Abstract
Previous studies had shown that the amplification factors for cannabinoid receptors, defined as the number of total G proteins activated per occupied receptor, differs between several rat brain regions. In this study, we sought to determine which specific Gi/Goα subunits were activated by CB1 receptors in several rat brain regions and if this coupling might explain the regional differences in receptor/G protein amplification factors. Furthermore, we examined whether cannabinoid agonists might activate different subtypes of Gα subunits with varying degrees of efficacy and/or potency. Activation of specific G proteins by cannabinoid receptors was evaluated by the ability of the agonist WIN 55212-2 to stimulate incorporation of [α-32P]azidoanilido-GTP into Gα subunits in membranes. Photolabeled G proteins were either directly resolved using urea/SDS-polyacrylamide gel electrophoresis or first immunoprecipitated with specific antisera for different Gα subunits before electrophoresis. Individual Gα subunits were separated into distinct bands on a single gel and the amount of agonist-induced increase in radioactivity was quantified by densitometry. Stimulation of CB1 receptors by WIN 55212-2 resulted in the activation of a distinct pattern of at least five different Giα/Goα subunits in several brain regions. Furthermore, although the pattern of G proteins activated by WIN 55212-2 appeared to be similar across brain regions, slight differences were observed in both the percentage of increase and the amount of the individual Gα subunits activated. Most importantly, the amount of WIN 55212-2 required to half-maximally activate individual G proteins in the cerebellum varied over a 30-fold range for different Gα subunits. These results suggest that cannabinoid receptors activate multiple G proteins simultaneously in several brain regions and both the efficacy and potency of cannabinoid agonists to activate individual Gα subunits may vary considerably.
Footnotes
-
Send reprint requests to: Paul L. Prather, Ph.D., Department of Pharmacology and Toxicology, Mail Slot 611, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. E-mail: pratherpaull{at}exchange.uams.edu
-
This study was supported in part by National Institute on Drug Abuse Grants DA10936 (to P.L.P.) and DA06784 and DA06634 (to S.R.C.).
- Abbreviations:
- GCPR
- G protein-coupled receptor
- PTX
- pertussis toxin
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- AA-GTP
- azidoanilido-guanosine-5′-O-(3-thio)triphosphate
- PAGE
- polyacrylamide gel electrophoresis
- IP
- immunoprecipitation
- AR
- autoradiography
- ECL
- enhanced chemiluminescence
- Received July 26, 1999.
- Accepted February 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|