Role of intracellular buffering power on the mitochondria-cytosol pH gradient in the rat liver perfused at 4°C

The factors regulating the amplitude and the pH gradient between cytosol and mitochondria (hpH(mito-cyt)) were investigated in the isolated rat liver perfused at 4 degrees C. Liver ATP content, pH, and buffering power of cytosolic and mitochondrial compartments were evaluated in situ using phosphorus-31 nuclear magnetic resonance spectroscopy. No Delta pH(mito-cyt) was detected in the liver perfused without bicarbonate. Permeant weak acid in the perfusate (H2CO3, 25 mM, or isobutyric acid, 25, 50, or 100 mM) acidified both cytosol and mitochondria and revealed a Delta pH(mito-cyt) from 0.06 to 0.31 pH unit Nevertheless, the manipulations of the Delta pH(mito-cyt) were more effective under bicarbonate-free conditions, due to the absence of buffering by H2CO3/HCO3-. In the absence of bicarbonate, the intracellular buffering power was threefold higher in the mitochondria (110 mmol/pH unit at pH(mito) 7.16) than in the cytosol (44 mmol/pH unit at pH(cyt) 7.30) and dependent on the matrix and cytosol pH, respectively. These buffering powers were almost double in the presence of bicarbonate. In the bicarbonate-free perfused liver, the respiratory activity was 0.08 +/- 0.02 mu mol O-2/min.g liver wet weight and the ATP turnover was only 40 +/- 7 nmol/min.g liver wet weight, indicating the weak activity of liver mitochondria when Delta pH(mito-cyt) was <0.05 pH unit. The ATP turnover during a 50 mM isobutyric acid load was 35 +/- 4 nmol/min g liver wet weight whereas Delta pH(mito-cyt) rose to 0.26 +/- 0.02 pH unit and pH(mito) remained alkaline. Hence, although Delta pH(mito-cyt) was increased the ATP turnover remained unchanged. This work is the first evaluation of the mitochondrial buffering power in the isolated liver. The Delta pH(mito-cyt) observed within various acid loads reflected the differential titration of cytosol and mitochondria containing proteins and H2CO3/HCO3- buffering systems. Moreover, no direct relationship between Delta pH(mito-cyt) and ATP turnover could be shown. (C) 1999 Academic Press.