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

Colonocytes must regulate intracellular pH (pH(i)) while they transpor t H+ and HCO3-. To investigate the membrane transport processes involv ed in pH(i) regulation, colonocyte pH(i) was measured with 2',7'-bis(2 -carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of r at distal colon mounted on a holder that fits into a standard fluorome ter cuvette and allows independent superfusion of mucosal and serosal surfaces. When BCECF-acetoxymethyl ester was in the mucosal solution o nly, BCECF loaded surface colonocytes with a high degree of selectivit y. 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 i nto the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Nawas absent from the mucosal solution. The presence or absence of Na+ i n the serosal solution did not affect pH(i). This indicated that the N a+ dependent pH(i) recovery process is located in the apical cell memb rane, but not in the basolateral membrane. Because amiloride (1 mM) in hibited Na+-dependent pH(i) recovery by 75%, Na+/H+ exchange appears t o be present in the apical membrane. Because Na+-independent pH(i) rec overy was not affected by K+-free media, 50 mu M SCH-28080, 100 nM baf ilomycin A(1), or Cl--free media, this transport mechanism does not in volve a gastriclike H+-K+-ATPase, a vacuolar H+ ATPase, or a Cl-/base exchanger. In summary, pH(i) was selectively measured in surface colon ocytes 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.