Guan Yanfang, Dong Jin, Tackett Lixuan, Meyer Jamie W, Shull Gary E, Montrose Marshall H
Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Medical Sciences Bldg., Cincinnati, OH 45267-0576, USA.
Am J Physiol Gastrointest Liver Physiol. 2006 Oct;291(4):G689-99. doi: 10.1152/ajpgi.00342.2005. Epub 2006 May 11.
The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.
结肠隐窝顶端依赖钠的氢离子排出机制存在争议。通过对装载有BCECF或SNARF-5F（荧光pH传感器）的活体小鼠远端结肠进行共聚焦显微镜观察，报告了隐窝底部或结肠表面上皮细胞内pH值（pH(i)）的测量结果。细胞酸化后，在野生型或钠/氢交换体亚型2（NHE2）基因敲除小鼠的远端结肠隐窝底部细胞中，添加管腔钠刺激了相似的pH(i)恢复速率。在野生型隐窝中，20微摩尔HOE694（NHE2抑制剂）阻断了68 - 75%的pH(i)恢复速率，而NHE2基因敲除的隐窝对HOE694、NHE3特异性抑制剂S-1611（20微摩尔）或碳酸氢盐转运抑制剂4-乙酰氨基-4'-异硫氰酸基芪-2,2'-二磺酸（SITS；1毫摩尔）不敏感。一种通用的NHE抑制剂5-(N-乙基-N-异丙基)氨氯吡脒（EIPA；20微摩尔）抑制了NHE2基因敲除小鼠中pH(i)的恢复（46%），但比野生型小鼠（74%）的抑制作用弱，提示存在EIPA敏感和不敏感的代偿机制。跨上皮钠泄漏随后激活基底外侧NHE1可能会混淆结果；然而，依赖钠的pH(i)恢复速率与跨上皮对荧光素黄的通透性无关，并在NHE1基因敲除小鼠中未发生改变。NHE2免疫定位在野生型隐窝的顶端膜上，但不在NHE2基因敲除组织中。在NHE2基因敲除小鼠中，NHE3免疫反应性在结肠表面附近但不在隐窝底部。野生型小鼠的结肠表面细胞对管腔钠的反应表现出对S1611和HOE694敏感的pH(i)恢复，证实了NHE3和NHE2在此部位的功能作用。我们得出结论，NHE2的组成性缺失导致了一种与NHE1、NHE3或SITS敏感转运体不同的、依赖钠的、EIPA敏感的酸排出体的代偿性增加。
