UREA PRODUCTION IN LONG-TERM CULTURES OF ADULT-RAT HEPATOCYTES

To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cy cle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with me dium containing either 0.4 mM arginine or 0.4 mM ornithine and in cult ures exposed to different concentrations of NH4Cl, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea productio n decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 da ys with medium containing ornithine or arginine. Hepatocytes exposed f or 24 hr to 1, 3 and 5 mM NH4Cl showed an average increase in urea pro duction of 25, 50 and 69%, respectively, above that of unexposed cultu res over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hep atocytes seeded on plastic substratum, urea production decreased to 24 % of the initial value after 8 days in culture. OTC and arginase activ ities also decreased to 13 and 10% of their initial values after 8 day s in culture. These results show that 3T3-hepatocyte cultures from adu lt rats produce urea from ornithine and/or arginine for at least 15 da ys and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC an d arginase activities, respectively, owing probably to an adaptative r esponse dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitab le in vitro system to study urea production, its regulation by substra tes and hormones and its alteration by drugs and toxic chemicals.