The role of mitochondrially bound arginase in the regulation of urea synthesis: Studies with [U-¹⁵N₄]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-¹⁵N ₄]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of ¹⁵N into various intermediate metabolites of the urea cycle. The results indicate that ∼8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that ∼60-70% of external [U- ¹⁵N₄]arginine catabolism was recovered as ¹⁵N-labeIed ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of ¹⁵N-Iabeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U_m+1) or two (U_m+2) ¹⁵N was generated from perfusate [U- ¹⁵N₄]arginine. The output of U_m+2 was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U_m+1 was formed following mitochondrial production of [¹⁵N]glutamate from [α,δ-¹⁵N ₂]ornithine and transamination of [¹⁵N]glutamate to [ ¹⁵N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic omithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-¹⁵N₄]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing omithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.