Mediation of adenylyl cyclase sensitization by PTX-insensitive GalphaoA, Galphai1, Galphai2 or Galphai3.
Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Galphai/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Galphai/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (Galpha ), were used to determine whether each of the Galphai/o subtypes is able to mediate sensitization of AC. Galpha , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Galphai/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). Each Galphai/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Galphai/o subtype, but the level of sensitization was increased in clones expressing higher levels of Galphai/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Galphai/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC activity.
Clark
Mary J
MJ
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan USA.
Traynor
John R
JR
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000701:Analgesics, Opioid / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects*; D005910:Glioma; D006801:Humans; D009154:Mutation / Q000502:physiology; D037342:Pertussis Toxin / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods
D000701:Analgesics, Opioid; D010726:Phosphodiesterase Inhibitors; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D003545:Cysteine; D015056:1-Methyl-3-isobutylxanthine