Molecular Pharmacology, Vol 17, 66-72, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

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

¹ Department of Pharmacology, School of Medicine, Mie University, Edobashi, Tsu 514, Japan

Reagents such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine, prenylamine, and N²-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 ³²P_i from [-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.

Note:
ACKNOWLEDGMENTS We thank M. Ohara, Kyoto University for critical reading of the manuscript, and Dr. T. Totsuka for helpful discussion.

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