MODULATION OF CALMODULIN FUNCTION AND OF CA-2+-INDUCED SMOOTH-MUSCLE CONTRACTION BY THE CALMODULIN ANTAGONIST, HT-74

  • 1 March 1986
    • journal article
    • research article
    • Vol. 29  (3) , 264-269
Abstract
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 .mu.M HT-74, in sharp contrast to phenothiazines and napthalenesulfonamides 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 .mu.M and 0.4 .mu.M, respectively. In the presence of 0.3 .mu.M 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 the heretofore reported. It inhibits Ca2+ binding to CaM and produces a competitive inhibition of Ca2+ induced contractions of depolarized vascular smooth muscle.