MCI-154 increases Ca2+ sensitivity of reconstituted thin filament. A study using a novel in vitro motility assay technique.
MCI-154 (6-[4-(4'-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca2+ sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca2+] of the medium (control, pH 7.2 at 25 degrees C). When [Ca2+] was low, the filaments showed only brownian motion. However, above a certain level of [Ca2+] (the threshold [Ca2+]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca2+]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca2+ regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca2+] to a lower level (sensitization) in a concentration-related manner. And 10(-4) mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15 degrees C), or the addition of inorganic phosphate (10 mmol/L).(ABSTRACT TRUNCATED AT 250 WORDS)
Sata
M
M
Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Sugiura
S
S
Yamashita
H
H
Fujita
H
H
Momomura
S
S
Serizawa
T
T
D008841:Actin Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D000199:Actins / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002316:Cardiotonic Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D008955:Models, Cardiovascular; D009068:Movement; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism*; D009218:Myosins / Q000187:drug effects; D011724:Pyridazines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling; D014335:Tropomyosin / Q000187:drug effects; D014336:Troponin / Q000187:drug effects
D000199:Actins; D002316:Cardiotonic Agents; D011724:Pyridazines; D014335:Tropomyosin; D014336:Troponin; C052228:MCI 154; D009218:Myosins; D002118:Calcium