We have previously shown that the isometric tension of a fully calcium-activated skinned rabbit psoas muscle fiber is reversibly depressed by increased hydrostatic pressure. We report here the characterization of tension transients induced by a rapid (
2,3-butanedione 2-monoxime (BDM) inhibits muscle contraction and actomyosin ATPase both in fibres and in solution. It is potentially useful as a tool for exploring weak interactions between actin and myosin. We have examined the effect of BDM on several key steps of the myosin subfragment-1 and actomyosin subfragment-1 ATPase in solution. These studies show that BDM shifts the equilibrium between two actomyosin states towards a more weakly bound form when the acto.myosin complex has ADP alone or ADP and phosphate bound. We also confirm the findings of Herrmann and colleagues (1993, Biochemistry, 31, 12227-32) that the main effect of BDM on the myosin subfragment-1 ATPase is to slow the release of phosphate following ATP hydrolysis. Skinned fibre studies show that the effects of BDM and phosphate on the steady isometric tension of the fibres are additive. This is consistent with the interpretation that BDM is reducing fibre tension either by increasing phosphate binding or by a direct effect on the crossbridge. Tension transients induced by rapid pressure release were examined in single muscle fibres; they showed that BDM reduces the rate of tension generation following pressure release. This result suggest that BDM directly affects the force generating event in the crossbridge.
The effect of changes in hydrostatic pressure (up to 10 MPa) on the maximally calcium-activated tension in glycerinated rabbit psoas fibres has been examined. The steady active tension was depressed by 0.8% per MPa pressure rise. This pressure sensitivity was enhanced by the pressure of millimolar phosphate and depressed by millimolar ADP. These results support the conclusions that increased pressure is perturbing a crossbridge event. The results are discussed in terms of a three state crossbridge model and are shown to be compatible with a pressure effect on the transition from an attached crossbridge state to a tension bearing state. This is compatible with the effects of pressure on the isolated proteins in solution.
1. Effects of hydrostatic pressure (range 0.1-10 MPa) on the isometric tension of skinned (rabbit psoas) muscle fibres were examined at 12°C and at different levels of Ca2" activation (pCa range 4-7); the effects on both the steady tension and the tension transients induced by rapid pressure release (< 1 ms) are described. 2. The steady tension was depressed by increased pressure (-1 % MPa1) at a high level of Ca2" activation (pCa -4) whereas it was potentiated at lower Ca2" levels (pCa > 6); the effects were reversible. 3. At maximal Ca2+ activation, the tension recovery following pressure release (10 MPa to atmospheric) consisted of a fast (-30 s') and a slow (2-3 s-) phase; the rate and the normalized amplitude (normalized to the steady tension at atmospheric pressure for a particular pCa) of the fast phase were invariant with changes in Ca2+ level. 4. The effects of changing Ca2+ level on the slow phase were complex; its positive amplitude at high Ca2" levels changed to negative and the rate decreased to -1 s-I at low Ca2" levels (pCa > 6 0). 5. Results are discussed in relation to previous studies on the effect of pressure on intact muscle fibres and the actin-myosin interaction. This work supports calcium regulation of cross-bridge recruitment rather than calcium regulation of the rate of a specific step in the cross-bridge cycle.
Glycerinated muscle fibers isolated from rabbit psoas muscle, and a number of other nonmuscle elastic fibers including glass, rubber, and collagen, were exposed to hydrostatic pressures of up to 10 MPa (100 Atm) to determine the pressure sensitivity of their isometric tension. The isometric tension of muscle fibers in the relaxed state (passive tension) was insensitive to increased pressure, whereas the muscle fiber tension in rigor state increased linearly with pressure. The tension of all other fiber types (except rubber) also increased with pressure; the rubber tension was pressure insensitive. The pressure sensitivity of rigor tension was 2.3 kN/m2/MPa and, in comparison with force/extension relation determined at atmospheric pressure, the hydrostatic compression in rigor muscle fibers was estimated to be 0.03% Lo/MPa. As reported previously, the active muscle fiber tension is depressed by increased pressure. The possible underlying basis of the different pressure-dependent tension behavior in relaxed, rigor, and active muscle is discussed.
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