Watts B A, Good D W
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555-0562.
J Gen Physiol. 1994 May;103(5):917-36. doi: 10.1085/jgp.103.5.917.
The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium-sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%.
(a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)-NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.
肾髓质厚升支（MTAL）可主动重吸收铵离子。为研究NH4+转运对细胞内pH（pHi）的影响以及顶端膜NH4+转运的机制，分离大鼠的MTAL并在体外用25 mM HCO3(-)缓冲溶液（pH 7.4）进行灌注。使用荧光染料BCECF监测pHi。在无NH4+时，平均pHi为7.16。向管腔中加入20 mM NH4+导致细胞内迅速酸化（dpHi/dt = 11.1 U/分钟），并使稳态pHi降至6.67（ΔpHi = 0.5 U），表明顶端NH4+的进入比NH3的进入更快。管腔中的呋塞米（10(-4) M）使细胞酸化的初始速率降低70%，稳态pHi的下降降低35%。呋塞米存在时观察到的残余酸化被管腔中的钡（12 mM）抑制，表明顶端NH4+的进入通过呋塞米（Na(+)-NH4(+)-2Cl-共转运）和钡敏感途径发生。在对称铵条件下评估了这些途径在NH4+吸收中的作用。当灌注液和浴液中均含有4 mM NH4+时，平均稳态pHi为6.61，净铵吸收为12 pmol/分钟/毫米。向管腔中加入呋塞米消除了净铵吸收，并使pHi突然升高（dpHi/dt = 0.8 U/分钟）至7.0。将管腔中的[K+]从4 mM增加到25 mM导致类似的快速细胞碱化。在无NH4+时未观察到呋塞米或增加[K+]时明显的细胞碱化。在对称的4 mM NH4+溶液中，向管腔中加入钡导致细胞内缓慢碱化，仅使净铵吸收降低14%。
（a）铵转运是MTAL中pHi的关键决定因素，NH4+吸收使细胞明显酸化，而抑制顶端NH4+摄取的操作（呋塞米或管腔[K+]升高）导致细胞内碱化；（b）大多数或所有跨细胞铵吸收由顶端膜Na(+)-NH4(+)-2Cl-共转运介导；（c）NH4+也可通过钡敏感的顶端膜转运途径（可能是顶端膜K+通道）渗透，但在生理（对称铵）条件下该途径对铵吸收的贡献不大。
