STIMULATION OF GUANOSINE-5'-O-(3-[S-35]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 C-6 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 stud ied, as well as two recently discovered endogenous opioids, endomorphi n-1 and -2, which are thought to represent a fourth family of endogeno us opioid peptides. Stimulation of guanosine-5'-O-(3-[S-35]thio)tripho sphate ([S-35]GTP gamma S) binding to membranes was used as a measure of G protein activation. It was possible to differentiate high efficac y compounds such as Tyr-D-Ala-Gly-(Me)Phe-Gly-ol from lower-efficacy a gonists 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 d ynorphin A-(1-13). Endomorphin-1 and -2 were found to be partial agoni sts, capable of both stimulating [S-35]GTP gamma S binding and antagon izing the stimulation produced by the higher-efficacy agonist Tyr-D-Al a-Gly-(Me)Phe-Gly-ol. Binding affinities for the opioid agonists at th e cloned mu receptor were measured by the displacement of radiolabeled antagonist. It was found that the Ki values closely matched the EC50 values for [S-35]GTP gamma S binding stimulation, indicating that a la rge receptor reserve does not exist for the complete activation of G p roteins in this system.