Binding of Trifluoperazine to the Calcium-Dependent Activator of Cyclic Nucleotide Phosphodiesterase

Abstract

The activity of one form of cyclic nucleotide phosphodiesterase is increased over 10-fold by the addition of an endogenous, calcium-dependent protein activator. This activation of phosphodiesterase is selectively inhibited by phenothiazine antipsychotics such as trifluoperazine. To gain insight into the specific mechanism of this inhibition, we examined the binding of trifluoperazine to various proteins, including the endogenous cyclic nucleotide activator prepared from bovine brain. Using equilibrium dialysis, we found that trifluoperazine binds to proteins at two distinctly different types of sites: a high-affinity, calcium-dependent binding site (Kd = 1 µM, N = 2) and a low-affinity, calcium-independent binding site (Kd = 5 mM, N = 24). All proteins examined, including catalase, cytochrome c, aldolase, chymotrypsinogen, egg albumin, bovine serum albumin, and the cyclic nucleotide activator, gave evidence of having low-affinity sites for trifluoperazine. However, only the activator had the calcium-dependent, high-affinity binding sites for the phenothiazine. At low concentrations of trifluoperazine (less than 1 µM), binding to the activator was 10-fold higher in the presence of calcium than in its absence. The binding of trifluoperazine to the other proteins showed no calcium dependence, and the degree of binding to these proteins was similar to the binding of trifluoperazine to activator when measured in the absence of calcium. The binding of trifluoperazine to activator in the presence of calcium markedly decreased when the pH was raised from 7.5 to 8.0, whereas the nonspecific binding was not altered by changes in pH between 6.5 and 8.5. A study of other divalent cations showed that calcium could be replaced by strontium, nickel, cobalt, zinc, and manganese in promoting the binding of trifluoperazine to activator; barium and magnesium were ineffective. These results suggest that the selective inhibition of the activatable form of phosphodiesterase by trifluoperazine is due to specific binding of the phenothiazine to the calcium-dependent protein activator of cyclic nucleotide phosphodiesterase. Since this activator apparently is identical with the activator of adenylate cyclase, our results might also provide the mechanism by which phenothiazines inhibit adenylate cyclase activity and might, in fact, provide a common explanation for some of the diverse physiological and pharmacological actions of the phenothiazine antipsychotics.