Effects of inorganic phosphate on endothermic force generation in muscle

Abstract

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 ¢bres when they were exposed to di¡erent 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 8C to ca. 12 8C) 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 ¢bres, 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-speci¢c to stereospeci¢c 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 ¢ndings from a variety of di¡erent studies, including pressure perturbation experiments and other studies where the e¡ect of phosphate on muscle force was studied.

Changes in myosin S1 orientation and force induced by a temperature increase

“…The origin of the temperature-induced rise in force may be a temperature-sensitive endothermal isomerization step in the actomyosin ADP⅐P i state, associated with no change (4) or a 20% increase in I M3 (25) over a temperature jump from 5 to 17°C, according to temperature-jump studies in skinned fibers (26). In this case, a rise in temperature would displace the isomerization step toward a high force state.…”

Coupling between phosphate release and force generation in muscle actomyosin

“…White et al (1997) found that P i release from actomyosin in solution is only 75 s À1 , not sufficiently faster than the ATP hydrolysis step to support the assumption of a rapid equilibrium described above. More complex schemes could, of course, be invoked, and integration of pressure-and temperature-jump and fibre transient data led Ranatunga (1999) to postulate yet another state in this part of the actomyosin cycle. If the 75 s À1 value for P i dissociation measured with isolated acto-S1 applies to experiments in which muscle fibres are activated by photolysis of caged ATP (Goldman et al 1984b;Sleep et al 2004), the rate of force development is too fast for it to follow P i release (Sleep et al 2004).…”

Simulation of molecular steps in muscle force generation

“…For comparison, the experimental data (mean AE s.e.m.) for 12 8C in ¢gure 2a of Ranatunga (1999) are also shown (¢lled symbols). Figure 2c,d shows two simulated tension transients generated by a standard temperature jump from ca.…”

“…Step II is the temperature-sensitive force generation step and the rate constants k 23 and k 32 were taken from a preceding (temperature-jump) paper (Ranatunga 1999). AM * .ADP.Pi and AM * .ADP are high (and equal) force states and the sum of their occupancy was considered to represent muscle force ( tension).…”

“…The particular reaction scheme used for illustration is that proposed in a preceding paper (Ranatunga 1999). In that paper, the rate of force generation induced by a standard temperature jump (endothermic force generation) was examined in single-skinned (rabbit psoas) muscle ¢bres when they were exposed to di¡erent levels of inorganic phosphate (Pi, a product released during ATP hydrolysis in active muscle).…”