ML-9 inhibits the vascular contraction via the inhibition of myosin light chain phosphorylation.
We investigated the effects of a newly synthesized compound, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor of superprecipitation of actomyosin, isometric tension development, and phosphorylation of the 20,000-Da myosin light chain (LC20) in vascular smooth muscle. Superprecipitation of actomyosin from bovine aorta was inhibited by the addition of ML-9 in a dose-dependent manner. In chemically skinned smooth muscles of the rabbit mesenteric artery, ML-9 inhibited the Ca2+-independent contraction provoked by application of trypsin-treated MLCK. In the intact rabbit mesenteric artery, increases in LC20 phosphorylation reached a maximal value of 0.49 mol of Pi/mol of LC20 within 10 sec from a resting value of 0.15 mol of Pi/mol of LC20 and then declined to near the basal level during the maintained isometric force developed in response to 50 mM KCl. Preincubation with 10-30 microM ML-9 for 30 min significantly inhibited both the maximal rate and extent of KCl-induced contraction and the phosphorylation of LC20, in a dose-dependent manner. There was a linear relationship between the initial rate of tension development and the extent of LC20 phosphorylation at 10 sec after stimulation. ML-9 nonspecifically antagonized the contraction induced by various contractile agonists, such as CaCl2, norepinephrine, serotonin, histamine, and angiotensin II. ML-9 dose dependently produced a shift to the right and down, in the dose-response curves, to all the agonists tested. These results suggest that ML-9 inhibits the actin-myosin interaction through the modulation of LC20 phosphorylation via the inhibition of MLCK activity. Thus, ML-9 may be a useful compound for investigating the physiologic role of myosin light chain phosphorylation by MLCK in living cells and tissues as well as in vitro.
Ishikawa
T
T
Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Japan.
Chijiwa
T
T
Hagiwara
M
M
Mamiya
S
S
Saitoh
M
M
Hidaka
H
H
D000818:Animals; D001381:Azepines / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D008297:Male; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors*; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D014661:Vasoconstriction / Q000187:drug effects*
D001381:Azepines; C056218:ML 9; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium