Kikeri D, Zeidel M L
Harvard Center for the Study of Kidney Diseases, Harvard Medical School, Boston, MA.
J Am Soc Nephrol. 1990 Dec;1(6):890-901. doi: 10.1681/ASN.V16890.
To define proton transport mechanisms involved in the regulation of intracellular pH (pHi) in cells of the inner medullary collecting duct (IMCD), pHi and cell membrane potential were estimated by using the fluorescent dyes 2,7-biscarboxyethyl-5(6)-carboxyfluorescein and 3,3'-dipropylthiadicarbocyanine iodide, respectively, in suspensions of freshly isolated rabbit IMCD cells. The resting pHi of IMCD cells in nonbicarbonate Ringer's solution (pH 7.4) was 7.21 +/- 0.03 (mean +/- SE). When cells were acidified by ammonium withdrawal, the initial pHi recovery rate was 0.33 +/- 0.02 pH unit/min; replacement of extracellular Na+ (130 mM) with N-methyl-D-glucamine+ reduced the pHi recovery rate to 0.08 +/- 0.02 pH unit/min, while addition of 0.1 mM amiloride in the presence of extracellular Na+ reduced the rate of pHi recovery to 0.02 +/- 0.02 pH unit/min. Similar results were obtained in cells acid loaded with HCl. Cells recovering from acidification exhibited 22Na+ uptake rates threefold higher than did nonacidified cells. The rate of Na(+)-dependent pHi recovery was independent of the cell membrane potential. In the absence of extracellular Na+, depolarizing cell membrane potential in a stepwise manner by increasing extracellular K+ concentrations from 1 to 130 mM resulted in graded increments in the rate of pHi recovery. In the presence of 130 mM K+, the pHi recovery rate in acidified cells was dependent on cellular ATP levels, sensitive to 1 mM N-ethylmaleimide, and insensitive to 0.01 mM oligomycin in the presence of glucose (control, 0.24 +/- 0.01; ATP-depleted, 0.13 +/- 0.02; addition of N-ethylmaleimide, 0.16 +/- 0.01; addition of oligomycin, 0.27 +/- 0.02 pH unit/min). ATP depletion markedly inhibited H+ extrusion from IMCD cells measured by using a pH stat. These results provide direct evidence in freshly isolated IMCD cells that both a Na+:H+ antiporter and a rheogenic H(+)-ATPase participate in pHi regulation.
为了确定参与调节髓质内集合管（IMCD）细胞内pH（pHi）的质子转运机制，分别使用荧光染料2,7-双（羧乙基）-5（6）-羧基荧光素和3,3'-二丙基硫代二碳菁碘化物，对新鲜分离的兔IMCD细胞悬液中的pHi和细胞膜电位进行了估计。在无碳酸氢盐的林格氏溶液（pH 7.4）中，IMCD细胞的静息pHi为7.21±0.03（平均值±标准误）。当细胞因铵去除而酸化时，初始pHi恢复率为0.33±0.02 pH单位/分钟；用N-甲基-D-葡糖胺+替代细胞外Na+（130 mM）可将pHi恢复率降低至0.08±0.02 pH单位/分钟，而在细胞外Na+存在的情况下添加0.1 mM氨氯吡咪可将pHi恢复率降低至0.02±0.02 pH单位/分钟。在用HCl酸负荷的细胞中也获得了类似的结果。从酸化中恢复的细胞表现出的22Na+摄取率比未酸化的细胞高两倍。Na+依赖性pHi恢复率与细胞膜电位无关。在无细胞外Na+的情况下，通过将细胞外K+浓度从1 mM逐步增加到130 mM来逐步使细胞膜电位去极化，导致pHi恢复率逐渐增加。在130 mM K+存在的情况下，酸化细胞中的pHi恢复率取决于细胞内ATP水平，对1 mM N-乙基马来酰亚胺敏感，在有葡萄糖存在的情况下对0.01 mM寡霉素不敏感（对照，0.24±0.01；ATP耗尽，0.13±0.02；添加N-乙基马来酰亚胺，0.16±0.01；添加寡霉素，0.27±0.02 pH单位/分钟）。ATP耗尽显著抑制了用pH计测量的IMCD细胞中H+的排出。这些结果在新鲜分离的IMCD细胞中提供了直接证据，表明Na+:H+反向转运体和生电H(+)-ATP酶都参与了pHi调节。
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