Stimulation of Guanosine-5′-O-(3-[35S]Thio)Triphosphate Binding by Endogenous Opioids Acting at a Cloned MuReceptor1
- Departments of 1Pharmacology (A.A., F.M., J.R.T., J.H.W.),2Biological Chemistry (F.M.), 3Psychology (J.H.W.), and 4Mental Health Research Institute (A.M., H.A.) University of Michigan, Ann Arbor, Michigan
Abstract
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 theKi 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.
Footnotes
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Send reprint requests to: Andrew Alt, 1301 Medical Science Research Building III, The University of Michigan Medical School, Department of Pharmacology, Ann Arbor, MI 48109-0632.
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↵1 This work was supported by National Institutes of Health Grants R01 DA04087, R01 DA02265, DA00254, T32-DA07281 and T32-GM07767
- Abbreviations:
- G protein
- GTP-binding protein
- GTP
- guanosine triphosphate
- GDP
- guanosine diphosphate
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- cAMP
- adenosine 3′,5′-cyclic monophosphate
- DAMGO
- Tyr-d-Ala-Gly-(Me)Phe-Gly-ol
- Tris
- tris(hydroxymethyl)-aminomethane
- EDTA
- ethylenediaminetetraacetic acid
- EGTA
- ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid
- HEPES
- (N-[2-hydroxethyl]piperazine-N′-[2-ethanesulfonic acid])
- PMSF
- phenylmethylsulfonyl flouride.
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- Received September 22, 1997.
- Accepted March 9, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
