Differential sensitivity of α o and α 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

Abstract

The guanine nucleotide regulatory proteins, α i and α 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 α i-like protein mediates GTP-dependent inhibition of adenylate cyclase activity. However, direct studies comparing the role of α i and α 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, α o was more sensitive to ADP-ribosylation in the presence of pertussis toxin than was α i. The T 1 2 for pertussis toxin-mediated ADP-ribosylation of α i was 199 ± 10 min (mean ± SE, N = 10) compared to 157 ± 7 min for α o. The IC 50 for pertussis toxin-induced ADP-ribosylation of α i was 158 ± 40 pg/ml(mean ± SE, N = 11) compared to 35 ± 8 pg/ml for α o. The differences in both T 1 2 and IC 50 for α i and α o were statistically significant (P < 0.001). Studies were carried out to determine whether α 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 α i, but differed significantly (P < 0.001) from the time course and dose dependence of the pertussis toxin mediated ADP-ribosylation of α o. The T 1 2 and IC 50 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 T 1 2 and IC 50 for pertussis toxin mediated ADP-ribosylation of α i. The data are consistent with the hypothesis that, in the intact cell, a pertussis toxin-sensitive α i−like protein, but not α o, couples muscarinic receptors to inhibition of adenylate cyclase activity.