NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice
NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice
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DOI：
10.1152/ajpgi.00342.2005
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发表时间：
2006-10-01
影响因子：
4.5
通讯作者：
Montrose, Marshall H.
中科院分区：
文献类型：
--
作者：
Guan, Yanfang;Dong, Jin;Montrose, Marshall H.
结肠隐窝顶端Na~+依赖性H~+排出的机制尚有争议。用共聚焦显微镜对装有BCECF或SNARF-5F(荧光pH传感器)的活体小鼠远端结肠进行了细胞内pH(pH(I))的测量。在细胞酸化后，在野生型或Na+/H+交换异构体2(NHE2)缺失小鼠的远端结肠隐窝底部加入鲁米那Na+，可刺激相似的phi恢复率。在野生型隐窝中，20 mM的HOE694(NH_2抑制剂)阻断了68-75%的PHI回收率，而NH_2阴性的隐窝对HOE694、NHE3特异性抑制剂S-1611(20 MM)或重碳酸盐转运抑制剂4-acetamido-4‘-isothiocyanostilbene-2，2’-disulfonic酸(1 MM)不敏感。一种通用的NHE抑制剂，5-(N-乙基-N-异丙基)阿米洛利(EIPA；20MU M)能抑制NHE2缺失型小鼠的PHI恢复(46%)，但不如野生型小鼠(74%)，这表明EIPA敏感和不敏感的代偿机制。跨上皮Na+渗漏和基底外侧NHE1的激活可以混淆结果；然而，依赖Na+的phi恢复率与跨上皮对荧光黄的泄漏无关，在NHE1缺失的小鼠中没有变化。NHE2免疫定位于野生型隐窝的顶膜，而不是NHE2缺失的组织。NHE2基因敲除小鼠的NHE3免疫反应位于结肠表面附近，但不在隐窝底部。野生型小鼠的结肠表面细胞对鲁米那钠的反应显示对S1611和HOE694敏感的pH(I)恢复，证实了NHE3和NHE2在该位点的功能作用。我们的结论是，结构性缺乏NHE2会导致有别于NHE1、NHE3或SIT敏感转运蛋白的Na+依赖的、EIPA敏感的酸挤出器的补偿性增加。
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 pHi 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 mu M HOE694 (NHE2 inhibitor) blocked 68-75% of the pHi recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 mu M), 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 mu M), inhibited pHi 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 pHi 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.