Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide phosphodiesterase by calmodulin

Experiments were designed to investigate the effect of inhibitors on calmodulin's hydrophobic sites and their consequences on the activation of a target enzyme, cyclic nucleotide phosphodiesterase. Two fluorescent probes, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS) and 9-anthroylcholine (9AC) were used to study the interactions with calmodulin of inhibitors devoid of direct effect on the probes. Contrary to W-7, nicergoline, nicardipine and quercetin, which decreased the fluorescence of the two probes bound to calmodulin, bepridil only decreased 9AC fluorescence but increased the fluorescence intensity at the wavelength of the emission maximum of TNS. In spite of this difference, bepridil as well as W-7 and nicergoline competitively inhibited calmodulin activation of phosphodiesterase. In addition, nicergoline also inhibited phosphodiesterase activity competitively to cyclic GMP. These results show differences in the interactions of inhibitors with calmodulin; these differences are not detected in functional studies of the effect of inhibitors on phosphodiesterase activation.