κ-Opioid receptors are differentially labeled by arylacetamides and benzomorphans
Introduction
The pharmacology of κ-opioid receptor agonists has been the target of a constant scientific inquiry due to increased evidence pointing towards κ-opioid receptor selective-compounds as possible treatments for drug abuse Mello and Negus, 1998, Negus et al., 1997. Indeed, it has been demonstrated that κ-opioid receptor selective-agonists are capable of reducing the increase in dopamine levels following cocaine administration Maisonneuve et al., 1994, Collins et al., 2001. In addition, the analgesia induced by κ-opioid receptor selective-agonists was found to be considerably more resistant to the induction of tolerance and withdrawal than analgesia, induced by μ-opioid receptor selective-agonists (Bhargava et al., 1989).
One of the challenges of studying the pharmacology of κ-opioid receptor selective-agonists consists of increased heterogeneity in results for compounds that have been postulated to be selective for κ-opioid receptors and induce similar degrees of analgesia. Several hypotheses were advanced and tested in order to clarify these inconsistencies. Jordan and Devi (1999) demonstrated the existence of homo/heterodimer formation between various opioid receptors, in transfected cell lines, which suggests the putative existence of additional binding pockets formed within the already known opioid receptors. On the other hand, apparent subtypes of κ-opioid receptors were pharmacologically identified in brain tissues of non-human primates and humans Zukin et al., 1988, Butelman et al., 1998, Caudle et al., 1998, Wollemann et al., 1993. κ1-Opioid receptors were functionally discovered due to their selective binding to arylacetamides, such as (5α,7α,8β)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro [4.5]dec-8-yl)-benzeneacetamide (U69,593) (Lahti et al., 1985) while κ2-opioid receptors were pharmacologically described due to their selective binding to benzomorphans such as bremazocine (Romer et al., 1980). Moreover, κ1-opioid receptor subtypes were pharmacologically defined as highly selective for the κ1-opioid antagonist nor-binaltorphimine Portoghese et al., 1987, Portoghese et al., 1991, while κ2-opioid receptor subtypes reportedly showed about a 100-fold lower affinity and selectivity for nor-binaltorphimine Zukin et al., 1988, Clark et al., 1989. The difference in their molecular structure remains unclear thus far, because only one κ-opioid receptor clone has been found within each species (Raynor et al., 1994). Another possible explanation for the pharmacological heterogeneity of κ-opioid receptors could be the putative existence of different affinity states (i.e., high affinity versus low affinity states) of the same receptor population. Thus, the previously described different subtypes of κ-opioid receptors may actually represent different affinity states or binding sites on the same receptor.
In order to test this hypothesis, we designed a series of experiments that compared and contrasted the pharmacological profiles of two structurally different κ-opioid receptor agonists: U69,593, an arylacetamide, and bremazocine, a benzomorphan. These two ligands have been previously associated with high selectivity for κ1- and κ2-opioid receptor subpopulations, respectively Lahti et al., 1985, Romer et al., 1980. In order to evaluate their binding affinity and selectivity for the different κ-opioid receptor subtypes, competition and saturation binding experiments were designed, in the presence and absence of GTPγS, a stable analog of GTP which is a G-protein uncoupling agent (Ofri et al., 1992). We further compared the efficacies of the two κ-opioid receptor agonists by determining the agonist-stimulated binding of [35S]GTPγS to the alpha subunit of the G protein (Traynor and Nahorski, 1995). The effect of increased GDP concentrations on the agonist-induced G-protein opioid receptor coupling and activation were also studied for both U69,593 and bremazocine. Also, the ability of the selective κ1-opioid receptor antagonist, nor-binaltorphimine (Marki et al., 2000) to differentiate between the different receptor binding and/or affinity states labeled by either bremazocine or U69,593 was investigated.
Section snippets
Materials and methods
Chinese Hamster Ovary cells stably expressing human κ-opioid receptors (hKOR-CHO) (L.Toll, SRI, Palo Alto, CA, USA) were used in all the experiments. These cells were cultured at 37 °C and 10% CO2, in a Dulbecco's modified Eagle's media (DMEM) enriched with 10% fetal bovine serum and penicillin–streptomycin (10,000 units/ml).
Effect of GTPγS on [3H]bremazocine and [3H]U69,593 binding
To determine if GTPγS altered either [3H]bremazocine or [3H]U69,593 binding to hKOR-CHO membranes, the binding of 0.5 nM [3H]U69,593 or 0.1 nM [3H]bremazocine in the presence of 12 different concentrations of GTPγS was determined. Fig. 1 shows that GTPγS inhibited [3H]U69,593 binding with an IC50value of 550±55 nM, the concentration needed to achieve half of the maximal inhibition (Imax=74±3%). GTPγS was relatively ineffective in reducing [3H]bremazocine binding to hKOR receptors. The maximal
Discussion
According to the current literature, there may be at least two different κ-opioid receptors functionally defined by their selective binding to either arylacetamides or benzomorphans Lahti et al., 1985, Romer et al., 1980. This current study was designed to test the hypothesis that the above differentiation in binding profiles between the two compound classes could be attributed to different affinity states of the same κ-opioid receptors.
Our results have shown that GTPγS inhibited the binding of
Acknowledgments
This work was supported by the U.S. Public Health Service Grants K05-DA00360, DA03742, U19-DA11007, and K05-DA00101 from the National Institute on Drug Abuse, National Institutes of Health.
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