MCI-154 INCREASES CA2- A STUDY USING A NOVEL IN-VITRO MOTILITY ASSAY TECHNIQUE( SENSITIVITY OF RECONSTITUTED THIN FILAMENT )

MCI-154 pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochlori de trihydrate) is a potent novel cardiotonic agent whose positive inot ropism 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 reconstitut ed thin filament on a myosin layer in vitro. Cardiac thin filaments we re reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the fil ament showed that tropomyosin-troponin complex was integrated along ac tin filament homogeneously. Thin filaments thus prepared were fluoresc ently 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, reachi ng the maximum velocity within a very narrow range of [Ca2+]. The regu lation was completely abolished by using simple actin filaments withou t tropomyosin-troponin complex, demonstrating that the regulatory prot eins are responsible for this Ca2+ regulation of the movement of the r econstituted thin filament. Under the control condition, addition of M CI-154 shifted the threshold [Ca2+] to a lower level (sensitization) i n a concentration-related manner. And 10(-4) mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low t emperature (15 degrees C), or the addition of inorganic phosphate (10 mmol/L). However, the maximum sliding Velocity was not affected by the drug under any condition. In conclusion, MCI-154 directly sensitized the contractile apparatus under not only physiological but also pathop hysiological conditions. This in vitro motility assay technique using reconstituted thin filaments is a useful tool for studying the mechani sm of action of Ca2+ sensitizers.