CELLULAR SOURCES OF GLUTATHIONE-S-TRANSFERASE-P IN PRIMARY CULTURED RAT HEPATOCYTES - LOCALIZATION BY IN-SITU HYBRIDIZATION

Hepatocytes in vivo express Alpha and Mu but not Pi forms of glutathione S-transferase (GST). GST P (a fetal Pi form) appears in rat hepatocytes after 2 days in primary culture, which suggests that hepatocytes may undergo dedifferentiation [Abramovitz, Ishigaki and Listowsky (1989) Hepatology 9, 235-239]. However, in this and other studies, primary rat hepatocyte cultures were shown by immunohistochemistry to contain significant numbers of lipocytes (Ito cells). Freshly isolated lipocytes contained GST activity when assayed with chlorodinitrobenzene (680 nmol/min per mg), and expression of Alpha, Mu and Pi forms of GST was detected by Western-blot analysis. Expression of GST P persisted during culture of the lipocytes. In situ hybridization of the cultured cells was performed to define whether hepatocytes, lipocytes or both expressed the enzyme. Lipocytes in culture contained abundant GST P transcripts. Hepatocytes contained no GST P transcripts after 12 h in culture, and after 24 h, only a few hepatocytes expressed this enzyme. After 48 h in culture all heptocytes contained GST P transcripts, and the number of transcripts continued to increase up until 72 h. Therefore, in freshly isolated preparations of hepatocytes and early in hepatocyte culture, measurable levels of GST P protein or message appeared to reflect the presence of lipocytes. After 48 h in culture almost all of the GST P reflected expression by the hepatocytes. Lipocytes constitutively expressed Alpha-, Mu- and Pi-class GSTs and had significant intracellular levels of GSH (5.2 nmol/mg of protein). Lipocytes are capable therefore of detoxifying a number of injurious compounds.