NMR-STUDIES OF INORGANIC-PHOSPHATE COMPARTMENTATION IN THE ISOLATED RAT-LIVER DURING ACIDIC PERFUSION

Mitochondrial inorganic phosphate has been shown to be undetectable by P-31 NMR in the isolated rat liver perfused under physiological condi tions. Cold perfusion (4 degrees C) with valinomycin (K+ ionophore) in duced the appearance of an additional resonance assigned to P-i from m itochondrial compartment (P-i,P-mito) (Thiaudiere et at, 1993, FEBS Le tt. 330, 232-235). Here we have demonstrated that P-i,P-mito can be de tected by NMR under normothermic conditions (37 degrees C) in acidic ( pH 6.5, bicarbonate-free) perfused liver using 50 nM valinomycin or 10 mu M N,N'-dicyclohexylcarbodiimide (DCCD, a mitochondrial H+-ATP synt hase inhibitor). These conditions resulted in a significant increase i n 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 cytos ol, 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 mitoch ondrial 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 perfusio n. 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 associa ted with proton influx, owing to the stability of transmembrane pH gra dient and matrix proton concentration. (C) 1997 Academic Press