The accumulation of pyrophosphate by rat hepatocytes

Hepatocytes that were isolated from 48 hr starved rats and incubated in Krebs-Henseleit bicarbonate buffer containing 10 microM A23187, 10mM l-lactate, 1mM pyruvate and 2mM l-lysine were found to contain 0.064 mumol of inorganic pyrophosphate/g wet wgt cells. Addition of either 20mM acetate or butyrate, which caused the formation of pyrophosphate in both the cytosol and the mitochondrial matrix or only the matrix, resulted in an increase of 0.915 and 1.91 mumol pyrophosphate/g wet wgt cells, respectively. The accumulation of pyrophosphate was shown to be non-linear with time and dependent on the calcium concentration of the incubation media. In contrast, incubations containing a combination of 10 mM NH4Cl and 5 mM ornithine, which resulted in the formation of pyrophosphate only in the cytosol, had a pyrophosphate content of 0.032 mumol/g wgt cells. When isolated hepatocytes that had been incubated with acetate or butyrate were subjected to digitonin fractionation, all of the recoverable pyrophosphate was present in the particulate fraction. It is concluded that pyrophosphate accumulates in isolated rat hepatocytes only in the presence of calcium and a calcium ionophore, only within the mitochondrial matrix and only when pyrophosphate is formed within the mitochondrial matrix.