1. Orthophosphate (P(i), 0.1-2.0 mM) was photogenerated within the filament lattice of isometrically contracting glycerinated fibres of rabbit psoas muscle at 10 and 20-degrees-C. The P(i) was produced by laser flash photolysis of the photolabile compound 1-(2-nitrophenyl)ethylphosphate (caged P(i)). Caged P(i) caused a depression of tension that was much smaller than that caused by P(i). 2. Photolysis of caged P(i) produced a decline in isometric force composed of four phases: phase 1, a lag phase (e.g. 1-4 ms at 10-degrees-C) during which force did not change; phase 11, an exponential decline by as much as 20 % of the pre-pulse force; phase III, a partial force recovery (0-3 % of the pre-pulse force); and phase IV, a further slow (0.5-3 s) decline to the steady value. Phases I, III and IV were largely independent of [P(i)] and are likely to be indirect effects caused by the caged P(i) photolysis. 3. Both the rate and amplitude of phase II depended markedly on [P(i)]. The amplitude of phase II was similar to the reduction of steady-state force by P(i). The rate of phase Il increased with increasing temperature and [P(i)]. At high [P(i)] the rate began to saturate, and approached limits of 123 s-1 at 10-degrees-C and 194 s-1 at 20-degrees-C. 4. The rate of phase II was independent of sarcomere overlap, while the amplitude was proportional to tension at partial filament overlap. A control experiment using caged ATP showed that phase II was not produced by the photolytic by-products or the light pulse. The results suggest phase II is associated with the force-generating transition of the cross-bridge cycle. 5. Sinusoidal length oscillations at 0.5 and 2 kHz were used to measure muscle stiffness during phase II. Stiffness declined in a single exponential phase, with the same time course as phase II of the tension transient. The change in stiffness was 83+/-6% (mean+/-S.E.M., n = 10, 0.5 kHz) of the change in tension when both signals were normalized to their pre-flash values. 6. Analysis of the data shows that two steps are involved in force generation and P(i) release. The non-force exerting AM-ADP-P(i) cross-bridge state first isomerizes to form a force-exerting cross-bridge state (AM'-ADP-P(i)). P(i) is then released to form a second force-generating state, AM'-ADP. The data suggest that all three states are significantly populated during an isometric contraction. 7. The forward rate constant of the force-generating isomerization is very temperature sensitive (temperature quotient, Q10 = 3-7), while the backward rate constant is not. A strain-dependent kinetic model predicts that the forward rate constant is probably more strain sensitive than the reverse rate constant.
