Department of Physiology, Li Ka Shing Faculty of Medicine and Institute of Cardiovascular Science and Medicine, University of Hong Kong, Pokfulam, Hong Kong SAR, China.
J Physiol. 2010 Nov 15;588(Pt 22):4563-78. doi: 10.1113/jphysiol.2010.195255. Epub 2010 Sep 6.
The present study was performed to investigate the effect of acidosis on the efflux of ATP from skeletal muscle. Infusion of lactic acid to the perfused hindlimb muscles of anaesthetised rats produced dose-dependent decreases in pH and increases in the interstitial ATP of extensor digitorum longus (EDL) muscle: 10 mM lactic acid reduced the venous pH from 7.22 ± 0.04 to 6.97 ± 0.02 and increased interstitial ATP from 38 ± 8 to 67 ± 11 nM. The increase in interstitial ATP was well-correlated with the decrease in pH (r(2) = 0.93; P < 0.05). Blockade of cellular uptake of lactic acid using α-cyano-hydroxycinnamic acid abolished the lactic acid-induced ATP release, whilst infusion of sodium lactate failed to depress pH or increase interstitial ATP, suggesting that intracellular pH depression, rather than lactate, stimulated the ATP efflux. Incubation of cultured skeletal myoblasts with 10 mM lactic acid significantly increased the accumulation of ATP in the bathing medium from 0.46 ± 0.06 to 0.76 ± 0.08 μM, confirming the skeletal muscle cells as the source of the released ATP. Acidosis-induced ATP efflux from the perfused muscle was abolished by CFTR(inh)-172, a specific inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), or glibenclamide, an inhibitor of both K(ATP) channels and CFTR, but it was not affected by atractyloside, an inhibitor of the mitochondrial ATP transporter. Silencing of the CFTR gene using an siRNA abolished the acidosis-induced increase in ATP release from cultured myoblasts. CFTR expression on skeletal muscle cells was confirmed using immunostaining in the intact muscle and Western blotting in the cultured cells. These data suggest that depression of the intracellular pH of skeletal muscle cells stimulates ATP efflux, and that CFTR plays an important role in the release mechanism.
本研究旨在探讨酸中毒对骨骼肌中 ATP 外排的影响。向麻醉大鼠的灌流后肢肌肉输注乳酸会导致 pH 值呈剂量依赖性降低，伸趾长肌（EDL）肌肉的间质 ATP 增加：10 mM 乳酸将静脉 pH 值从 7.22±0.04 降低至 6.97±0.02，并将间质 ATP 从 38±8 增加至 67±11 nM。间质 ATP 的增加与 pH 值的降低密切相关（r²=0.93；P<0.05）。使用α-氰基-羟基肉桂酸阻断细胞对乳酸的摄取会消除乳酸引起的 ATP 释放，而输注乳酸钠未能降低 pH 值或增加间质 ATP，表明细胞内 pH 值降低而不是乳酸刺激了 ATP 外排。用 10 mM 乳酸孵育培养的骨骼肌成肌细胞会显著增加在浴液中积累的 ATP，从 0.46±0.06 增加至 0.76±0.08 μM，证实骨骼肌细胞是释放的 ATP 的来源。CFTR（inh）-172，囊性纤维化跨膜电导调节剂（CFTR）的特异性抑制剂，或格列本脲，K（ATP）通道和 CFTR 的抑制剂，均可消除灌流肌肉中酸中毒诱导的 ATP 外排，而阿曲霉素，一种线粒体 ATP 转运体的抑制剂，对其没有影响。使用 siRNA 沉默 CFTR 基因可消除培养的成肌细胞中酸中毒引起的 ATP 释放增加。在完整肌肉中使用免疫染色和在培养细胞中使用 Western blot 证实了骨骼肌细胞上的 CFTR 表达。这些数据表明，骨骼肌细胞细胞内 pH 值的降低会刺激 ATP 外排，而 CFTR 在释放机制中发挥重要作用。
