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MUCOSAL BIOLOGY
Departments of 1Molecular and Cellular Physiology and 3Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 2Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana
Submitted 22 July 2005 ; accepted in final form 9 May 2006
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 (pHi) 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 µM 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 µM), 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 µM), 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 pHi 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.
Slc9a2; Slc9a3; BCECF; SNARF-5F; intracellular pH; laser scanning confocal microscopy; colon; Na+/H+ exchanger
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