Opioid inhibition of adenylyl cyclase in membranes from pertussis toxin-treated NG108-15 cells.
Gi/Go proteins are uncoupled from receptors by ADP-ribosylation with pertussis toxin (PTX). However, PTX treatment of delta opioid receptor-containing NG108-15 cells reduces, but does not eliminate, opioid inhibition of adenylyl cyclase. The present study explored potential mechanisms of this residual inhibition. Overnight treatment of NG108-15 cells with 100 ng/ml PTX eliminated both PTX-catalyzed [adenylyl-32P]NAD+-labeling of G proteins and agonist stimulation of low Km GTPase in membranes. Although PTX-treatment decreased the maximal opioid inhibition of adenylyl cyclase by 50-65%, the inhibition that remained was concentration-dependent and antagonist-reversible. This inhibition persisted in the absence of GTP (even though opioid inhibition of adenylyl cyclase in untreated membranes was GTP-dependent), but was eliminated by hydrolysis-resistant guanine nucleotide analogs, indicating that G-proteins were still involved in the coupling mechanism. However, assays of agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP indicated that PTX pretreatment eliminated stimulation of guanine nucleotide exchange by opioid agonists. These results suggest that in membranes from PTX-treated NG108-15 cells, a subpopulation of G proteins may transduce an inhibitory signal from agonist-bound opioid receptors without involvement of guanine nucleotide exchange.
Selley
D E
DE
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.
Breivogel
C S
CS
Childers
S R
SR
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009294:Narcotics / Q000819:agonists / Q000494:pharmacology*; D009447:Neuroblastoma; D037342:Pertussis Toxin; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D006150:Guanine Nucleotides; D009294:Narcotics; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C055382:naltrindole