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Abstract

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.

Drug Effect Unveils Inter-head Cooperativity and Strain-dependent ADP Release in Fast Skeletal Actomyosin

“…These results showed that amrinone reduces the sliding velocity ( V max ) at saturating MgATP concentrations but not at MgATP concentrations close to, or below, the K m value for the hyperbolic relationship between MgATP concentration and sliding velocity. Such a combination of effects is consistent with a reduced MgADP release rate (24) but not with competitive inhibition of substrate binding. However, effects of amrinone on the MgADP release rate have not been directly demonstrated.…”

“…It has been proposed (20–22) that amrinone might competitively inhibit the MgATP binding by myosin. However, more recently, results from in vitro motility assay experiments (24) challenged this idea. These results showed that amrinone reduces the sliding velocity ( V max ) at saturating MgATP concentrations but not at MgATP concentrations close to, or below, the K m value for the hyperbolic relationship between MgATP concentration and sliding velocity.…”

“…Additionally, in view of the uncertainty about what step actually determines the sliding velocity at saturating [MgATP] (see above and Refs. 7–9), it is of interest to consider other possible drug effects that could account for the data of Klinth et al (24). These include the following: 1) an increased drag force, e.g.…”

Self-Organization of Motor-Propelled Cytoskeletal Filaments at Topographically Defined Borders

“…The methods for protein preparations, in vitro motility assays and recording of in vitro motility assay data have been described in detail elsewhere [10, 29–31]. The in vitro motility assays were performed at 25°C, pH 7.4, 1 mM MgATP, and 40 mM ionic strength.…”

New Inotropic Pharmacologic Strategies Targeting the Failing Myocardiumin the Newborn and Infant

“…The main mechanism of action of milrinone is, as in most of the here discussed agents, multiple. The increase in myocardial contraction is result of an altered trans-sacrolemal Ca + flux [5], the decrease in peripheral vascular resistance is a result of an increased uptake of Ca in the sarcoplasmatic reticulum and therefore a decrease in free intracellular Ca + in the smooth muscle cells of the vasculature [6] and finally, some improvement in lusitropy is considered to be a result of an enhanced dissociation of actin and myosion during diastole [7]. The vasodilatory effects of milrinone are magnified in the presence of andadrenergic agents such as phenylephrine, norepinephrine, or dopamine because of the combined activation of adenylate cyclase and inhibition of phosphodiesterase III [8,9],.…”