Endogenous regulator of G protein signaling proteins suppress Galphao-dependent, mu-opioid agonist-mediated adenylyl cyclase supersensitization.
Chronic mu-opioid agonist treatment leads to dependence with withdrawal on removal of agonist. At the cellular level withdrawal is accompanied by a supersensitization of adenylyl cyclase, an effect that requires inhibitory Galpha proteins. Inhibitory Galpha protein action is modulated by regulator of G protein signaling (RGS) proteins that act as GTPase activating proteins and reduce the lifetime of Galpha-GTP. In this article, we use C6 glioma cells expressing the rat mu-opioid receptor (C6mu) to examine the hypothesis that Galphao alone can mediate mu-opioid agonist induced adenylyl cyclase supersensitivity and that endogenous RGS proteins serve to limit the extent of this supersensitization. C6mu cells were stably transfected with pertussis toxin (PTX)-insensitive Galphao that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin or morphine. Supersensitization was observed in cells expressing wild-type Galpha, but this was lost on PTX treatment. In cells expressing PTX-insensitive Galphao supersensitization was recovered, confirming that Galphao alone can support supersensitization. In cells expressing the RGS-insensitive mutant Galphao, there was a greater degree of supersensitization and the concentration of micro-agonist needed to achieve half-maximal supersensitization was reduced by 10-fold. The amount of supersensitization seen did not directly relate to the degree of acute inhibition of adenylyl cyclase. These results demonstrate a role for Galphao in adenylyl cyclase supersensitization after mu-agonist exposure and show that this action is modulated by endogenous RGS proteins.
Clark
Mary J
MJ
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0632, USA.
Neubig
Richard R
RR
Traynor
John R
JR
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051379:Mice; D037342:Pertussis Toxin / Q000494:pharmacology; D020710:RGS Proteins / Q000378:metabolism; D051381:Rats; D017450:Receptors, Opioid, mu / Q000819:agonists*; D014407:Tumor Cells, Cultured
D044385:GTP-Binding Protein alpha Subunits; D020710:RGS Proteins; D017450:Receptors, Opioid, mu; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases