical modification of unnatural opiates as a means to produce a PCP antagonist, or PCP agonists with properties different than PCP, has not been fully explored. Towards this end, we determined the equilibrium dissociation constants of eight enantiomeric pairs of opiates for the rat brain PCP receptor.

Tritiated-6-beta-fluoro-6-desoxy-oxymorphone ([3H]FOXY): a new ligand and photoaffinity probe for the mu opioid receptors.

6-Beta-fluoro-6-desoxy-oxymorphone (FOXY) is a fluorinated derivative of oxymorphone originally developed as a potential PET scanning ligand. Preliminary work (Rothman et al., Neuropeptides 4: 311-317, 1984) demonstrated that [3H]FOXY selectively labeled mu opioid binding sites with low levels of nonspecific binding. In this study the opiate receptor subtypes labeled by [3H]FOXY and [3H]D-ala2-MePhe4, Gly-ol5-enkephalin ([3H] DAGO) were compared using site directed acylating agents and binding surface analysis. Although the data indicated that both ligands selectively label mu opiate receptors, other experiments demonstrated that [3H]DAGO and [3H]FOXY labeled mu binding sites differently. Additional experiments demonstrated that [3H]FOXY can be used as a high yield photoaffinity label for the mu opiate receptor subtype.

Pretreatment of rats with the irreversible μ-receptor antagonist, β-FNA, fails to prevent naltrexone-induced upregulation of μ-opioid receptors

Abstract This study examined the effect of β-funaltrexamine (β-FNA), an irreversible μ-receptor antagonist, on naltrexone-induced upregulation of μ -( μ cx + μ ncx ) and δ ncx -opioid receptors. [The subscripts ‘ex’ and ‘ncx’ denote binding sites ‘in’ (cx) and ‘not in’ (ncx) the opioid receptor complex.] Rats were treated according to the following protocol. Two naltrexone or two placebo pellets were implanted subcutaneously in a nylon mesh on day 1, and were removed intact on day 8. Rats were given either saline or 20 nmol of β-FNA in 10 μ1 of saline (i.c.v.) on days 1, 3, 5 and 6, 60 min prior to implantation of the pellet. On day 9 frozen lysed-P2 membranes were prepared for assay of μ binding sites. In other experiments, membranes were depleted of μ-receptors by pretreatment with the site-directed acylating agent 2-(4-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCl (BIT) for assay of δ ncx binding sites, using [ 3 H][ d -ala 2 , d -leu 5 ]enkephalin. The results demonstrated that β-FNA did not upregulate the μ binding sites and also did not prevent naltrexone-induced upregulation of μ binding sites. Both μ-FNA and naltrexone increased the B - max of δ ncx binding sites and their effects were additive. These data suggest that the mechanism(s) responsible for antagonist-induced upregulation of opioid receptors are more complex than previously appreciated.

The potent opioid agonist, (+)-cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: Evidence for a novel mechanism of action

The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl [(+)-cis-MF], followed by extensive washing of the membranes, produces a wash-resistant decrease in the binding of [3H]-[D-ala2,D-leu5]enkephalin to the d binding site of the opioid receptor complex (delta cx binding site). Intravenous administration of (+)-cis-MF (50 micrograms/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of delta cx and mu binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior to the preparation of membranes) completely reversed the catatonia and restored masked delta cx binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.