Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

Abstract

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.

Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein

“…Acidification was followed by partial recovery in HEK293 cells ( Figure 5 ), as well as in CHO and Caco-2 cells (data not shown), as a result of pHi-regulatory mechanisms. [18][19][20] As a control, the injection of buffer containing 30 mM NaCl, instead of 30 mM propionate Na + , had no effect on pHi. …”

Differential Cl-and HCO3-mediated anion secretion by different colonic cell types in response to tetromethylpyrazine

“…The observed difference between T84 and Caco-2 cells in their I SC responses to different concentrations of TMP suggested that colonic Cl -and HCO 3 -mediated secretions might be differentially elicited by the same agent depending on the concentrations used, probably due to differential expression of receptors, cotranspoters, exchangers and ion channels [10][11][12][13] leading to differential responses to TMP. This notion is supported by the observation that TMP at different concentrations could elicit different I SC responses from freshly isolated rat colon mucosa with distinct current characteristics.…”

“…T84 cell line is a well-characterized colonic epithelial model that maintains a secretory phenotype similar to crypt base cells, and has been widely used to examine the regulation of intestinal Cl -secretion since CFTR and Na + -K + -Cl -cotransporter (NKCC) are highly expressed in this cell line [1,10,11] . The properties of human colonic Caco-2 cells are mostly close to those of mature absorptive villus/surface cells but they possess a certain CFTR characteristic of secretory cells [1,12,13] . However, few studies have been conducted to compare the secretory properties of these cell lines.…”

General – Epithelia/GI Tract

“…This differential localization reflects the major roles (pHi homeostasis and transcellular Na+ absorption) of the two transporters. Studies using the human intestinal epithelial cell line Caco-2, however, provide conflicting evidence for NHE3 expression at the apical surface (Watson et al 1991 ;Osypiw et al 1994). In this study we have determined the expression of NHE isoforms in Caco-2 cells using a combination of molecular and cellular biological techniques (RT-PCR, 22Na+ influx and measurement of intracellular pH).…”