Intracellular pH in isolated Necturus duodenal mucosa exposed to luminal acid

Regulation of intracellular pH (pHi) and its maintenance within physiological ranges during exposure to luminal acid was studied in isolated Necturus duodenal mucosa using liquid sensor microelectrodes. Exposure of the mucosa to luminal pH 2.7 caused significant intraepithelial acidification. Subsequent removal of HCO3-/CO2 (HEPES/O2 substitution) from the serosal perfusate caused a further decrease of pHi. Blocking of HCO3- transport across the basolateral cell membrane by addition of 4-acetamido-4,isothiosyanostilbene-2,2-disulfonic acid (SITS) to serosal perfusate also caused a slight but significant decrease of pHi. Removal of Na+ (choline substitution) from the serosal perfusate during acid exposure likewise caused a significant decrease in pHi, as did serosal addition of an inhibitor of Na+/H+ antiport, 1 mmol/L amiloride. When Na+ was removed from the serosal perfusate after HCO3- removal, pHi first rapidly acidified; this was followed after an initial 5-minute steady state by an uncontrolled progressive acidification at a rate of 0.33 pH unit/15 min without any further steady state. A similar but weaker effect could also be shown with amiloride addition. The epithelial surface pH was 7.13 +/- 0.08 at the apex of mucosal villus and 7.42 +/- 0.11 (n = 5) in the cryptal area between the villi, i.e., greater than 1 pH unit higher than that of the luminal bulk solution (pH 6), thus suggesting active alkalization of the epithelial surface. Removal of serosal HCO3-/CO2 decreased surface pH significantly both at the villus apex and at the cryptal area, suggesting that the surface alkalization is mediated by transport of serosal HCO3- to the epithelial surface. The data suggest that pHi in acid-exposed duodenal mucosa is primarily maintained within physiological range by an HCO3(-)-dependent mechanism, which, at least in part, exerts its action extracellularly by forming an alkaline buffer layer at the epithelial surface. If adequate serosal (or systemic) HCO3- is not available, a second-line Na(+)-dependent and amiloride-sensitive pHi-regulatory mechanism, presumably an Na+/H+ antiport, becomes the main regulator of pHi.