Cardiotonic bipyridine amrinone slows myosin-induced actin filament sliding at saturating [MgATP].
Previously reported effects of amrinone on skeletal muscle function suggest that the drug reduces the rate constant of myosin cross-bridge dissociation. We have used the in vitro motility assay to further elucidate the mechanism underlying this effect and to aid these studies a new, improved, filament tracking software was developed in the Matlab environment. The experiments were carried out at 30 degrees C using heavy meromyosin from fast rabbit muscle and rhodamine-phalloidin labeled actin filaments. A slowing effect of amrinone on filament sliding velocity at 1 mM MgATP was observed at drug concentrations >0.3 mM. This effect showed signs of saturation at the highest drug concentrations (1-2 mM) that could be readily tested. The sliding velocity exhibited hyperbolic dependence on [MgATP] with a Vmax of 7.2 +/- 0.9 microm/s and a KM of 0.18 +/- 0.02 mM. Amrinone (1 mM) reduced Vmax by 32 +/- 5% (P < 0.01) and KM by 42 +/- 8% (P < 0.05; n=4). These results are accounted for in the most straightforward way by a model where amrinone acts directly on the actomyosin system and reduces the rate constant of MgADP release. Such a well-defined effect on the myosin cross-bridge cycle makes the drug a potentially useful pharmacological tool for further studies of myosin function both in vitro and in the ordered filament array of a living muscle fiber.
Klinth
J
J
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
Arner
A
A
Månsson
A
A
D008841:Actin Cytoskeleton / Q000187:drug effects* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000676:Amrinone / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D003627:Data Interpretation, Statistical; D001330:Electronic Data Processing / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D018482:Muscle, Skeletal / Q000502:physiology; D009206:Myocardium / Q000378:metabolism; D015879:Myosin Subfragments / Q000502:physiology; D009218:Myosins / Q000502:physiology*; D011817:Rabbits; D015203:Reproducibility of Results; D012984:Software
D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D009218:Myosins; D000676:Amrinone