A CE assay for the detection of agonist-stimulated adenylyl cyclase activity.
A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two- to three-fold maximum increase in AC activity with EC50s of 4.2 +/- 0.7 and 2.4 +/- 0.7 microM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the beta2-adrenergic receptor (beta2AR) fused to the stimulatory G protein. Terbutaline (beta2AR agonist) increased the basal rate of cAMP formation 1.7 +/- 0.1-fold resulting in an EC50 of 62 +/- 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC50 of terbutaline by the beta2AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.
Cunliffe
Jennifer M
JM
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA. rtkenn@umich.edu
Whorton
Matthew R
MR
Sunahara
Roger K
RK
Kennedy
Robert T
RT
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D001681:Biological Assay; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D020536:Enzyme Activators / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D015813:Substance Abuse Detection / Q000379:methods; D013379:Substrate Specificity; D013726:Terbutaline / Q000494:pharmacology
D020536:Enzyme Activators; D043562:Receptors, G-Protein-Coupled; D005576:Colforsin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D013726:Terbutaline