Na(+)-H+ exchange activity and cellular pH regulation in enterocytes isolated from chick small intestine

Intracellular pH (pHi) and Na+/H+ exchange activity have been examined in isolated chicken enterocytes using pH sensitive fluorescence dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and in a nominally bicarbonate-free buffer. Under resting conditions the pHi (7.18) was higher than that observed in the presence of the proton ionophore FCCP (6.98), indicating that [H+] is below the value predicted for electrochemical equilibrium across the plasma membrane, i.e., pHi is regulated. Removal of extracellular Na+ lowered pHi by 0.28 units and subsequent addition of 80 mM Na+ rapidly increased pHi towards the control value. The acidification induced by Na(+)-removal was prevented by 1 mM amiloride. After an intracellular acidification by exposure to 30 mM NH4Cl during 5 min, the pHi decreased from approx. 7.18 to approx. 6.86. Subsequent alkalinization of cells back to control pHi was observed after addition of Na+ or Li+ but not TEA+. Na(+)-dependent recovery of pHi after an acid-load was unaffected by valinomycin, and was 82% reduced by 1 mM amiloride. The inhibitory action of amiloride was abolished by 10 microM monensin. The initial rate of pHi recovery from an acid-load following exposure to Na+ exhibited simple saturation kinetics, with an apparent Km of 12.5 mM Na+ and maximum velocity of alkalinization of approx 0.2 pH units.min-1. The rate of pHi recovery was inversely proportional to pHi. The 'set point' for the exchanger is approx. 7.35. It is concluded that in chicken enterocytes the Na+/H+ exchange system is not quiescent at resting pHi and, thus, contributes to the maintenance of a steady-state pHi at neutral or slightly alkaline levels.