Modulation of calmodulin function and of Ca2+-induced smooth muscle contraction by the calmodulin antagonist, HT-74.
The relationship between the functions of calmodulin (CaM) and Ca2+-induced smooth muscle contraction was investigated using a newly synthesized CaM antagonist, 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4- -phenylpiperidinyl)propyl]benzenesulfonamide (HT-74). We noted a selectivity of HT-74 for CaM, compared to other calcium-binding proteins and target enzymes of CaM. As HT-74 had no significant effect on the intensity of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence in the presence of the Ca2+-CaM complex, the HT-74-binding sites may differ from those of naphthalenesulfonamides and phenothiazines which decrease ANS fluorescence. The Ca2+ binding to CaM was inhibited significantly by 1.0 microM HT-74, in sharp contrast to phenothiazines and naphthalenesulfonamides which increase the extent of the Ca2+ binding to CaM. Increasing CaM concentrations reversed the HT-74-induced inhibition of CaM-dependent enzymes such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase, with Ki values of 0.5 microM and 0.4 microM, respectively. In the presence of 0.3 microM HT-74, potassium-depolarized rabbit aortic strips pre-contracted with 0.3 mM CaCl2 relaxed, and this relaxation was completely reversed by the addition of an excess amount of CaCl2 (10 mM). This compound shifted the dose-response curve for CaCl2 to the right, in a competitive manner. However, HT-74 inhibited the phenylephrine-induced contraction elicited in Ca2+-free solution and the calcium ionophore A23187-induced contraction in the presence of calcium ion. Therefore, this agent affects intracellular actions of Ca2+ rather than membrane receptors or the influx of Ca2+. HT-74 is a CaM antagonist which binds to CaM in a manner different from that heretofore reported. It inhibits Ca2+ binding to CaM and produces a competitive inhibition of Ca2+-induced contractions of depolarized vascular smooth muscle.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000032:analysis; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D009219:Myosin-Light-Chain Kinase; D011494:Protein Kinases / Q000494:pharmacology; D011817:Rabbits; D013449:Sulfonamides / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology*; D000096926:Benzenesulfonamides
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D000817:Anilino Naphthalenesulfonates; D052160:Benzothiazoles; D002118:Calcium; D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D011494:Protein Kinases; D013449:Sulfonamides; D013844:Thiazoles; C017967:W 7; C048411:HT 74; D000096926:Benzenesulfonamides