Characterization of endomorphin-1 and -2 on [35S]GTPgammaS binding in the mouse spinal cord

In the present study, G-protein activation by newly-isolated opioid peptides, endomorphin-1 and -2, was examined in the mouse spinal cord by monitoring the binding of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). Both endomorphin-1 and -2 increased [35S]GTPgammaS binding to mouse spinal cord membranes in a concentration-dependent and saturable manner and reached a maximal stimulation of 57.3+/-5.0 and 60.2+/-3.2%, respectively, at 10 microM. In contrast, the synthetic selective micro-opioid receptor agonist [D-Ala2,NHPhe4,Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced 103.4+/-5.4% of the maximal stimulation. The receptor specificity of endomorphin-stimulated [35S]GTPgammaS binding was verified by co-incubating membranes with endomorphins in the presence of specific micro-(beta-funaltrexamine and D-Phe-Cys-D-Tyr-Om-Thr-Pen-Thr-NH2 (CTOP)), delta-(naltrindole) or K-(nor-binaltorphimine) opioid receptor antagonists. Co-incubation with either beta-funaltrexamine or CTOP blocked both endomorphin-1- and-2-stimulated [35S]GTPgammaS binding in a concentration-dependent manner, whereas neither naltrindole nor nor-binaltorphimine had any effect on the [35S]GTPgammaS binding stimulated by either endomorphin-1 or -2. The data presented indicate that either endomorphin-1 or -2 activate G-proteins by specific stimulation of micro-opioid receptors, and may act as partial agonists with moderate catalytic efficacies in the mouse spinal cord.