Elsevier

Biochemical Pharmacology

Volume 59, Issue 11, 1 June 2000, Pages 1395-1401
Biochemical Pharmacology

Neuroscience
Relationship of mu opioid receptor binding to activation of G-proteins in specific rat brain regions

https://doi.org/10.1016/S0006-2952(00)00272-0Get rights and content

Abstract

This study investigated the relationship between mu receptor binding and mu agonist activation of G-proteins in the rat brain. To directly compare agonist potencies in receptor binding (Ki values) and G-protein activation (Ks values), both agonist-stimulated [35S]guanosine-5′-O-(γ-thio)-triphosphate ([35S]GTPγS) and [3H]naloxone binding assays were conducted under identical conditions, using the full mu agonist [d-Ala2, N-Me4, Gly5-ol]-enkephalin (DAMGO). DAMGO exhibited biphasic competition of [3H]naloxone binding and stimulation of [35S]GTPγS binding in most regions. Whereas the high-affinity component represented a large percentage (50–80%) of total receptor sites, the high-affinity component of DAMGO-stimulated [35S]GTPγS binding was much lower, <30% of the total, and in most regions significant stimulation of [35S]GTPγS binding did not occur until the high-affinity binding sites were completely occupied. Moreover, the low-affinity potencies for DAMGO in receptor binding and G-protein activation were the same across different regions. Receptor–transducer amplification factors were calculated by the ratio of the apparent Bmax of net agonist-stimulated [35S]GTPγS binding to the Bmax of receptor binding. Amplification factors for the nine regions examined were relatively high and varied significantly across regions, from a ratio of 8 in the thalamus to 38 in the cortex, suggesting that the efficiency of mu opioid receptor coupling to G-proteins varies across brain regions.

Section snippets

Materials

Male Sprague–Dawley rats (150–200 g) were purchased from Zivic Miller. [35S]GTPγS (1250 Ci/mmol) and [3H]naloxone (57.5 Ci/mmol) were purchased from the New England Nuclear Corp. Guanosine-5′-diphosphate and GTPγS were obtained from Boehringer Mannheim. DAMGO, naloxone, naltrindole, and adenosine deaminase were purchased from the Sigma Chemical Co. Ecolite scintillation fluid was purchased from Fisher Scientific. All other reagent grade chemicals were purchased from Sigma or Fisher Scientific.

Membrane preparations

Results

Several binding assays were used to compare the parameters of mu opioid receptor binding and mu opioid activation of G-proteins in nine rat brain regions: amygdala, brainstem, colliculus, frontal cortex, hippocampus, hypothalamus, sensomotor cortex, striatum, and thalamus. First, the potency (G-Ks) and the efficacy (G-Emax) of DAMGO in stimulating [35S]GTPγS binding to membranes were measured by constructing DAMGO concentration–effect curves. Second, DAMGO potencies in receptor binding (R-Ki)

Discussion

The present study explored three questions about the relationship between mu opioid receptor binding and activation of G-proteins in brain membranes. First, what is the relationship between agonist potencies in binding to receptors versus their potencies in activating G-proteins? Second, does agonist potency in activating G-proteins vary across brain regions? Third, does the amplification factor between mu receptor number and mu agonist-stimulated G-proteins vary across brain regions? To answer

Acknowledgements

These studies were supported by DA-02904, DA-06634 (S. R. C.), DA-07625 and DA-05957 (C. E. M.) and DA-10770 (D. E. S.) from the National Institute on Drug Abuse.

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