Regulation of cyclic AMP metabolism in the rat erythrocyte during chronic beta-adrenergic stimulation. Evidence for calmodulin-mediated alteration of membrane-bound phosphodiesterase activity.

The regulation of cyclic AMP metabolism in the rat erythrocyte has been investigated during chronic exposure to the beta agonist isoproterenol. A triphasic response is observed: 1) an acute increase in cyclic AMP to levels four- to fivefold greater than basal, maximal by 1 minute (Phase I); 2) a gradual decline in cAMP content to levels near basal during the next 15-20 minutes (Phase II) and a second sustained rise in cAMP, maximal by 60 minutes, to a concentration greater than that observed during the first minute (Phase III). Extensively washed Phase II and Phase III cells are refractory to a second challenge by isoproterenol. In phosphodiesterase-inhibited intact Phase II and III cells adenylate cyclase activity is maximally activated. Isoproterenol has no effect on soluble phosphodiesterase activity but increases membrane-bound phosphodiesterase activity 3- and 2.2-fold in Phase II and Phase III cells, respectively. The activation of this membrane-bound enzyme activity appears to be mediated by the calcium-dependent regulatory protein, calmodulin, because 1) the amount of exogenous calmodulin required to achieve half-maximal activation of membrane-bound phosphodiesterase is 3.7, 2.0, and 1.2 micrograms in control, Phase III and Phase II membranes, respectively; and 2) there is less calmodulin in membrane-free lysates prepared from Phase II cells than control cells. These data support the idea that the major mechanism regulating cAMP content in the rat erythrocyte during chronic isoproterenol stimulation is the membrane-bound phosphodiesterase and that there is a translocation of calmodulin from the cytoplasm to the membrane during hormone stimulation.