Ellis D, MacLeod K T
J Physiol. 1985 Feb;359:81-105. doi: 10.1113/jphysiol.1985.sp015576.
Intracellular pH (pHi) of Purkinje fibres from sheep heart was recorded with pH-sensitive glass micro-electrodes. The cells were acidified by one of three methods: (1) exposure to and subsequent removal of NH4Cl, (2) exposure to solutions containing 5% CO2 or (3) exposure to an acidic Tyrode solution. The pHi recovery from these acidifications was studied. The time constant of recovery from an acidification induced by NH4Cl was almost twice as long as that from one induced by CO2 or acid extracellular pH. Following an acidification induced by exposure to CO2 the time constant of pHi recovery was not changed when the cell was depolarized to -40 mV (by replacement of some Na+ by K+). An intracellular acidification was produced when extracellular Na+ was removed and replaced by quaternary ammonium ions or K+. Such Na+-free solutions also inhibited pHi recovery from an acidification. A 50% inhibition of the rate of recovery was produced by lowering the [Na+]o to 8 mM. When used as a Na+ substitute, Li+ could permit recovery. Tris (22 mM) changed pHi in the alkaline direction. Amiloride (1 mM) or a decrease in temperature slowed the recovery from an acidification (Q10 = 2.65). There was no effect of SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid disodium salt; 100 microM) on the recovery. Na+-sensitive glass micro-electrodes were used to measure the intracellular Na+ activity when [Na+]o was lowered to levels used in our pHi recovery experiments. From these data we have calculated the apparent Na+ electrochemical gradient at different values of [Na+]o. If this gradient is responsible for H+ efflux from the cell then, by applying thermodynamic considerations, it can be shown that only low concentrations (1-2mM) of extracellular Na+ are required. Solutions containing a very low [Ca2+]o (less than 10(-8) M, buffered with EGTA) were used to prevent large rises of [Ca2+]i which may occur on removal of external Na+. Under these conditions pHi recovery is still dependent upon [Na+]o, and the apparent inhibition of pHi recovery by removal of Na+ is not simply due to rises in [Ca2+]i. The intracellular acidification which occurs on removal of Na+ does not occur when [Ca2+]o is very low (less than 10(-8) M).(ABSTRACT TRUNCATED AT 400 WORDS)
用对pH敏感的玻璃微电极记录绵羊心脏浦肯野纤维的细胞内pH（pHi）。细胞通过以下三种方法之一进行酸化：（1）暴露于NH4Cl并随后去除；（2）暴露于含5%CO2的溶液；（3）暴露于酸性台氏液。研究了这些酸化后pHi的恢复情况。由NH4Cl诱导的酸化恢复的时间常数几乎是由CO2或酸性细胞外pH诱导的酸化恢复时间常数的两倍。在暴露于CO2诱导酸化后，当细胞去极化至-40mV（通过用K+替代一些Na+）时，pHi恢复的时间常数不变。当细胞外Na+被去除并用季铵离子或K+替代时，会产生细胞内酸化。这种无Na+溶液也抑制了酸化后pHi的恢复。将细胞外[Na+]o降至8mM可使恢复速率受到50%的抑制。当用作Na+替代物时，Li+可允许恢复。Tris（22mM）使pHi向碱性方向变化。氨氯吡咪（1mM）或温度降低会减慢酸化后的恢复（Q10 = 2.65）。4-乙酰氨基-4'-异硫氰酸基芪-2,2'-二磺酸二钠盐（SITS；100μM）对恢复没有影响。当细胞外[Na+]o降至我们pHi恢复实验中所用水平时，用对Na+敏感的玻璃微电极测量细胞内Na+活性。根据这些数据，我们计算了不同细胞外[Na+]o值下的表观Na+电化学梯度。如果这个梯度负责细胞内H+外流，那么通过应用热力学原理可以表明，仅需要低浓度（1-2mM）的细胞外Na+。使用含有极低细胞外[Ca2+]o（小于10(-8)M，用EGTA缓冲）的溶液来防止去除细胞外Na+时可能发生的细胞内[Ca2+]i大幅升高。在这些条件下，pHi恢复仍然依赖于细胞外[Na+]o，并且去除Na+对pHi恢复的明显抑制并非仅仅由于细胞内[Ca2+]i升高。当细胞外[Ca2+]o非常低（小于10(-8)M）时，去除Na+时发生的细胞内酸化不会出现。（摘要截于4
