Mickelson J R, Ross J A, Reed B K, Louis C F
Biochim Biophys Acta. 1986 Nov 17;862(2):318-28. doi: 10.1016/0005-2736(86)90234-8.
To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.
为了进一步确定肌浆网钙的积累和释放可能与骨骼肌疾病恶性高热（MH）有关，我们检测了从正常猪肌肉和MH易感性猪肌肉中分离出的肌浆网部分的各种特性。一种富含源自终池的囊泡的肌浆网制剂，在不连续蔗糖密度梯度上进一步分级分离（迈斯纳，G.（1984年）《生物化学杂志》259卷，2365 - 2374页）。所得的MH易感性和正常肌浆网部分，命名为F0 - F4，在产量、胆固醇和磷脂含量或尼群地平结合能力方面没有差异。MH易感性和正常肌浆网部分F3的钙积累（22℃时为0.27微摩尔钙/毫克每分钟）、Ca2 + - ATP酶活性（22℃时为0.98微摩尔无机磷/毫克每分钟）和肌集钙蛋白含量也相似。为了检测肌浆网钙释放，F3部分的囊泡被动加载45Ca（约40纳摩尔钙/毫克），并迅速稀释到特定Ca2 + 浓度的介质中。稀释到1微摩尔/升Ca2 + 后，5秒后测量的Ca2 + 依赖性钙释放程度，MH易感性肌浆网比正常肌浆网显著更高，分别为加载钙的65.9±2.8%和47.7±3.9%。与正常肌浆网相比，MH易感性肌浆网这种钙释放（5秒值）的Ca2 + 刺激的半数最大浓度（C1/2）似乎也向更高的Ca2 + 敏感性偏移。丹曲林对F3部分的钙释放没有影响，然而，氟烷（0.1 - 0.5毫摩尔）在MH易感性和正常肌浆网中同样增加了钙释放程度（5秒）。此外，与正常肌浆网相比，Mg2 + 在抑制方面效果较差，而ATP和咖啡因在刺激来自MH易感性的这种Ca2 + 依赖性钙释放方面效果更好。我们的结果表明，虽然在MH中肌浆网钙积累似乎未受影响，但肌浆网Ca2 + 诱导的钙释放机制的某些方面发生了改变。虽然肌浆网Ca2 + 诱导的钙释放机制在原位的作用尚不清楚，但我们的结果表明，肌浆网钙释放调节异常可能在MH综合征中起重要作用。
