Acid-base transport systems in a polarized human intestinal cell monolayer: Caco-2.
Acid-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 HCO(3-)-buffered media. Intracellular pH (pHi) 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 HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl 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 (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi 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.
Osypiw
J C
JC
Department of Gastroenterology, Royal Infirmary, Manchester.
Gleeson
D
D
Lobley
R W
RW
Pemberton
P W
PW
McMahon
R F
RF
D000136:Acid-Base Equilibrium; D000584:Amiloride / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D016764:Cell Polarity; D002712:Chlorides / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000648:ultrastructure; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology*; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001639:Bicarbonates; D002712:Chlorides; D015222:Sodium Channels; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; D011188:Potassium