Na+-dependent recovery of intracellular pH from acid loading in mouse colonic crypt cells

The membrane transport mechanism for regulating the intracellular pH value (pHi) was investigated in mouse distal colon crypt cells. pHi was measured by microfluorometry in an isolated crypt fragment loaded with the pH-sensitive fluoroprobe, 2',7'-bis-(2-carboxyethyl)-5-(6) carboxyfluorescein. The pHi recovery process after acid loading induced by a 40 mM NH4Cl prepulse was almost totally dependent on Na+ in both the presence and absence of CO2/HCO3- in the perfusion solution. In the CO2/HCO3(-)-free, HEPES-buffered solution, amiloride partially inhibited the pHi recovery rate from acid loading with an ED50 value of 15 microM and maximum inhibition of 83%. In a CO2/HCO3- solution, amiloride inhibited the pHi recovery rate with an ED50 value of 18 microM, which was similar to that in the HEPES-buffered solution, while the rate of pHi recovery remaining in the presence of the maximum effective concentration of amiloride was significantly larger than that in the HEPES-buffered solution. The Na+-dependent pHi recovery from the acid loading was significantly less (by 18%) in the presence of forskolin. These results suggest that the pHi recovery from acid loading was mediated by 1) amiloride-sensitive Na+/H+ exchanger, 2) the amiloride-insensitive Na+/H+ exchanger, and 3) the Na+- and HCO3(-)-dependent acid extruder. The pHi recovery could be inhibited by cAMP.