The development of esophagitis is the result of hydrogen ion diffusion into the mucosa leading to cellular acidification and necrosis. In these studies, whether esophageal cells possess transport system(s) that can respond to cytoplasmic acidification was assessed; specifically, whether esophageal cells possess an Na+,H+ antiport was determined. Nucleated esophageal cells were isolated from rabbit esophagi using a trypsin-digestion technique that yielded 5-8 x 10(6) cells per esophagus, of which 74% +/- 3% were basal and 26% +/- 8% were squamous. Trypan blue was excluded by 95% +/- 2% of the cells. Cytoplasmic pH (pHi) was measured using the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester. Cells were acidified to the desired pHi by suspension in solutions with varying external pH (pHo) in the presence of nigericin. When cells acidified to pHi 6.3 were suspended in a choline chloride solution (pHo 7.4), cytoplasmic pHi did not increase. In contrast, Nao+ caused a concentration-dependent increase in the rate of cytoplasmic alkalinization with saturation occurring above 50 mmol/L Nao+. The transporter behaved according to first-order Michaelis-Menten type kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to cytoplasmic hydrogen ion concentration. Amiloride (10(-4) mol/L) caused a reversible inhibition of Na(+)-dependent alkalinization. Amiloride-sensitive cytoplasmic alkalinization was not observed when either cholineo or Ko+ was substituted for Nao+, while Lio+ resulted in alkalinization that was 60% +/- 8% of that seen with equimolar concentrations of Nao+. The basal pHi of cells suspended in a bicarbonate-free 130 mmol/L NaCl solution (pHo 7.4) averaged 7.42 +/- 0.03 (n = 10); amiloride (10(-4) mmol/L caused the basal pHi to decrease to 7.26 +/- 0.05 (n = 10; P less than 0.0025). When cells were suspended in a choline chloride (pHo 7.4) solution, pHi averaged 7.29 +/- 0.06 (n = 10) (P less than 0.0025 compared with Nao+). These studies indicate that nucleated esophageal cells obtained from rabbits possess an amiloride-sensitive Na+,H+ antiport that functions to regulate basal pHi and responds to intracellular acidification.