Posterino G S, Lamb G D
Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia.
J Physiol. 2003 Aug 15;551(Pt 1):219-37. doi: 10.1113/jphysiol.2003.040022. Epub 2003 Jul 4.
This study examined the relationship between the level of Ca2+ loading in the sarcoplasmic reticulum (SR) and the amount of Ca2+ released by an action potential (AP) in fast-twitch skeletal muscle fibres of the rat. Single muscle fibres were mechanically skinned and electric field stimulation was used to induce an AP in the transverse-tubular system and a resulting twitch response. Responses were elicited in the presence of known amounts (0-0.38 mM) of BAPTA, a fast Ca2+ buffer, with the SR Ca2+ pump either functional or blocked by 50 microM 2,5-di-tert-butyl-1,4-hydroquinone (TBQ). When Ca2+ reuptake was blocked, an estimate of the amount of Ca2+ released by an AP could be derived from the size of the force response. In a fibre with the SR loaded with Ca2+ at the endogenous level (approximately 1.2 mM, expressed as total Ca2+ per litre fibre volume; approximately one-third of maximal loading), a single AP triggered the release of approximately 230 microM Ca2+. If a second AP was elicited 10 ms after the first, only a further approximately 60 microM Ca2+ was released, the reduction probably being due to Ca2+ inactivation of Ca2+ release. When Ca2+ reuptake was blocked, APs applied 15 s apart elicited similar amounts of Ca2+ release (approximately 230 microM) on the first two or three occasions and then progressively less Ca2+ was released until the SR was fully depleted after a total of approximately eight APs. When the SR was loaded to near-maximal capacity (approximately 3-4 mM), each AP (or pair of APs 10 ms apart) still only released approximately the same amount of Ca2+ as that released when the fibre was endogenously loaded. Consistent with this, successive APs (15 s apart) elicited similar amounts of Ca2+ release approximately 10-16 times before the amount released declined, and the SR was fully depleted of Ca2+ after a total release calculated to be approximately 3-4 mM. When the SR was loaded maximally, increasing the [BAPTA] above 280 microM resulted in an increase in the amount of Ca2+ released per AP, probably because the greater level of cytoplasmic Ca2+ buffering prevented Ca2+ inactivation from adequately limiting Ca2+ release. These results show that the amount of Ca2+ released by AP stimulation in rat fast-twitch fibres normally stays virtually constant over a wide range of SR Ca2+ content, in spite of the likely large change in the electrochemical gradient for Ca2+. This was also found to be the case in toad twitch fibres. This constancy in Ca2+ release should help ensure precise regulation of force production in fast-twitch muscle in a range of circumstances.
本研究检测了大鼠快肌骨骼肌纤维肌浆网（SR）中Ca2+负荷水平与动作电位（AP）释放的Ca2+量之间的关系。对单根肌纤维进行机械去膜处理，并利用电场刺激在横管系统中诱发AP及产生相应的抽搐反应。在存在已知量（0 - 0.38 mM）的快速Ca2+缓冲剂BAPTA的情况下引发反应，此时SR Ca2+泵功能正常或被50 microM 2,5 - 二叔丁基对苯二酚（TBQ）阻断。当Ca2+再摄取被阻断时，可根据力反应的大小推算出AP释放的Ca2+量。在内源性Ca2+负荷水平（约1.2 mM，以每升纤维体积中的总Ca2+表示；约为最大负荷的三分之一）下加载Ca2+的纤维中，单个AP触发释放约230 microM Ca2+。如果在第一个AP后10 ms引发第二个AP，则仅再释放约60 microM Ca2+，这种减少可能是由于Ca2+释放的失活所致。当Ca2+再摄取被阻断时，间隔15 s施加的AP在前两三次引发相似量的Ca2+释放（约230 microM），随后释放的Ca2+逐渐减少，直至在总共约8个AP后SR中的Ca2+完全耗尽。当SR被加载至接近最大容量（约3 - 4 mM）时，每个AP（或间隔10 ms的一对AP）仍然仅释放与纤维内源性加载时释放的Ca2+量大致相同。与此一致的是，连续的AP（间隔15 s）在释放量下降之前引发相似量的Ca2+释放约10 - 16次，并且在计算得出的总释放量约为3 - 4 mM后，SR中的Ca2+完全耗尽。当SR被最大程度加载时，将[BAPTA]增加至280 microM以上会导致每个AP释放的Ca2+量增加，这可能是因为更高水平的细胞质Ca2+缓冲作用阻止了Ca2+失活充分限制Ca2+释放。这些结果表明，尽管Ca2+的电化学梯度可能发生很大变化，但在大鼠快肌纤维中，AP刺激释放的Ca2+量在广泛的SR Ca2+含量范围内通常实际上保持恒定。在蟾蜍抽搐纤维中也发现了这种情况。Ca2+释放的这种恒定性应有助于确保在一系列情况下对快肌中力的产生进行精确调节。
