Peroxynitrite generated from constitutive nitric oxide synthase mediates the early biochemical injury in short-term cultured hepatocytes

Early loss of P450 in rat hepatocyte cultures appears directly related to n itric oxide (NO) overproduction. This study provides experimental evidence for the induction - shortly after isolation through the classical procedure - of strong oxidative stress that involves both oxygen-derived and NO-deri ved species. NO formation at this stage is due to the early activation of l iver constitutive NO synthase (cNOS), Immunodetection of nitrated proteins provides direct evidence of endogenous peroxynitrite (PN) formation upon he patocyte isolation. On the basis of the combined use of dihydrorhodamine 12 3 and NOS inhibitors, the analysis of the amount, time course and nature of the species involved supports the view that PN generated from cNOS-derived NO, while not affecting cell viability and hepatocyte monolayer developmen t, is the main species likely responsible for the early biochemical injury commonly observed in hepatocyte cultures. (C) 2000 Federation of European B iochemical Societies.