Pertussis toxin-treated dog: a whole animal model of impaired inhibitory regulation of adenylate cyclase.
We have shown previously that stimulation of high-affinity GTP hydrolysis and inhibition of adenylate cyclase activity by muscarinic agonists are mediated by pertussis toxin (IAP) substrates (Gi and Go) in canine cardiac sarcolemma. We have now used the pertussis toxin-treated dog as a whole animal model in which Gi- and Go-mediated biochemical mechanisms can be studied. Mongrel dogs were injected intravenously with IAP 48 hours prior to death and isolation of left ventricular sarcolemma. Treatment of the animal in vivo with the toxin prevented subsequent in vitro IAP-catalyzed [32P]ADP-ribosylation of substrates in cardiac, erythrocytic, and renal cortical plasma membranes, suggesting that ADP-ribosylation occurred in vivo from endogenous substrate. Consistent with our previous results obtained by treating sarcolemma in vitro with IAP, muscarinic receptor-mediated stimulation of high-affinity GTP hydrolysis and inhibition of GTP-activated adenylate cyclase activity were attenuated in sarcolemma purified from the toxin-treated animals. Proximal to adenylate cyclase, guanine nucleotide regulation of muscarinic receptor affinity for agonists was also abolished in membranes from the toxin-treated animals. In addition, the ability of oxotremorine to attenuate GTP regulation of stimulation of adenylate cyclase activity by magnesium ions was abolished in sarcolemma from the IAP-treated dogs. Thus, cardiac sarcolemma isolated from the IAP-treated animals displayed biochemical characteristics of an adenylate cyclase system in which inhibitory regulatory pathways had been attenuated. The cardiac biochemical studies and the in vivo ADP-ribosylation of noncardiac IAP substrates also suggests considerable potential use of this model in the physiological and biochemical study of regulatory mechanisms mediated by GTP-binding proteins in other systems.
Fleming
J W
JW
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis 46223.
Hodges
T D
TD
Watanabe
A M
AM
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D004285:Dogs; D008954:Models, Biological; D010095:Oxotremorine / Q000378:metabolism; D037342:Pertussis Toxin; D012508:Sarcolemma / Q000187:drug effects; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D010566:Virulence Factors, Bordetella; D010095:Oxotremorine; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases