Two distinct HCO 3 − -dependent H⁺efflux pathways in human vascular endothelial cells

Intracellular pH (pH(i)) regulation in human umbilical vein endothelial cells (HUVEC) was investigated. The pH(i) was recorded using seminaphthorhodafluor-1 (SNARF-1). Cells were intracellularly acid loaded with NH(4)Cl prepulse. In HEPES-buffered Tyrode (nominally HCO(-)(3) free), pH(i) recovery from acid load was inhibited by 1.5 mM amiloride or Na(+)-free solution. Additionally, in HCO(-)(3)-buffered Tyrode, a HCO(-)(3)-dependent pH(i) recovery from acidosis was evident in the presence of 1.5 mM amiloride, which mediated complete recovery of pH(i) (7.26). In Na(+)-free solution, the HCO(-)(3)-dependent acid extruder mediated pH(i) recovery after an acid load but only back to 7.09. These results suggest that there are two HCO(-)(3)-dependent acid extruders in the HUVEC. One is Na(+) dependent, and the other is Na(+) independent. The former was further shown to be completely inhibited by 0.5 mM DIDS, whereas the latter was only inhibited by 24.6%. In Cl(-)-free solution, both of the HCO(-)(3)-dependent pathways were inhibited. In conclusion, one HCO(-)(3)-dependent acid extruder in the HUVEC resembles the Na(+)-dependent Cl(-)/HCO(-)(3) exchange found in other tissues, and the other is Cl(-) dependent but Na(+) independent.