Adenylate cyclase activity during growth and maturation of keratinocytes: comparison of two methods of study

The adenylate cyclase activity of primary cultures of guinea-pig ear keratinocytes was studied at different times by challenge with various agonists after labelling of ATP by exposure of the cells to [3H]-adenine. The [3H]-cAMP (cyclic adenosine-3',5-monophosphate) formed was assayed by scintillation counting. In other experiments, cells were exposed to the equivalent amount of unlabelled adenine, challenged and cAMP was then assayed by radioimmunoassay. These absolute amounts were compared to [3H]-cAMP levels expressed as percentage conversion of [3H]-ATP. Comparison of the two methods suggested that the use of the prelabelling method would result in underestimation of cAMP formed. It was found that the response of the enzyme to different agonists changed with the length of time of the cells in culture.