Effects of sarcomere length and temperature on the rate of ATP utilisation by rabbit psoas muscle fibres.
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1. The steady state rate of ATP utilisation by single permeabilised fibres from rabbit psoas muscle immersed in silicone oil was measured using a linked enzyme assay that coupled ADP production to the oxidation of NADH.2. At sarcomere length 2.5 microm, at 10 degrees C, the rate of ATP utilisation in relaxing conditions was 6 +/- 1 microM s-1 (mean +/- S.E.M., n = 8 fibres); during isometric contraction it was 310 +/- 10 microM s-1 (mean +/- S.E.M., n = 11). Assuming a myosin active site concentration of 150 microM, these values correspond to rates of ATP utilisation per active site of about 0.04 and 2.1 s-1, respectively. 3. The rate of ATP utilisation in relaxing conditions was independent of sarcomere length in the range 2.5-4.0 microm. The rate of ATP utilisation during isometric contraction had a dependence on resting sarcomere length similar to that of isometric force in the range 2.5-4.0 microm, but was less strongly dependent on sarcomere length than was isometric force in the range 1.5-2.5 microm. 4. The rate of ATP utilisation in relaxing conditions had a Q10 of 2.5 in the temperature range 7-25 degrees C, but this increased to 9.7 in the range 25-35 degrees C, suggesting that some active force may have been generated in relaxing solution at temperatures above 25 degrees C. 5. The rate of ATP utilisation during isometric contraction had a Q10 of 3.6 throughout the temperature range 7-25 degrees C; this was similar to the Q10 for isometric force at low temperature (3.5 at 7-10 degrees C) but much larger than that for isometric force at higher temperature (1.3 at 20-25 degrees C). 6. Application of the NADH-linked assay to single muscle fibres in oil improves the effective sensitivity and time resolution of the method, and allows continuous measurements of the rate of ADP production during active contraction. (+info)
Effect of active shortening on the rate of ATP utilisation by rabbit psoas muscle fibres.
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1. The rate of ATP utilisation during active shortening of single skinned fibres from rabbit psoas muscle at 10 degrees C was measured using an NADH-linked assay. Fibres were immersed in silicone oil and illuminated with 365 nm light. The amounts of NADH and carboxytetramethylrhodamine (CTMR) in the illuminated region of the fibre were measured simultaneously from fluorescence emission at 425-475 and 570-650 nm, respectively. The ratio of these two signals was used to determine the intracellular concentration of NADH, and thus the ATP utilisation, without interference from movements of the fibre with respect to the measuring light beam. 2. The total extra ATP utilisation due to shortening (ATP) was determined by extrapolation of the steady isometric rates before and after shortening to the mid-point of the shortening period. ATP had a roughly linear dependence on the extent of shortening in the range 1-15 % fibre length (L0) at a shortening velocity of 0.4 L0 s-1 from initial sarcomere length 2.7 microm. For shortening of 1 % L0, ATP was 21 +/- 1 M (mean +/- S.E.M., n = 3). 3. The mean rate of ATP utilisation during ramp shortening of 10 % L0 had a roughly linear dependence on shortening velocity in the range 0.05-1.2 L0 s-1. During unloaded shortening at 1.2 L0 s-1 the mean rate of ATP utilisation was 1.7 mM s-1, about 9 times the isometric rate. ATP was roughly independent of shortening velocity, and was 84 +/- 9 microM (mean +/- S.E.M., n = 6) for shortening of 10 % L0. 4. The implications of these results for mechanical-chemical coupling in muscle are discussed. The total ATP utilisation associated with shortening of 1 % L0 is only about 17 % of the concentration of the myosin heads in the fibre, suggesting that during isometric contraction either less than 17 % of the myosin heads are attached to actin, or that heads can detach without commitment to ATP splitting. The fraction of myosin heads attached to actin during unloaded shortening is estimated from the rate of ATP utilisation to be less than 7 %. (+info)
Strong binding of myosin increases shortening velocity of rabbit skinned skeletal muscle fibres at low levels of Ca(2+).
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1. At low levels of activation, unloaded shortening of skinned skeletal muscle fibres takes place in two phases: an initial phase of high-velocity shortening followed by a phase of low-velocity shortening. The basis for Ca(2+) dependence of unloaded shortening velocity (V(o)) in the low-velocity phase was investigated by varying the level of thin filament activation with Ca(2+) and N-ethyl-maleimide myosin subfragment-1 (NEM-S1), a non-tension-generating, strong binding derivative of subfragment-1. V(o) was measured with the slack-test method. 2. Treatment of skinned fibres with 5 microM NEM-S1 eliminated the low-velocity phase of shortening but had no effect on the high-velocity phase of shortening during submaximal activation with Ca(2+), or on V(o) during maximal activation with Ca(2+). 3. Extensive washout of NEM-S1 from the treated fibres restored the low-velocity phase of shortening and returned low-velocity V(o) to pre-treatment values. 4. The effect of NEM-S1 to increase low-velocity V(o) can be explained in terms of a model in which strong binding myosin cross-bridges activate the thin filament to a state in which the rate of ADP release from the actin-myosin-ADP complex and the rate of cross-bridge detachment from actin are accelerated during unloaded shortening. (+info)
Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.
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The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle. (+info)
Retroperitoneal hematoma associated with femoral neuropathy: a complication under antiplatelets therapy.
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We report a case of retroperitoneal hematoma presenting as femoral nerve pulsy on antiplatelet therapy. The patient, a 78-year-old man who had undergone antiplatelet treatment using ticlopidine, was admitted to our hospital with complaints of sudden-onset low abdominal and back pain. Computed tomography showed an iso-density mass in the right retroperitoneum within the psoas muscle. We made a diagnosis of retroperitoneal hematoma compressing the femoral nerve and performed an operation to remove the hematoma in order to decompress the femoral neuropathy. Postoperatively, the patient rapidly recovered from the femoral neuropathy. In the particular case in which no antagonist against the ticlopidine is available, surgical decompression could produce a good outcome. (+info)
Structural characterization of weakly attached cross-bridges in the A*M*ATP state in permeabilized rabbit psoas muscle.
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It is well established that in a skeletal muscle under relaxing conditions, cross-bridges exist in a mixture of four weak binding states in equilibrium (A*M*ATP, A*M*ADP*P(i), M*ATP, and M*ADP*P(i)). It has been shown that these four weak binding states are in the pathway to force generation. In the past their structural, biochemical, and mechanical properties have been characterized as a group. However, it was shown that the myosin heads in the M*ATP state exhibited a disordered distribution along the thick filament, while in the M*ADP*P(i) state they were well ordered. It follows that the structures of the weakly attached states of A*M*ATP and A*M*ADP*P(i) could well be different. Individual structures of the two attached states could not be assigned because protocol for isolating the two states has not been available until recently. In the present study, muscle fibers are reacted with N-phenylmaleimide such that ATP hydrolysis is inhibited, i.e., the cross-bridge population under relaxing conditions is distributed only between the two states of M*ATP and A*M*ATP. Two-dimensional x-ray diffraction was applied to determine the structural characteristics of the attached A*M*ATP state. Because the detached state of M*ATP is disordered and does not contribute to layer line intensities, changes as a result of increasing attachment in the A*M*ATP state are attributable to that state alone. The equilibrium toward the attached state was achieved by lowering the ionic strength. The results show that upon attachment, both the myosin and the first actin associated layer lines increased intensities, while the sixth actin layer line was not significantly affected. However, the intensities remain weak despite substantial attachment. The results, together with modeling (see J. Gu, S. Xu and L. C. Yu, 2002, Biophys. J. 82:2123-2133), suggest that there is a wide range of orientation of the attached A*M*ATP cross-bridges while the myosin heads maintain some degree of helical distribution on the thick filament, suggesting a high degree of flexibility in the actomyosin complex. Furthermore, the lack of sensitivity of the sixth actin layer line suggests that the binding site on actin differs from the putative site for rigor binding. The significance of the flexibility in the A*M*ATP complex in the process of force generation is discussed. (+info)
A model of cross-bridge attachment to actin in the A*M*ATP state based on x-ray diffraction from permeabilized rabbit psoas muscle.
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A model of cross-bridges binding to actin in the weak binding A*M*ATP state is presented. The modeling was based on the x-ray diffraction patterns from the relaxed skinned rabbit psoas muscle fibers where ATP hydrolysis was inhibited by N-phenylmaleimide treatment (S. Xu, J. Gu, G. Melvin, L. C. Yu. 2002. Biophys. J. 82:2111-2122). Calculations included both the myosin filaments and the actin filaments of the muscle cells, and the binding to actin was assumed to be single headed. To achieve a good fit, considerable flexibility in the orientation of the myosin head and the position of the S1-S2 junction is necessary, such that the myosin head can bind to a nearby actin whereas the tail end was kept in the proximity of the helical track of the myosin filament. Hence, the best-fit model shows that the head binds to actin in a wide range of orientations, and the tail end deviates substantially from its lattice position in the radial direction (approximately 60 A). Surprisingly, the best fit model reveals that the detached head, whose location thus far has remained undetected, seems to be located close to the surface of the myosin filament. Another significant requirement of the best-fit model is that the binding site on actin is near the N terminus of the actin subunit, a position distinct from the putative rigor-binding site. The results support the idea that the essential role played by the weak binding states M*ATP <--> A*M*ATP for force generation lies in its flexibility, because the probability of attachment is greatly increased, compared with the weak binding M*ADP*P(i) <--> A*M*ADP*P(i) states. (+info)
Titin-based contribution to shortening velocity of rabbit skeletal myofibrils.
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The shortening velocity of skeletal muscle fibres is determined principally by actomyosin cross-bridges. However, these contractile elements are in parallel with elastic elements, whose main structural basis is thought to be the titin filaments. If titin is stretched, it may contribute to sarcomere shortening simply because it can recoil 'passively'. The titin-based contribution to shortening velocity (V(p)) was quantified in single rabbit psoas myofibrils. Non-activated specimens were rapidly released from different initial sarcomere lengths (SLs) by various step amplitudes sufficient to buckle the myofibrils; V(p) was calculated from the release amplitude and the time to slack reuptake. V(p) increased progressively (upper limit of detection, approximately 60 microm s(-1) sarcomere(-1)) between 2.0 and 3.0 microm SL, albeit more steeply than passive tension. At very low passive tension levels already (< 1-2 mN mm(-2)), V(p) could greatly exceed the unloaded shortening velocity measured in fully Ca(2+)-activated skinned rabbit psoas fibres. Degradation of titin in relaxed myofibrils by low doses of trypsin (5 min) drastically decreased V(p). In intact myofibrils, average V(p) was faster, the smaller the release step applied. Also, V(p) was much higher at 30 degrees C than at 15 degrees C (Q(10): 2.0, 3.04 or 6.15, for release steps of 150, 250 or 450 nm sarcomere(-1), respectively). Viscous forces opposing the shortening are likely to be involved in determining these effects. The results support the idea that the contractile system imposes a braking force onto the passive recoil of elastic structures. However, elastic recoil may aid active shortening during phases of high elastic energy utilization, i.e. immediately after the onset of contraction under low or zero load or during prolonged shortening from greater physiological SLs. (+info)