Amorena C E, Wilding T J, Manchester J K, Roos A
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Gen Physiol. 1990 Nov;96(5):959-72. doi: 10.1085/jgp.96.5.959.
We examined the effect of depolarization on intracellular pH (pHi) of normal (pHi approximately 7.37) and acidified (pHi 5.90-6.70) frog semitendinosus muscle using microelectrodes. A small bundle was superfused with a Na(+)-free buffered solution (10 mM HEPES, 100% O2, pH 7.35) containing either 2.5 or 25 mM K+. An NH4Cl prepulse was used to lower pHi. At normal pHi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi of approximately 0.2. In contrast, in all 25 acidified bundles pHi rose by 0.1-0.7. The rise was greater the lower the initial pHi. It could be imitated by caffeine and blocked by tetracaine and thus was, most likely, initiated by release of calcium. We ascribed the alkalinization to hydrolysis of phosphocreatine (PCr); 2,4-dinitrofluorobenzene abolished it. Biochemical analysis on fibers at the peak of alkalinization showed PCr to be reduced by one-half, while PCr in normal fibers that had been depolarized for the same period (4-6 min) showed no change. We postulated that low pHi slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-1,6-diphosphate through inhibition of phosphofructokinase (PFK), an enzyme which is known to be highly pH sensitive. Thus PCr hydrolysis would be required to replace much of the hydrolyzed ATP. This postulated effect on PFK is in agreement with the finding that glucose-6-phosphate (in near-equilibrium with fructose-6-phosphate) was increased nearly fivefold in the depolarized acid fibers, but not in the depolarized normal fibers. However, fructose-1,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced bottleneck between the latter two substrates. We measured the intrinsic buffering power, beta, of frog semitendinosus muscle with small pulses of NH4Cl. It was found to vary with pHi according to beta = 144.6 - 17.2 (pHi).
我们使用微电极研究了去极化对正常（细胞内pH值约为7.37）和酸化（细胞内pH值为5.90 - 6.70）青蛙半腱肌细胞内pH值（pHi）的影响。一小束肌肉用不含钠的缓冲溶液（10 mM HEPES，100% O₂，pH 7.35）进行灌流，该溶液含有2.5或25 mM的钾离子。用氯化铵预脉冲来降低细胞内pH值。在正常细胞内pH值时，去极化通常会产生轻微的（0.04）碱化，随后细胞内pH值下降约0.2。相比之下，在所有25个酸化的肌束中，细胞内pH值上升了0.1 - 0.7。初始细胞内pH值越低，上升幅度越大。它可以被咖啡因模拟，并被丁卡因阻断，因此很可能是由钙的释放引发的。我们将碱化归因于磷酸肌酸（PCr）的水解；2,4 - 二硝基氟苯消除了这种碱化。在碱化峰值时对纤维进行生化分析表明，磷酸肌酸减少了一半，而在相同时间段（4 - 6分钟）去极化的正常纤维中的磷酸肌酸没有变化。我们推测，低细胞内pH值通过减少糖原分解，特别是通过抑制磷酸果糖激酶（PFK）来减缓糖酵解及其相关的ATP形成，已知该酶对pH高度敏感。因此，需要磷酸肌酸水解来补充大量水解的ATP。这种对磷酸果糖激酶的推测作用与以下发现一致：在去极化的酸性纤维中，葡萄糖 - 6 - 磷酸（与果糖 - 6 - 磷酸接近平衡）增加了近五倍，但在去极化的正常纤维中没有增加。然而，果糖 - 1,6 - 二磷酸也显著增加；3 - 磷酸甘油酸不受影响。这表明在这两种底物之间存在额外的酸诱导瓶颈。我们用氯化铵小脉冲测量了青蛙半腱肌的固有缓冲能力β。发现它根据β = 144.6 - 17.2（pHi）随细胞内pH值变化。
