Mu and kappa1 opioid-stimulated [35S]guanylyl-5′-o-(γ-thio)-triphosphate binding in cynomolgus monkey brain
Section snippets
Materials
[35S]GTPγS (1250 Ci/mmol) was purchased from New England Nuclear Corporation (Boston, MA). [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)-benzeneacetamide (U50,488H), naloxone, and guanosine 5′-diphosphate (GDP) were obtained from Sigma Chemical Co. (St Louis, MO). Nor-binaltorphimine (nor-BNI) was purchased from Research Biochemicals International (Natick, MA). GTPγS and GDP for membrane assays were obtained from Boehringer Mannheim (New York,
Opioid-stimulated [35S]GTPγS binding in membranes
To determine optimal concentrations of agonists and antagonists, concentration–effect curves were generated in membranes from the frontal pole of the brain (Fig. 1). Maximal stimulation of [35S]GTPγS binding by DAMGO and U50,488H was 33% and 51%, respectively. Maximal stimulation was produced by 3 μM DAMGO and 1 μM U50,488H. The ec50 values for stimulation of [35S]GTPγS binding were 0.28 μM and 0.025 μM for DAMGO and U50,488H, respectively. In both cases, the addition of the appropriate antagonist
Discussion
The present study was designed to apply, for the first time, agonist-stimulated [35S]GTPγS binding to localize receptor-coupled G-protein activity in primate brain, thus extending previous studies regarding opioid receptor function in rodents. These studies provide new information regarding the functional neuroanatomy of mu and kappa1 opioid receptors that may be relevant to the understanding of such complex processes as reinforcement and analgesia. The ability to measure the functional
Conclusions
This study demonstrates the feasibility of using [35S]GTPγS autoradiography to examine receptor-activated G-proteins in the primate brain. The distribution of mu and kappa1-stimulated [35S]GTPγS binding is consistent with that previously reported for receptor binding, although some differences may exist in the relative levels of receptor versus [35S]GTPγS binding. Mu and kappa1 opioid-stimulated [35S]GTPγS binding were found in regions including the limbic and association cortices, amygdala,
Acknowledgments
Mack Miller and Ruoyu Xiao provided excellent technical assistance. Drs David Friedman and Dana E. Selley provided helpful discussions regarding this manuscript. These studies were supported by PHS grants DA-00287 (LJS), DA-02904 (SRC), DA-9085 (LJP) and DA-07246 (JBD) from the National Institute on Drug Abuse.
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