IDENTIFICATION OF NA+/H+ EXCHANGE ON THE APICAL SIDE OF SURFACE COLONOCYTES USING BCECF

Colonocytes must regulate intracellular pH (pH(i)) while they transport H+ and HCO3-. To investigate the membrane transport processes involved in pH(i) regulation, colonocyte pH(i) was measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of rat distal colon mounted on a holder that fits into a standard fluorometer cuvette and allows independent superfusion of mucosal and serosal surfaces. When BCECF-acetoxymethyl ester was in the mucosal solution only, BCECF loaded surface colonocytes with a high degree of selectivity. In HEPES-buffered solutions, basal pH(i) was 7.31 +/- 0.01 (n = 68), and pH(i) was dependent on extracellular Na+. Cells acidified in Na+-free solution, and pH(i) rapidly corrected when Na+ was returned. pH(i) recovered at 0.22 +/- 0.01 pH/min (n = 6) when Na+ was introduced into the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Na+ was absent from the mucosal solution. The presence or absence of Na+ in the serosal solution did not affect pH(i). This indicated that the Na+ dependent pH(i) recovery process is located in the apical cell membrane, but not in the basolateral membrane. Because amiloride (1 mM) inhibited Na+-dependent pH(i) recovery by 75%, Na+/H+ exchange appears to be present in the apical membrane. Because Na+-independent pH(i) recovery was not affected by K+-free media, 50 mu M SCH-28080, 100 nM bafilomycin A(1), or Cl--free media, this transport mechanism does not involve a gastriclike H+-K+-ATPase, a vacuolar H+ ATPase, or a Cl-/base exchanger. In summary, pH(i) was selectively measured in surface colonocytes by this technique. In these cells, the Na+/H+ exchange activity involved in pH(i) regulation was detected in the apical membrane, but not in the basolateral membrane.