NHE2 is the main apical NHE in mouse colonic crypts but an alternative [Na.sup.+]-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical [Na.sup.+]-dependent [H.sup.+] 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.sub.i]) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal [Na.sup.+] stimulated similar rates of [pH.sub.i] recovery in cells at the base of distal colonic crypts of wild-type or [Na.sup.+]/[H.sup.+] exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 [micro]M HOE694 (NHE2 inhibitor) blocked 68-75% of the [pH.sub.i] recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 [micro]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 [micro]M), inhibited [pH.sub.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.sup.+] leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of [Na.sup.+]-dependent [pH.sub.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.sub.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.sup.+]-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters. S1c9a2; S1c9a3; BCECF; SNARF-5F; intracellular pH; laser scanning confocal microscopy; colon; [Na.sup.+]/[H.sup.+] exchanger