Differential sensitivity of alpha o and alpha i to ADP-ribosylation by pertussis toxin in the intact cultured embryonic chick ventricular myocyte. Relationship to the role of G proteins in the coupling of muscarinic cholinergic receptors to inhibition of adenylate cyclase activity.
The guanine nucleotide regulatory proteins, alpha i and alpha o, coexist in a variety of tissues, including heart, brain, and adipose tissues and are ADP-ribosylated by pertussis toxin (Gilman AG, G-proteins and dual control of adenylate cyclase. Cell 26: 577-579, 1984). Previous studies in which purified G proteins were reconstituted with cell membranes and/or phospholipid vesicles have suggested that an alpha i-like protein mediates GTP-dependent inhibition of adenylate cyclase activity. However, direct studies comparing the role of alpha i and alpha o in mediating the inhibition of adenylate cyclase activity in the intact cell have not appeared. In the present study, we demonstrated that, in the intact cell, alpha o was more sensitive to ADP-ribosylation in the presence of pertussis toxin than was alpha i. The T1/2 for pertussis toxin-mediated ADP-ribosylation of alpha i was 199 +/- 10 min (mean +/- SE, N = 10) compared to 157 +/- 7 min for alpha o. The IC50 for pertussis toxin-induced ADP-ribosylation of alpha i was 158 +/- 40 pg/ml (mean +/- SE, N = 11) compared to 35 +/- 8 pg/ml for alpha o. The differences in both T1/2 and IC50 for alpha i and alpha o were statistically significant (P less than 0.001). Studies were carried out to determine whether alpha o was involved in coupling the muscarinic cholinergic receptor to inhibition of adenylate cyclase activity in intact cells. The time course and dose dependence of the pertussis toxin-induced uncoupling of the muscarinic receptor from inhibition of adenylate cyclase closely paralleled the time course and dose dependence for the ADP-ribosylation of alpha i but differed significantly (P less than 0.001) from the time course and dose dependence for the ADP-ribosylation of alpha i but differed significantly (P less than 0.001) from the time course and dose dependence of the pertussis toxin mediated ADP-ribosylation of alpha o. The T1/2 and IC50 values for the pertussis toxin-induced decrease in the inhibition of adenylate cyclase activity were 210 +/- 6 min (mean +/- SE, N = 11) and 169 +/- 25 pg/ml (mean +/- SE, N = 12), respectively, which were not significantly different from the T1/2 and IC50 for pertussis toxin mediated ADP-ribosylation of alpha i. The data are consistent with the hypothesis that, in the intact cell, a pertussis toxin-sensitive alpha i-like protein, but not alpha o, couples muscarinic receptors to inhibition of adenylate cyclase activity.
Liang
B T
BT
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Galper
J B
JB
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006321:Heart / Q000196:embryology; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D037342:Pertussis Toxin; D011976:Receptors, Muscarinic / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins