Na+-HCO3(-) cotransporter and intracellular pH regulation in chicken enterocytes.

Abstract

The current studies examine the presence of the Na+-HCO3(-) cotransporter in chicken enterocytes and its role in cytosolic pH (pHi) regulation. The pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pHi. Under resting conditions, pHi was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO3(-). Removal of external Na+ decreased pHi and readdition of Na+ rapidly increased pHi towards the control values. These Na+-dependent changes were greater in HCO3(-)- than in HEPES-buffered solutions. In HCO3- - free solutions the Na+-dependent changes in pHi were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4'-diisothiocyanatostilbene disulphonic acid (H2-DIDS). In the presence of HCO3-, the Na+-induced changes in pHi were sensitive to both EIPA and H2-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na+ and HCO3- were present. This pHi recovery, which was EIPA resistant, and dependent on Na+ and HCO3-, was inhibited by H2-DIDS and occurred at equal rates in both Cl--containing and Cl--free solutions. Kinetic analysis of the rate of HCO3- and Na+-dependent pHi recovery from an acid load as a function of the Na+ concentration revealed first-order kinetics with a Michaelis constant, Km, of 11 mmol/l Na+. It is concluded that in HCO3(-) buffered solutions both the Na+/H+ exchanger and the Na+-HCO3(-) cotransporter participate in setting the resting pHi in isolated chicken enterocytes and help the recovery from acid loads.