μ-Opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPγS binding assays
Introduction
Opioid drugs and opioid peptides produce their pharmacological effects, including antinociception, by interaction with opioid receptors in the central nervous system. Opioid receptors are known to be a heterogeneous population consisting of at least three major types (μ, δ and κ) which exhibit different ligand selectivity profiles (Borsodi and Tóth, 1995). Most endogenous opioids and synthetic ligands do not possess absolute specificity for a given receptor type, but interact with more than one opioid receptor. The situation is further complicated by the fact that multiple receptor types may coexist within a single tissue, or even a single cell (Borsodi, 1991).
The multiplicity of opioid receptors is generally accepted and the primary structure of the δ-opioid receptor Kieffer et al., 1992, Evans et al., 1993, μ-opioid receptor (Chen et al., 1993) and κ-opioid receptor (Reisine and Bell, 1993) are known. The further development of highly selective ligands remains a challenge for better characterization for each receptor type and possible subtypes.
Opioid receptor antagonists have been indispensable pharmacological tools for identifying receptor types involved in the actions of endogenous and synthetic opioid receptor agonists. Antagonists are especially useful when the pharmacological endpoints are identical (e.g., antinociception or the inhibition of a smooth muscle contractions), and when it is not easy to distinguish among μ-, δ- and κ-opioid receptor mediated effects. Matthes et al. have recently reported that the μ-opioid receptor gene product is the molecular target of morphine in vivo and that it is a mandatory component for the main pharmacological responses of this opioid (Matthes et al., 1996).
It is known that opioid receptors exert their biological functions by interacting with GTP binding proteins. Gi/Go proteins to which the opioid receptors are coupled regulate effector molecules such as adenylyl cyclase and/or ion channels (Standifer and Pasternak, 1997). Signal transduction can be monitored in membrane preparations by measuring the binding of the non-hydrolysable GTP analogue, guanosine-5′-O-(γ-thio)triphosphate (GTPγS) as a function of the amount of a given ligand (Traynor and Nahorski, 1995).
Cyprodime (N-cyclopropylmethyl-4,14-dimethoxymorphinan-6-one) has been shown to be a selective μ-opioid receptor antagonist by using guinea pig ileal longitudinal muscle preparations, rat and mouse vas deferentia and acetic-acid writhing tests (Schmidhammer et al., 1989).
In the present study, we have further defined the in vitro ligand-binding profile of cyprodime and described the biochemical characterization of its tritiated derivative, [3H]cyprodime. We have also evaluated the functional effectiveness of cyprodime to alter [35S]GTPγS binding and to inhibit morphine-stimulated [35S]GTPγS binding.
Section snippets
Chemicals
Cyprodime was synthesized as previously reported (Schmidhammer et al., 1989). d-Phe-Cys-Tyr-d-Arg-Thr-Pen-Thr-NH2 (CTAP) and [d-Ser2, Leu5, Thr6]enkephalin (DSLET) were a generous gift from the National Institute of Drug Abuse Drug Supply System (Rockville, MD). (±)Ethylketocyclazocine methanesulfonate was supplied by Sterling Winthrop Research Institute (Rennsealeer, New York). Dihydromorphine, deltorphin II, Tyr-Tic-Phe-Phe-OH (TIPP) and Ile5,6deltorphin II were synthesized as previously
Competition assays
The selectivity of unlabelled cyprodime was tested in rat brain membranes using highly selective radioligands for each receptor (μ, δ and κ) (Table 1). The μ-opioid receptor selective peptide, [3H]DAMGO, was readily displaced by cyprodime (Ki value 5.4 nM). Cyprodime showed much less affinity for δ binding sites, which were labelled with [3H]DPDPE. More than 40-fold difference was observed (244.6 for DPDPE vs. 5.4 nM for DAMGO Ki values) when compared with binding to [3H]DAMGO. Almost a similar
Discussion
The present findings with [3H]cyprodime strongly support previous bioassay data which indicated cyprodime to be a highly selective μ-opioid receptor antagonist (Schmidhammer et al., 1989). The μ/κ selectivity ratio in the guinea pig ileum was found to be 37, while in the isolated mouse vas deferens preparation it was 28, which were 2 to 3× greater values than with naloxone. In the mouse vas deferens preparation the μ/δ selectivity ratio was 15× greater than with naloxone (Schmidhammer et al.,
Acknowledgements
Helpful discussion of Dr. Sándor Benyhe and excellent technical assistance of Ms. Zsuzsanna Canjavec are greatly acknowledged. This work was supported by COPERNICUS CIPA CT 94 0226, Hungarian National Committee for Technical Development No. 94-97-48-0767, PECO ERB 3530 PL 941123, Biomed II BMH4-95-0510, 0511 and BMH4-98-2267, National Committee for Technical Development OMFB 96-47-48/1205, and National Scientific Research Foundation (OTKA: T022104).
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Present address: University of Michigan Medical School, Ann Arbor, MI 48104, USA.