Graber M, Pastoriza-Munoz E
Veterans Affairs Medical Center, Northport 11768.
Am J Physiol. 1993 Dec;265(6 Pt 2):F773-83. doi: 10.1152/ajprenal.1993.265.6.F773.
Acid-loaded opossum kidney (OK) cells secrete H+ by Na+/H+ exchange and by a Na(+)- and HCO3(-)-independent pathway that has not been fully characterized. We studied the Na(+)-independent component by measuring H+ flux using the pH-sensitive trapped indicator 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein. Two Na(+)-independent H(+)-transport systems were identified in acid-loaded cells perfused with HCO3(-)-free buffers. The minor component appears to be a conductive pathway for H+, over 90% inhibitable by 5 mM barium. The major component is stimulated by extracellular K+ and was fully active in the presence of barium, amiloride, ouabain, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and bumetanide and in the absence of Cl-. Ammonium inhibited the H+ flux by 72% at 50 mM, and the H+ flux could be accelerated two- to threefold by limited proteolysis of intact cells using kallikrein or papain. In cells pretreated with barium, the K(+)-induced H+ flux caused no change of bis-oxonol fluorescence, suggesting an electroneutral pathway. The H+ flux was a saturable function of extracellular K+ (Michaelis constant 55 mM), and flux reversed when the K+ gradient was reversed. Similarly, the H+ flux was a linear function of the H+ gradient and reversed when the H+ gradient reversed. Evidence for ongoing K(+)-induced H+ flux was also found in nonacidified cells. First, changing perfusate K+ from 5 to 50 mM alkalinized baseline cell pH, an effect not reproduced by barium despite an equivalent depolarizing effect. Second, increasing perfusate K+ from 5 to 50 mM completely eliminated the acidification produced by 1 mM amiloride. We conclude that the OK cell expresses two Na(+)-independent acid-base transport systems. One is a barium-sensitive electrogenic H+ conductance and the other functions as an electroneutral K+/H+ antiporter. The antiporter is capable of H+ extrusion from acid-loaded cells but in normal cells functions in the reverse direction, as an H+ loader. The K+/H+ antiporter appears to be one of the major systems regulation cell pH in these cells, balancing the H+ efflux mediated by Na+/H+ exchange.
酸负荷的负鼠肾（OK）细胞通过Na⁺/H⁺交换和一条尚未完全明确的不依赖Na⁺和HCO₃⁻的途径分泌H⁺。我们使用pH敏感的捕获指示剂2',7'-双（2-羧乙基）-5（6）-羧基荧光素测量H⁺通量，研究了不依赖Na⁺的成分。在用不含HCO₃⁻的缓冲液灌注的酸负荷细胞中鉴定出两种不依赖Na⁺的H⁺转运系统。次要成分似乎是H⁺的传导途径，5 mM钡可抑制超过90%。主要成分受细胞外K⁺刺激，在钡、氨氯吡脒、哇巴因、4,4'-二异硫氰基芪-2,2'-二磺酸和布美他尼存在以及无Cl⁻的情况下仍完全活跃。50 mM时，铵抑制H⁺通量72%，使用激肽释放酶或木瓜蛋白酶对完整细胞进行有限的蛋白水解可使H⁺通量加速两到三倍。在用钡预处理的细胞中，K⁺诱导的H⁺通量未引起双羟萘酚荧光变化，提示为电中性途径。H⁺通量是细胞外K⁺的饱和函数（米氏常数55 mM），当K⁺梯度反转时通量也反转。同样，H⁺通量是H⁺梯度的线性函数，当H⁺梯度反转时通量也反转。在未酸化的细胞中也发现了持续的K⁺诱导H⁺通量的证据。首先，将灌注液K⁺从5 mM变为50 mM可使基线细胞pH碱化，尽管钡有等效的去极化作用，但该效应未被钡重现。其次，将灌注液K⁺从5 mM增加到50 mM可完全消除1 mM氨氯吡脒产生的酸化。我们得出结论，OK细胞表达两种不依赖Na⁺的酸碱转运系统。一种是对钡敏感的生电性H⁺电导，另一种作为电中性K⁺/H⁺反向转运体起作用。反向转运体能够将酸负荷细胞中的H⁺挤出，但在正常细胞中以相反方向起作用，作为H⁺装载器。K⁺/H⁺反向转运体似乎是这些细胞中调节细胞pH的主要系统之一，平衡由Na⁺/H⁺交换介导的H⁺外流。
