SUBPOPULATIONS OF RAT HEPATOCYTES SEPARATED BY PERCOLL DENSITY-GRADIENT CENTRIFUGATION SHOW CHARACTERISTICS CONSISTENT WITH DIFFERENT ACINAR LOCATIONS

Freshly isolated viable rat hepatocytes were separated into five subpo pulations on shallow discontinuous Percoll density gradients. The peri portal marker enzymes alanine aminotransferase (ALT), malate dehydroge nase (MDH) and lactate dehydrogenase (LDH) showed gradients of increas ing activity from the subpopulation of least density (band 1, rho = 1. 07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamat e dehydrogenase (GDH) showed gradients of decreasing activity from ban d-1 cells to band-5 cells. Glutamine synthetase (GS), which is confine d to the two or three cell layers around the hepatic venule, was almos t entirely restricted to band-1 hepatocytes. Band-5:band-1 ratios of e nzyme activity were as follows. ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7 ; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions fo r up to 72 h, whereas the ratios for MDH and GDH decreased and increas ed respectively towards unity. Band-1 hepatocytes exhibited greater cy totoxicity than band-5 cells after incubation with carbon tetrachlorid e or paracetamol. These perivenous-selective toxins produced greater d ecreases in cell viability and greater release of ALT and LDH from ban d-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepat ocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal -selective toxins). It is concluded that band-5 hepatocytes are enrich ed in periportal cells, whereas band-1 hepatocytes are enriched in per ivenous cells. Isolation of hepatocyte subpopulations by Percoll densi ty-gradient centifugation has the considerable advantage that periport al and perivenous cells can be obtained from the same liver.