Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding by endogenous opioids acting at a cloned mu receptor

The ability of endogenous opioids to activate G proteins was measured in membranes from C6 rat glioma cells stably expressing a cloned rat mu receptor. Peptides representing each of the three known families of endogenous opioids (enkephalins, endorphins and dynorphins) were studied, as well as two recently discovered endogenous opioids, endomorphin-1 and -2, which are thought to represent a fourth family of endogenous opioid peptides. Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding to membranes was used as a measure of G protein activation. It was possible to differentiate high efficacy compounds such as Tyr-D-Ala-Gly-(Me)Phe-Gly-ol from lower-efficacy agonists such as morphine or meperidine. Met- and leu-enkephalin, beta endorphin and dynorphin A were all found to have high efficacy at the mu receptor, as were the peptide fragments beta endorphin-1(1-27) and dynorphin A-(1-13). Endomorphin-1 and -2 were found to be partial agonists, capable of both stimulating [35S]GTP gamma S binding and antagonizing the stimulation produced by the higher-efficacy agonist Tyr-D-Ala-Gly-(Me)Phe-Gly-ol. Binding affinities for the opioid agonists at the cloned mu receptor were measured by the displacement of radiolabeled antagonist. It was found that the Ki values closely matched the EC50 values for [35S]GTP gamma S binding stimulation, indicating that a large receptor reserve does not exist for the complete activation of G proteins in this system.