NMR studies of inorganic phosphate compartmentation in the isolated rat liver during acidic perfusion.

Mitochondrial inorganic phosphate has been shown to be undetectable by 31P NMR in the isolated rat liver perfused under physiological conditions. Cold perfusion (4 degrees C) with valinomycin (K+ ionophore) induced the appearance of an additional resonance assigned to P(i) from mitochondrial compartment (P(i,mito)) (Thiaudière et al., 1993, FEBS Lett. 330, 232-235). Here we have demonstrated that P(i,mito) can be detected by NMR under normothermic conditions (37 degrees C) in acidic (pH 6.5, bicarbonate-free) perfused liver using 50 nM valinomycin or 10 microM N,N'-dicyclohexylcarbodiimide (DCCD, a mitochondrial H+-ATP synthase inhibitor). These conditions resulted in a significant increase in mitochondrial P(i) content. 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 P(i) resonances. Electron microscopy demonstrated a large matrix swelling under valinomycin perfusion, explaining the increased level of mitochondrial P(i). The amount of mitochondrial P(i) measured by NMR increased linearly with the cellular ATP depletion, suggesting a mitochondrial influx of P(i) from the cytosolic compartment with valinomycin perfusion. Moreover, the level of matrix P(i) was dependent on the cytosolic pH value, the resonance being not detectable at physiological cytosolic pH. During mitochondrial swelling, P(i) influx was likely to be associated with proton influx, owing to the stability of transmembrane pH gradient and matrix proton concentration.