Mechanisms of cyclic AMP phosphodiesterase regulation.

The mechanisms of regulation of cyclic AMP phosphodiesterases were studied using the cytoplasmic fraction of PC-12 cells sensitive to the action of nerve growth factor. The cells contain phosphodiesterases of two types. One of them possesses a high affinity for cyclic AMP (Km = 2.46 mM), whereas the other has the affinity by an order worse (Km = 37.1 mM). PC-12 cell differentiation under the action of nerve growth factor is connected with the cyclic nucleotide elevation; however, activities of both phosphodiesterases remain unchanged. This indicates that the regulation of activity of these enzymes in PC-12 cells is mediated by second messenger effects. The main object of cell regulation is phosphodiesterase with low affinity for the substrate. Its activity is modulated by the calmodulin-Ca2+ complex, cyclic GMP and NAD+ at micromolar concentrations. The effect on the phosphodiesterase system of both a "quick" messenger, Ca2+ and "slow" messengers, cyclic GMP and NAD+, has the same consequences: the turnover number of the enzymic reaction increases that is accompanied by a proportional decrease in the enzyme affinity for cyclic AMP so that the ratio Vmax/Km remains constant. A possible explanation of functional significance of such an activity modulation may be the necessity to maintain the conditions for phosphodiesterase functioning when Km much greater than [cyclic AMP] and the reaction rate are directly proportional to the substrate concentration: v = Vmax/Km [cyclic AMP]. Then the cells are transferred into such a mode when autoregulation of the cyclic nucleotide level takes place. Besides the transient effects causing changes in phosphodiesterase activity, studies of PC-12 cells revealed a chronic effect of phosphodiesterase activity change under the action of staphylococcal enterotoxin A. This protein which induces differentiation of PC-12 cells and possesses a NAD+-glycohydrolase activity is translocated into cytoplasm of cells in the presence of NAD+ and accomplishes ADP-ribosylation of phosphodiesterase. As a result, the enzyme activity falls, cyclic AMP level increases and cell differentiation starts. The activity of soluble phosphodiesterase of PC-12 cells also decreases under the effect of two neurotoxins from bee venom, melittin and tertiapin. Both the toxins at concentration of 10 microM completely block calcium regulation of the enzyme. The mechanism of tertiapin action was investigated on a model system of calmodulin-bovine brain phosphodiesterase. It appeared that inhibition of Ca2+ action is achieved as the result of binding of two toxin molecules with Kd = 2 mM to the activated calmodulin molecule.(ABSTRACT TRUNCATED AT 400 WORDS)