Efflux of protons from acidic vesicles contributes to cytosolic acidification of hepatocytes during ATP depletion

The objective of this study was to determine the relationship between cytosolic pH and vesicular pH during ATP depletion. Using digitized video microscopy and single, cultured rat hepatocytes, cytosolic pH and vesicular pH were quantitated by ratio imaging of BCECF (2′, 7′ biscarboxyethyl‐5, 6‐carboxyfluorescein) fluorescence and fluoresceindextran fluorescence, respectively. Basal value for cytosolic pH was 7.26 and basal value for vesicular pH was 4.86. During ATP depletion by metabolic inhibition with KCN plus iodoacetic acid or antimycin A, cytosolic pH decreased 0.71 units to 6.55. In separate experiments under identical conditions, vesicular pH increased 1.59 units to 6.45, suggesting that protons were leaking from acidic vesicles during ATP depletion. Fluorescein‐dextran fluorescence remained punctate, indicating that the rise in vesicular pH was due to an efflux of protons from vesicles and not loss of vesicle integrity. To determine whether efflux of protons from acidic vesicles can acidify cytosolic pH, we used two maneuvers that result in leakage of protons from acidic vesicles without significantly decreasing cellular ATP: (a) hypotonic stress in K⁺ free media and (b) exposure of the cells to the H⁺ ‐ATPase inhibitor NBD‐Cl. Both hypotonic stress and NBD‐Cl decreased cytosolic pH 0.4 units to 6.86 and increased vesicular pH 2.0 units to 6.76, resulting in near‐equilibration of cytosolic pH and vesicular pH. Thus an efflux of protons from intracellular compartments will acidify cytosolic pH of hepatocytes (pH 6.86), but not to the same degree as ATP depletion (pH 6.55). Calculations based on buffering capacities and relative volumes of the cytosol and acidic compartments suggest that efflux of protons from acidic compartments into the cytosol may account for up to 20% of the decrease of cytosolic pH during the ATP depletion of anoxia observed in hepatocytes. (HEPATOLOGY 1991;14:626–633.)