Weissberg P L, Little P J, Cragoe E J, Bobik A
Baker Medical Research Institute, Melbourne, Australia.
Circ Res. 1989 Apr;64(4):676-85. doi: 10.1161/01.res.64.4.676.
We investigated the mechanisms by which spontaneously beating cultured rat ventricular cells regulate intracellular pH (pHi). Specifically, the relative contributions of the Na+/H+ antiport, Cl-/HCO3- exchange, ATP, and calmodulin-dependent processes in regulating the pHi of cells loaded with the intracellular fluorescent pH indicator BCECF were investigated. The pHi of ventricular cells bathed in HEPES-buffered medium averaged 7.30 +/- 0.02. Subsequent exposure of the cells to CO2-HCO3- -buffered medium resulted in intracellular acidification followed by recovery to pHi levels approximately 0.1 pH units lower than in controls. Recovery was inhibited by the Na+/H+ antiport inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The recovery from intracellular acidification, induced by a 15-mM ammonium chloride prepulse, was also dependent solely upon activation of the Na+/H+ antiport. Recovery was dependent upon extracellular sodium, was completely inhibited by EIPA, and could be modulated by changes in extracellular pH (pHo). At low pHo values (6.3) the recovery of pHi was greatly attenuated, while at high pHo (8.0) the recovery process was accelerated. The final pHi to which the cells recovered was also dependent upon pHo. Preincubation of the cells with 2-deoxy-D-glucose to deplete cellular ATP levels reduced pHi by approximately 0.2 pH units and greatly impaired the cells' ability to recover from 15-mM ammonium chloride-induced acid load. Similarly, preincubation of cells with the calmodulin inhibitors W-7 and trifluoperazine also impaired their ability to recover from the acid load. The Cl- -HCO3- exchange played no role in the cells' ability to recover from intracellular acidosis. However, the presence of HCO3- significantly increased the resistance of myocardial cells to changes in pHi by approximately doubling their buffer capacity. These results demonstrated that a Na+/H+ antiport is the major pHi-regulating system in spontaneously beating rat ventricular cells. The ability of the Na+/H+ antiport to regulate myocardial pHi is dependent upon the cells' ability to maintain adequate levels of ATP. The antiport's dependency on ATP, in conjunction with its dependency on calmodulin, suggests that activation of the antiport in ventricular cells involves phosphorylation processes.
我们研究了自发性搏动的培养大鼠心室细胞调节细胞内pH值（pHi）的机制。具体而言，研究了Na+/H+逆向转运体、Cl-/HCO3-交换体、ATP以及钙调蛋白依赖性过程在调节加载了细胞内荧光pH指示剂BCECF的细胞pHi中的相对贡献。置于HEPES缓冲培养基中的心室细胞的pHi平均为7.30±0.02。随后将细胞暴露于CO2-HCO3-缓冲培养基中导致细胞内酸化，随后恢复到比对照低约0.1个pH单位的pHi水平。Na+/H+逆向转运体抑制剂5-(N-乙基-N-异丙基)amiloride（EIPA）抑制了恢复过程。由15 mM氯化铵预脉冲诱导的细胞内酸化的恢复也仅依赖于Na+/H+逆向转运体的激活。恢复依赖于细胞外钠，被EIPA完全抑制，并且可以通过细胞外pH值（pHo）的变化进行调节。在低pHo值（6.3）时，pHi的恢复大大减弱，而在高pHo（8.0）时，恢复过程加速。细胞恢复到的最终pHi也依赖于pHo。用2-脱氧-D-葡萄糖预孵育细胞以耗尽细胞ATP水平，使pHi降低约0.2个pH单位，并大大损害了细胞从15 mM氯化铵诱导的酸负荷中恢复的能力。同样，用钙调蛋白抑制剂W-7和三氟拉嗪预孵育细胞也损害了它们从酸负荷中恢复的能力。Cl--HCO3-交换在细胞从细胞内酸中毒中恢复的能力中不起作用。然而，HCO3-的存在通过使心肌细胞的缓冲能力增加约一倍，显著提高了心肌细胞对pHi变化的抵抗力。这些结果表明，Na+/H+逆向转运体是自发性搏动的大鼠心室细胞中主要的pHi调节系统。Na+/H+逆向转运体调节心肌pHi的能力依赖于细胞维持足够ATP水平的能力。逆向转运体对ATP的依赖性，连同其对钙调蛋白的依赖性，表明心室细胞中逆向转运体的激活涉及磷酸化过程。
