The long term objective of this program is to determine the consequences of hormone-cell interaction at the level of cAMP accumulation and cellular responses to altered levels of the cyclic nucleotide. In this proposal we will examine the state of cells (in terms of the cAMP system) under conditions approximating those found in vivo. To do that, we will define the activities of adenylate cyclase, cAMP elimination mechanisms, relative rates of desensitization, and the protein kinase activity ratios (as an index of cAMP action) in cells incubated under a variety of conditions. It is our contention that the """"""""basal"""""""" state, that is, incubation in the absence of hormone never exists in vivo. Thus, we will approximate the in vivo basal state by incubation with concentrations of hormone appropriate to the resting state in intact animals. In the case of the catecholamines, levels of 1- 3nM have been reported (1). We will also examine the parameters of cAMP metabolism and action under levels of stimulation approximating those found in vivo, which are rarely greater than EC50. The experiments proposed test the following hypotheses: (1) That the cells in vivo are in a continuous state of partial desensitization. (2) That desensitization is a functional useful entity in the control of cAMP levels in cells. (3) That cAMP elimination is under elaborate and finely tuned control in intact cells. In the present period of the program we have established the procedures, assays, and models required for the measurement of the components cited above in cells going through the transition from zero levels to extremely high concentrations of hormone. The new program, which will yield quantitation of what happens as cells go from a continuous, low level of stimulation to one slightly higher, will provide us with a much better understanding of hormone and drug actions as they obtain in the living state.
