Mu and kappa1 opioid-stimulated [35S]guanylyl-5′-o-(γ-thio)-triphosphate binding in cynomolgus monkey brain

doi:10.1016/S0306-4522(99)00344-9

Neuroscience

Volume 94, Issue 2, September 1999, Pages 651-662

https://doi.org/10.1016/S0306-4522(99)00344-9 Get rights and content

Abstract

Agonist-stimulated [³⁵S]GTPγS binding allows the visualization of receptor-activated G-proteins, thus revealing the anatomical localization of functional receptor activity. In the present study, agonist-stimulated [³⁵S]GTPγS binding was used to demonstrate mu and kappa₁ opioid-stimulated [³⁵S]GTPγS binding in tissue sections and membranes from cynomolgus monkey brain using DAMGO and U50,488H, respectively. Concentrations of agonists required to produce maximal stimulation of [³⁵S]GTPγS binding were determined in membranes from the frontal poles of the brain. Receptor specificity was verified in both membranes and sections by inhibiting agonist-stimulated [³⁵S]GTPγS binding with the appropriate antagonist. Mu opioid-stimulated [³⁵S]GTPγS binding was high in areas including the amygdala, ventral striatum, caudate, putamen, medial thalamus and hypothalamus. Dense mu-stimulated [³⁵S]GTPγS binding was also found in brainstem nuclei including the interpeduncular nucleus, parabrachial nucleus and nucleus of the solitary tract. Kappa₁ opioid-stimulated [³⁵S]GTPγS binding was high in limbic and association cortex, ventral striatum, caudate, putamen, globus pallidus, claustrum, amygdala, hypothalamus and substantia nigra.

These results demonstrate the applicability of [³⁵S]GTPγS autoradiography to examine receptor-activated G-proteins in the primate brain and reveal functional mu and kappa₁ opioid receptor activity that may contribute to the reported central nervous system effects of opiates.

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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Mu and kappa1 opioid-stimulated [35S]guanylyl-5′-o-(γ-thio)-triphosphate binding in cynomolgus monkey brain

Abstract

Section snippets

Materials

Opioid-stimulated [35S]GTPγS binding in membranes

Discussion

Conclusions

Acknowledgments

Brain Res.

Analyt. Biochem.

Biochem. Pharmac.

Brain Res.

Peptides

Neuropharmacology

J. biol. Chem.

Brain Res. Bull.

Brain Res.

Brain Res.

Brain Res. Bull.

Brain Res.

Brain Res. Bull.

Neuropeptides

Brain Res.

Neuroscience

Pain

Tolerance of locus coeruleus neurones to morphine and suppression by clonidine

Science

Central cardiovascular effects of narcotic analgesics and enkephalins in rats

Br. J. Pharmac.

Regional differences in cannabinoid receptor/G-protein coupling in rat brain

J. Pharmac. exp. Ther.

Mu and kappa₁ opioid-stimulated [³⁵S]guanylyl-5′-o-(γ-thio)-triphosphate binding in cynomolgus monkey brain

Opioid-stimulated [³⁵S]GTPγS binding in membranes