ACID-BASE TRANSPORT-SYSTEMS IN A POLARIZED HUMAN INTESTINAL-CELL MONOLAYER - CACO-2

Acid-base transport systems have been incompletely characterized in in tact 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 progre ssive enrichment in microvillar hydrolases. Monolayers (16- to 25-day- old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyeth yl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvett e to allow selective superfusion of apical and basolateral surfaces wi th Hepes- or HCO3--buffered media. Intracellular pH (pH(i)) was measur ed by dual-excitation spectrofluorimetry; calibration was with standar ds containing nigericin and 110 mM K+, corresponding to measured intra cellular [K+] in Caco-2 cell monolayers. In HCO3--free (Hepes-buffered ) media, bilateral superfusion with 1 mM amiloride or with Na+-free me dia reversibly inhibited pH(i) recovery from an intracellular acid loa d (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na + to the apical or basolateral superfusate also induced a pH(i) recove ry, which was inhibited by ipsilateral but not by contralateral amilor ide (1 mM). The pH(i) recovery induced by apical Na+ readdition had a Michaelis constant (K-m) for Na+ of 30 mM and a relatively high inhibi tor constant (K-i) for amiloride of 45.5 mu M. Initial pH(i) in HCO3-- buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pH(i) recovery from an acid load in HCO3- was Na+ dependent but was i nhibited only 18% by 1 mM amiloride. The amiloride-independent pH(i) r ecovery 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 resist ant to amiloride, similar to apical Na+-H+ exchangers in several norma l epithelia; and (b) a Na+-dependent HCO3- transport system, either Na +HCO3- cotransport or Na+-dependent Cl--HCO3- exchange.