Azide- and vanadate-sensitive M-phase alkalinity and cytosolic acidification of Chang liver cells

Abstract

Flow cytometric cell-by-cell evaluation of NH4Cl acidification of human Chang cells showed that at steady state, 3% of the cells remained alkalinized (> PH(i) 7) over an extended period (up to 80 min) despite the absence of extracellular Na+ and HCO3 -. In fluorescence microscopy, the acidification-resistant cells were characteristically rounded M-phase cells. Both mean cytosolic pH and M-phase alkalinity were however sensitive to (a) azide and oligomycin, inhibitors of F-ATPase (ATP synthase), and to (b) vanadium ions, the phosphate analogue of P-ATPase (ATP-hydrolyzing), in dose-dependent and time-dependent manners. Dead cell indices were constant at ≃ 10%. Thiocyanate chaotrophic anions, which cleave the V-ATPase structure, had no effect. Since ATP synthesizing F-ATPase (ATP synthase) is coupled to ATP-hydrolyzing P-ATPase as 'master-and-slave', azide- and oligomycin-sensitivity corroborated with vanadate-sensitivity in suggesting energized proton pumping modulating (a) M-phase alkalinity and (b) cytosolic pH, against acidification.