文摘
The factors regulating the amplitude and the pH gradient between cytosol and mitochondria (ΔpHmito-cyt) were investigated in the isolated rat liver perfused at 4°C. Liver ATP content, pH, and buffering power of cytosolic and mitochondrial compartments were evaluatedin situusing phosphorus-31 nuclear magnetic resonance spectroscopy. No ΔpHmito-cytwas 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 ΔpHmito-cytfrom 0.06 to 0.31 pH unit. Nevertheless, the manipulations of the ΔpHmito-cytwere more effective under bicarbonate-free conditions, due to the absence of buffering by H2CO3/HCO−3. In the absence of bicarbonate, the intracellular buffering power was threefold higher in the mitochondria (110 mmol/pH unit at pHmito7.16) than in the cytosol (44 mmol/pH unit at pHcyt7.30) 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 μmol O2/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 ΔpHmito-cytwas <0.05 pH unit. The ATP turnover during a 50 mM isobutyric acid load was 35 ± 4 nmol/min · g liver wet weight whereas ΔpHmito-cytrose to 0.26 ± 0.02 pH unit and pHmitoremained alkaline. Hence, although ΔpHmito-cytwas increased the ATP turnover remained unchanged. This work is the first evaluation of the mitochondrial buffering power in the isolated liver. The ΔpHmito-cytobserved within various acid loads reflected the differential titration of cytosol and mitochondria containing proteins and H2CO3/HCO−3buffering systems. Moreover, no direct relationship between ΔpHmito-cytand ATP turnover could be shown.