In situ analysis of adenylate cyclase activity in permeabilized rat adipocytes: sensitivity to GTP, isoproterenol, and N6-(phenylisopropyl)adenosine.
Adenylate cyclase activity in rat adipocyte suspensions was assayed in situ using a digitonin permeabilization technique. Recovery of activity was dependent on digitonin concentration, reaching a maximum at 20 micrograms/ml digitonin and paralleling the effect on cell permeability. Maximum adenylate cyclase activity recovered in permeabilized cells was 75% of that in comparable homogenates. Isoproterenol, a beta-adrenergic agonist, activated adenylate cyclase by 1.4, 2.2 and 4.5 fold at 10(-6), 10(-5) and 10(-3) M, respectively, despite perturbation of the plasma membrane. Exogenous GTP was not required for expression of beta-adrenergic activation, but 10(-5) M GTP maximally increased both basal and isoproterenol-dependent activity. The response to 10(-6) M isoproterenol was increased 2.1 fold by 10(-5) M GTP. N6-(Phenylisopropyl)adenosine at 10(-6) M inhibited both basal and isoproterenol-dependent adenylate cyclase activity by approximately 30%, demonstrating that the adenosine-dependent inhibitory pathway (Ni) remained functional in the digitonin-permeabilized cells. In situ analysis of adenylate cyclase is not only simple and rapid, but provides a unique approach to studying regulation of this key enzyme.
D000241:Adenosine / Q000031:analogs & derivatives*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D002463:Cell Membrane Permeability; D004072:Digitonin / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D010660:Phenylisopropyladenosine / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D010660:Phenylisopropyladenosine; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D000241:Adenosine; D004072:Digitonin; D007545:Isoproterenol