Interrelationships between gluconeogenesis and ureogenesis in isolated hepatocytes.

or,-Cycloserine (5 to 10 mvl), when added to isolated hepatocytes, is shown to inhibit aspartate aminotransferase predominantly in the cytosol. At low concentrations (0.1 to 0.2 mu) it selectively inhibits alanine aminotransferase. Aminooxyacetate (0.5 mu) irreversibly inhibits all transaminase activity provided the inhibitor is added prior to substrate addition. Neither cycloserine nor aminooxyacetate caused appreciable inhibition of gluconeogenesis from pyruvate, but both inhibitors greatly decreased gluconeogenesis from lactate. These observations confirm earlier findings that malate efflux from mitochondria predominates with pyruvate as glucose precursor while aspartate efflux predominates with lactate as substrate. Urea synthesis from ammonia by isolated hepatocytes was limited by endogenous ornithine. Half-maximal stimulation of urea synthesis in the presence of pyruvate was obtained with 0.5 mu exogenous ornithine. Measurements of citrulline accumulation showed (a) that carbamyl phosphate formation was not rate-limiting and (6) that the apparent K, of argininosuccinate synthetase for citrulline was about 1.5 mu. Urea formation in the presence of ornithine and either lactate or pyruvate was limited by the activity of argininosuccinate synthetase. Urea synthesis was completely inhibited by aminooxyacetate, but was largely unaffected by cycloserine. These data show that aspartate required for urea synthesis must be generated in the mitochondria. Accumulation of aspartate showed that its rate of formation was not limiting for urea synthesis. Cycloserine addition in the absence of ornithine stimulated urea formation from ammonia. This effect was caused by an increase of intracellular citrulline levels because of inhibition of ornithine transaminase. Ammonia addition to hepatocytes incubated with oleate