Effects of inorganic phosphate on endothermic force generation in muscle

Using a rapid (ca. 0.2 ms) laser temperature jump technique, the rate of endothermic force generation was examined in single-skinned (rabbit psoas) muscle fibres when they were exposed to different levels of inorganic phosphate (a product released during ATP hydrolysis in active muscle). The steady force is reduced by increased phosphate but the apparent rate constant of force generation induced by a standard temperature jump (from ca. 9 degrees C to ca. 12 degrees C) increases two- to threefold when the phosphate added is increased from zero to ca. 25 mM. The increase in the apparent rate constant also exhibits saturation at higher phosphate levels and the relation is hyperbolic. Detailed examination of the data, particularly in relation to our pressure release experiments, leads to a scheme for the molecular steps involved in phosphate release and force generation in active muscle fibres, where phosphate release from attached cross-bridges involves three reversible and sequentially faster molecular steps. Step one is a moderately slow, pre-force generation step that probably represents a transition of cross-bridges from non-specific to stereospecific attached states. Step two is moderately fast and represents endothermic cross-bridge force generation (temperature sensitive) and step three is a very rapid phosphate release. Such a scheme accommodates findings from a variety of different studies, including pressure perturbation experiments and other studies where the effect of phosphate on muscle force was studied.