The Accumulation of Pyrophosphate By Rat Hepatocytes
Hepatocytes that were isolated from 48 hr starved rats and incu bated in Krebs-Henseleit bicarbonate buffer containing 10 μM A23187, 10mM l-lactate, 1 mM pyruvate and 2mM l-lysine were found to contain 0.064 μmol 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 μmol 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 incu bation media. In contrast, incubations containing a combination of 10 mM NH4 Cl and 5 mM ornithine, which resulted in the forma tion of pyrophosphate only in the cytosol, had a pyrophosphate content of 0.032 μmol/g wgt cells. When isolated hepatocytes that had been incubated with acetate or butyrate were subjected to dig itonin fractionation, all of the recoverable pyrophosphate was pres ent in the particulate fraction. It is concluded that pyrophosphate accumulates in isolated rat hepatocytes only in the presence of cal cium and a calcium ionophore, only within the mitochondrial matrix and only when pyrophosphate is formed within the mito chondrial matrix.