Calcium-Regulated Modulator Protein Interacting Agents Inhibit Smooth Muscle Calcium-Stimulated Protein Kinase and ATPase

Reagents such as N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine, prenylamine, and N 2-dansyl-L-arginine-4-t-butylpiperidine amide (No. 233) that interact with Ca2+-regu1ated modulator protein (modulator protein, calmodulin) were found to inhibit dose dependently not only Ca2+-dependent protein kinase (myosin light chain kinase), but also Ca2+-dependent ATPase of chicken gizzard actomyosin. Inhibition of Ca2+-dependent ATPase by these agents was prevented by the addition of modulator protein. These agents did not inhibit calcium-independent Mg2+-ATPase of actomyosin. Ca2+-dependent transfer of 32 P i from [γ-32P]ATP to the 20,000-dalton light chain of the gizzard myosin in the presence of Mg2+ was also inhibited dose dependently by these agents. The concentrations of these agents producing 50% inhibition of the Ca2+-dependent ATPase activity were found to be similar to concentrations producing 50% inhibition of myosin light chain phosphorylation, thereby suggesting that the inhibition of Ca2+-dependent ATPase of actomyosin by these drugs is due to their inhibition of myosin light chain phosphorylation. W-7 bound to Ca2+ modulator protein complex, but not to the modulator protein in the presence of EGTA. No. 233 and chlorpromazine inhibited the binding of W-7 to the Ca2+-modulator complex, suggesting that No. 233 and chlorpromazine bind to modulator protein. The modulator protein has two classes of W-7 binding sites: three functional sites with a high affinity for W-7 ( K W-7 = 11 µM) and nine sites with a low affinity for the drug ( K W-7 = 200 µM). W-7 did not show a significant binding to actin, myosin, tropomyosin, and bovine serum albumin at the concentration of the drug capable of binding to modulator protein. Troponin C was the only protein other than modulator protein that bound W-7 significantly but the affinity ( K W-7 = 25 µM) of this protein for W-7 was lower than that of modulator protein. These results suggest that agents that interact with modulator protein produce relaxation of smooth muscle by inhibition of modulator protein-dependent myosin light chain phosphorylation thus suppressing the actin-myosin interaction and concomitant myosin ATPase activation. ACKNOWLEDGMENTS We thank M. Ohara, Kyoto University for critical reading of the manuscript, and Dr. T. Totsuka for helpful discussion.