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]GTPγS) 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-(1-27) and dynorphin A-(1-13). Endomorphin-1 and -2 were found to be partial agonists, capable of both stimulating [³⁵S]GTPγS 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 K(i) values closely matched the EC₅₀ values for [³⁵S]GTPγS binding stimulation, indicating that a large receptor reserve does not exist for the complete activation of G proteins in this system.