Adler S, Shoubridge E, Radda G K
Am J Physiol. 1984 Sep;247(3 Pt 1):C188-96. doi: 10.1152/ajpcell.1984.247.3.C188.
To determine intracellular pH gradients rabbit renal cortical tubular cells were prepared by collagenase separation, suspended in a Krebs-Ringer buffer solution, and gassed with 95% O2-5% CO2 in a special nuclear magnetic resonance (NMR) probe. Renal tubular cellular pH was determined simultaneously from the distribution of 14C-dimethadione (DMO) (pHDMO) or the chemical shift of inorganic phosphate (pHNMR). Experiments were performed at different external pH values (pHe) ranging between 6.52 and 7.20. pHNMR, a measure of cytoplasmic pH, changed by an amount equal to the change in pHe. pHDMO, however, a measure of cytoplasmic plus mitochondrial pH, changed less than pHe as the latter increased. pHDMO, higher than pHNMR at low pHe, became equal to pHNMR at higher pHe values. By use of assumed mitochondrial volumes of 30-40% mitochondrial pH was calculated from pHDMO and pHNMR. Mitochondrial pH remained relatively constant over the entire pHe range studied. Since cytoplasmic pH fell as pHe was lowered, the transmitochondrial pH gradient increased at low pHE values. These findings suggest that the transmitochondrial pH gradient may be important in regulating metabolism.
为了测定细胞内pH梯度，通过胶原酶分离制备兔肾皮质肾小管细胞，将其悬浮于Krebs-Ringer缓冲溶液中，并在特殊的核磁共振（NMR）探头中用95% O₂-5% CO₂进行通气。肾小管细胞内pH通过¹⁴C-二甲双酮（DMO）的分布（pHDMO）或无机磷酸盐的化学位移（pHNMR）同时测定。实验在6.52至7.20范围内的不同外部pH值（pHe）下进行。pHNMR作为细胞质pH的一种测量方法，其变化量与pHe的变化量相等。然而，pHDMO作为细胞质加线粒体pH的一种测量方法，随着pHe升高，其变化小于pHe。在低pHe时，pHDMO高于pHNMR，在较高pHe值时，pHDMO与pHNMR相等。利用假定的线粒体体积为30%-40%，从pHDMO和pHNMR计算出线粒体pH。在所研究的整个pHe范围内，线粒体pH保持相对恒定。由于随着pHe降低细胞质pH下降，在低pHe值时跨线粒体pH梯度增加。这些发现表明跨线粒体pH梯度可能在调节代谢中起重要作用。
