Measurement of activation energy and oxidative phosphorylation onset kinetics in isolated muscle fibers in the absence of cross-bridge cycling.
Measurement of activation energy and oxidative phosphorylation onset kinetics in isolated muscle fibers in the absence of cross-bridge cycling.
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DOI：
10.1152/ajpregu.00687.2005
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发表时间：
2006-06
期刊：
影响因子：
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通讯作者：
B. Walsh;R. A. Howlett;C. Stary;C. A. Kindig;Michael C. Hogan
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文献类型：
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作者：
B. Walsh;R. A. Howlett;C. Stary;C. A. Kindig;Michael C. Hogan
本研究利用N-苄基-对甲苯磺酰胺（BTS），一种有效的跨桥循环抑制剂，测量1）收缩过程中跨桥循环的相对代谢成本和活化能，以及2）在存在和不存在肌球蛋白ATP酶活性的情况下的氧摄取动力学，在离体非洲爪蟾肌纤维中。在20 ℃的对照条件下（Con）和暴露于12.5 μ M BTS后，在收缩期间测量等长张力发展和细胞溶质Ca 2+浓度（[Ca 2 +]c）或细胞内Po 2（PiO 2）。BTS将张力发展衰减至Con的5+/-0.4%，但在重复等长收缩期间不影响静息或峰值[Ca 2 +]c。为了确定跨桥循环的相对代谢成本，我们测量了Con和BTS组收缩期间PiO 2的下降（Δ PiO 2; Vo 2的代表）。BTS使DeltaP（iO 2）衰减55+/-6%，反映了过桥循环的相对ATP成本。因此，外推DeltaPiO 2到0%张力下的值表明，在混合纤维类型的等长收缩期间，肌动球蛋白ATP需求约为总ATP消耗的58%。BTS还减缓了PiO 2（达到总Δ PiO 2的63%的时间）从75+/-9 s（Con）下降到101+/-9 s（BTS）（P<0.05），表明ATP水解产物在确定Vo 2起始动力学中的重要作用。这些结果表明，在分离的骨骼肌纤维，1）激活能占相当大的比例（约42%）的总ATP的成本在等长收缩，和2）尽管不变的[Ca 2 +]c瞬变，ATP消耗率降低导致较慢的Vo 2开始动力学。
This study utilized N-benzyl-p-toluene sulfonamide (BTS), a potent inhibitor of cross-bridge cycling, to measure 1) the relative metabolic costs of cross-bridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca2+ concentration ([Ca2+]c) or intracellular Po2 (PiO2) were measured during contractions at 20 degrees C in control conditions (Con) and after exposure to 12.5 microM BTS. BTS attenuated tension development to 5+/-0.4% of Con but did not affect either resting or peak [Ca2+]c during repeated isometric contractions. To determine the relative metabolic cost of cross-bridge cycling, we measured the fall in PiO2) (DeltaPiO2; a proxy for Vo2) during contractions in Con and BTS groups. BTS attenuated DeltaP(iO2) by 55+/-6%, reflecting the relative ATP cost of cross-bridge cycling. Thus, extrapolating DeltaPiO2 to a value that would occur at 0% tension suggests that actomyosin ATP requirement is approximately 58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in PiO2) (time to 63% of overall DeltaPiO2) from 75+/-9 s (Con) to 101+/-9 s (BTS) (P<0.05), suggesting an important role of the products of ATP hydrolysis in determining the Vo2 onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (approximately 42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca2+]c transients, a reduced rate of ATP consumption results in slower Vo2 onset kinetics.