Regional Differences in Cannabinoid Receptor/G-protein Coupling in Rat Brain 1 CHRISTOPHER S. BREIVOGEL, LAURA J. SIM and STEVEN R. CHILDERS Department of Physiology and Pharmacology, and Center for Investigative Neuroscience, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina Accepted for publication May 2, 1997 ABSTRACT Cannabinoid receptor activation of G-proteins can be mea- sured by WIN 55212–2-stimulated [ 35 S]GTPS binding. Recep- tor/transducer amplification factors, interpreted as the number of G-proteins activated per occupied receptor, are the ratio of the apparent B max of net agonist-stimulated [ 35 S]GTPS bind- ing to the B max of receptor binding. The present study exam- ined whether amplification factors for cannabinoid receptors differ among various rat brain regions. In autoradiographic studies with [ 3 H]WIN 55212–2 and WIN 55212–2-stimulated [ 35 S]GTPS binding, some regions displayed different relative levels of agonist-stimulated [ 35 S]GTPS binding than receptor binding. To quantify amplification factors, membranes from different brain regions were assayed by saturation binding analysis of net WIN 55212–2-stimulated [ 35 S]GTPS, [ 3 H]SR141716A (antagonist) and [ 3 H]WIN 55212–2 (agonist) binding. For [ 3 H]SR141716A binding, amplification factors var- ied significantly from 2.0 (frontal cortex) to 7.5 (hypothalamus). For [ 3 H]WIN 55212–2 binding, amplification factors ranged from 2.4 (hippocampus) to 5.5 (thalamus). Comparison of receptor binding and G-protein activation at subsaturating concentra- tions of WIN 55212–2 indicated that amplification factors may vary with receptor occupancy in some regions like cerebellum. Ratios between high-affinity [ 3 H]WIN 55212–2 B max and [ 3 H]SR141716A B max also differed significantly among brain regions. These results demonstrate that G-protein coupling by cannabinoid receptors differs among brain regions, and there- fore depends on the cellular environment. Cannabinoid receptors of the CB1 type mediate the central nervous system actions of cannabimimetic compounds (Dewey 1986; Martin 1986). CB1 receptors belong to the superfamily of seven transmembrane-spanning domain, G- protein-coupled receptors (Matsuda et al., 1990) and are neg- atively coupled to adenylyl cyclase (Howlett, 1984) and Ca ++ channels (Mackie and Hille, 1992; Mackie et al., 1995) and positively coupled to K + channels (Hampson et al., 1995; Mackie et al., 1995) via pertussis-toxin sensitive (G i/o ) G- proteins (Howlett and Fleming, 1984; Howlett, 1985; Howlett et al., 1986; Bidaut-Russell et al., 1990). Two splice variants of CB1 mRNA (CB1 and CB1A) have been identified in hu- man and rat brain (Shire et al., 1995). CB1 receptors are widely distributed with relatively high density in mamma- lian brain (Herkenham et al., 1991b; Jansen et al., 1992). Cannabinoid effects on memory, body temperature and motor function (Dewey, 1986) are consistent with the distribution of CB1 receptors in the hippocampus, hypothalamus and basal ganglia (Herkenham et al., 1991b; Jansen et al., 1992; Sim et al., 1995), yet the signal transduction mechanisms which mediate these biological actions are not well characterized. For G-protein-coupled receptors such as CB1, the initial step in the signal transduction cascade which determines agonist efficacy is the activation of the G-protein (Kenakin, 1993). This step can be measured effectively by use of net agonist-stimulated [ 35 S]GTPS binding in the presence of excess GDP (Hilf et al., 1989; Offermanns et al., 1991; Loren- zen et al., 1993; Sim et al., 1995; Traynor and Nahorski, 1995; Selley et al., 1996). Agonist-stimulated [ 35 S]GTPS autora- diography and membrane binding assays are useful in deter- mining relative levels of G-protein activation by agonists acting through a given G-protein-coupled receptor. When the apparent B max of [ 35 S]GTPS binding and B max of receptor binding are compared, a receptor/transducer amplification factor can be calculated as the relative number G-proteins activated on a per receptor molecule basis (Gierschik et al., 1991; Sim et al., 1996c). With such analyses, we recently demonstrated in rat striatal membranes that cannabinoid receptors are less efficiently coupled to G-proteins when com- pared with opioid receptors. In those studies, cannabinoid receptors activated only twice as many G-proteins as mu and delta opioid receptors, despite the 10-fold greater abundance Received for publication December 16, 1996. 1 This research was supported by U.S. Public Health Service grant DA- 06784 from the National Institute on Drug Abuse. ABBREVIATIONS: CB1, brain cannabinoid receptor subtype; GTPS, guanosine 5'-O-(3-thiotriphosphate); BSA, bovine serum albumin; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, 4-(2-hyroxyethyl)-1-piperazineethanesulfonic acid; ANOVA, analysis of variance. 0022-3565/97/2823-1632$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:1632–1642, 1997 1632