Bidet M, Tauc M, Merot J, Vandewalle A, Poujeol P
Institut National de la Santé et de la Recherche Médicale, Unité 246, Centre d'Etudes Nucléaires de Saclay, Gif-Sur-Yvette, France.
Am J Physiol. 1987 Nov;253(5 Pt 2):F935-44. doi: 10.1152/ajprenal.1987.253.5.F935.
The purpose of this study was to investigate the characteristics of the Na+-H+ exchange in isolated proximal cells from rabbit kidney cortex. The cells were prepared by mechanical dissociation and sequential passages through nylon meshes. The intracellular pH (pHi) was measured in a bicarbonate-free medium [extracellular pH (pHe) = 7.30], using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Resting pHi was 7.13 +/- 0.04 (n = 11) at 20-22 degrees C. Cells were acid loaded with nigericin in choline solution and H+ efflux, induced by extracellular Na+ (Nae), was calculated using a buffering power of 23.6 +/- 0.6 mmol.1-1.pH unit-1 (n = 19) estimated by NH4Cl exposure. In isolated proximal cells, the Na+-H+ antiporter had an apparent Km for Nae of 86.7 +/- 1.5 mM (n = 4) and was competitively inhibited by amiloride with a Ki of 33.3 +/- 6.4 X 10(-6) M (n = 3). Lowering pHe, inhibited the Na+-H+ exchanger. This inhibition was not purely competitive and the Ki was 40.4 +/- 12.7 nM (n = 3). The Na+-H+ exchange was greatly activated when the cytoplasm was acidified. The intracellular H+ concentration dependence did not follow simple Michaelis-Menten kinetics. Of the different cations tested on pHi recovery, such as Li+, choline+, K+, and tetramethylammonium, only Li+ induced an alkalinization of acidified cells similar to that of Na+. 22Na influx measurements indicated that cellular depletion of Na+ stimulated Na+-H+ exchange. The results permit the conclusion that the isolation procedures did not impair the main features of the Na+-H+ antiporter, at least as compared with those previously described in renal brush-border membrane vesicles or in other cellular systems. The integrity of the transporter in isolated proximal cells would permit the direct study of its hormonal and metabolic control.
本研究的目的是调查兔肾皮质分离的近端细胞中Na⁺-H⁺交换的特征。细胞通过机械解离并依次通过尼龙网制备。使用荧光染料2,7-双（羧乙基）-5（6）-羧基荧光素（BCECF）在无碳酸氢盐培养基[细胞外pH（pHe）= 7.30]中测量细胞内pH（pHi）。在20 - 22℃时，静息pHi为7.13±0.04（n = 11）。细胞在胆碱溶液中用尼日利亚菌素进行酸负荷处理，由细胞外Na⁺（Nae）诱导的H⁺外流通过NH₄Cl暴露估计的23.6±0.6 mmol·L⁻¹·pH单位⁻¹的缓冲能力来计算（n = 19）。在分离的近端细胞中，Na⁺-H⁺反向转运体对Nae的表观Km为86.7±1.5 mM（n = 4），并被氨氯地平竞争性抑制，Ki为33.3±6.4×10⁻⁶ M（n = 3）。降低pHe会抑制Na⁺-H⁺交换体。这种抑制并非纯粹竞争性的，Ki为40.4±12.7 nM（n = 3）。当细胞质酸化时，Na⁺-H⁺交换被极大激活。细胞内H⁺浓度依赖性不遵循简单的米氏动力学。在测试的不同阳离子如Li⁺、胆碱⁺、K⁺和四甲基铵对pHi恢复的影响中，只有Li⁺能诱导酸化细胞碱化，类似于Na⁺的作用。²²Na内流测量表明细胞内Na⁺耗竭会刺激Na⁺-H⁺交换。结果得出结论，至少与先前在肾刷状缘膜囊泡或其他细胞系统中描述的相比，分离程序并未损害Na⁺-H⁺反向转运体的主要特征。分离的近端细胞中转运体的完整性将允许对其激素和代谢控制进行直接研究。
