SUMMARYAcid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16-to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes-or HCO3--buffered media. Intracellular pH (pH.) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mm K+, corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO3--free (Hepes-buffered) media, bilateral superfusion with 1 mm amiloride or with Na+-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4C1 pulse) by 86 and 98 % respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (I mM). The pH. recovery induced by apical Na+ readdition had a Michaelis constant (K,R) for Na+ of 30 mm and a relatively high inhibitor constant (K,) for amiloride of 45 5 /lM. Initial pHi in HCO3--buffered media was lower than in the absence of HCO3-(7 35 vs. 7 80). pH, recovery from an acid load in HCO3-was Na+ dependent but was inhibited only 18 % by I mM amiloride. The amiloride-independent pH. recovery was inhibited 49 % by pre-incubation of cells in 5 mm DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na+-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na+-H+ exchangers in several normal epithelia; and (b) a Na+-dependent HCO3-transport system, either Na+-HCO3-cotransport or Na+-dependent Cl--HCO3-exchange.

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