NMR Studies of Inorganic Phosphate Compartmentation in the Isolated Rat Liver during Acidic Perfusion

Mitochondrial inorganic phosphate has been shown to be undetectable by31P NMR in the isolated rat liver perfused under physiological conditions. Cold perfusion (4°C) with valinomycin (K+ionophore) induced the appearance of an additional resonance assigned to Pifrom mitochondrial compartment (Pi,mito) (Thiaudièreet al.,1993,FEBS Lett.330, 232–235). Here we have demonstrated that Pi,mitocan be detected by NMR under normothermic conditions (37°C) in acidic (pH 6.5, bicarbonate-free) perfused liver using 50 nmvalinomycin or 10 μmN,N′-dicyclohexylcarbodiimide (DCCD, a mitochondrial H+-ATP synthase inhibitor). These conditions resulted in a significant increase in mitochondrial Picontent. In the presence of valimomycin, pH values of 7.00 ± 0.07 and 6.60 ± 0.10 (n= 7) for mitochondria and cytosol, respectively, were determined from the chemical shift values of Piresonances. Electron microscopy demonstrated a large matrix swelling under valinomycin perfusion, explaining the increased level of mitochondrial Pi. The amount of mitochondrial Pimeasured by NMR increased linearly with the cellular ATP depletion, suggesting a mitochondrial influx of Pifrom the cytosolic compartment with valinomycin perfusion. Moreover, the level of matrix Piwas dependent on the cytosolic pH value, the resonance being not detectable at physiological cytosolic pH. During mitochondrial swelling, Piinflux was likely to be associated with proton influx, owing to the stability of transmembrane pH gradient and matrix proton concentration.