Reversal of the cross‐bridge force‐generating transition by photogeneration of phosphate in rabbit psoas muscle fibres.

Abstract

SUMMARY1. Orthophosphate (Pi, 041-2-0 mM) was photogenerated within the filament lattice of isometrically contracting glycerinated fibres of rabbit psoas muscle at 10 and 200C. The Pi was produced by laser flash photolysis of the photolabile compound 1-(2-nitrophenyl)ethylphosphate (caged Pi). Caged Pi caused a depression of tension that was much smaller than that caused by Pi.2. Photolysis of caged P1 produced a decline in isometric force composed of four phases: phase I, a lag phase (e.g. 1-4 ms at 1000) during which force did not change; phase II, 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 [Pi] and are likely to be indirect effects caused by the caged Pi photolysis.3. Both the rate and amplitude of phase II depended markedly on [Pi]. The amplitude of phase II was similar to the reduction of steady-state force by Pi.

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

“…For step 2, the data fit better to the reversible model in muscle fibres, and the cause of its difference with solution data may reside in the vast difference in the experimental materials and conditions. Steps 1-2 are consistent with those deduced by using caged ATP (Goldman et al 1984), and steps 4-5 are consistent with those deduced by using caged Pi (Dantzig et al 1992;Araujo and Walker 1996) or pressure-release (Fortune et al 1991) on rabbit psoas fibres. Figure 3A is an electron micrograph of the control fibre showing the cross section in which both thick and thin filaments can be seen in hexagonal lattice.…”

“…Other examples are temperature jump (Goldman et al 1987;Bershitsky and Taturyan 1992), pressure release (Fortune et al 1991), and a use of caged compounds which releases a specific ligand on photoflash (Goldman et al 1984;Dantzig et al 1992;Araujo and Walker 1996). The released ligand binds to a contractile protein to initiate a transient.…”

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi]

“…It is generally accepted that the first and major force-generating step is not coupled to the cleavage of ATP into ADP and inorganic phosphate (P i ) but rather to the subsequent, reversible release of P i , e.g., through the backdoor of the myosin molecule (1)(2)(3)(4). To explore the chemomechanical coupling, force changes were induced in muscle fibers by rapid length changes (5-10), temperature jumps (5,11), pressure jumps (12), or photolytic release of caged-P i (13)(14)(15)(16)(17), and the kinetics of these force changes were interpreted in terms of cross-bridge models. The rapid change in [P i ] provides a well-defined approach because it specifically perturbs the reversible equilibrium of the P i -release.…”

A New and Improved View of Force Production

“…Kinetic analyses of the rate of force decline with variation of the final phosphate concentration were consistent with a hypothesis in which the Pi transient (k þPi2 ) behaved as if it were produced by a two-step process. This is one in which the first step Pi bound to a force-exerting actomyosin*-ADP state and formed a force-exerting actomyosin*-ADP-Pi state, followed by an isomerization in which the force step was reversed with some actomyosin-ADP-Pi dissociating to actin and myosin -ADP-Pi (6). There are however, two significant drawbacks associated with the use of the caged Pi in skinned fibers.…”

“…The first is that one could only increase Pi (and reduce force) but one could not suddenly reduce the Pi and increase force. Second, only average sarcomere spacing can be monitored in skinned muscle fiber experiments (6).…”