LOSS OF SUPPRESSION OF GSH SYNTHESIS AT LOW CELL-DENSITY IN PRIMARY CULTURES OF RAT HEPATOCYTES

Primary cultures of adult rat hepatocytes shift into the growth phase when plated at low density (LD). We used this model to examine changes in glutathione (GSH) metabolism, since cells undergoing active growth may be more susceptible to environmental toxins. When primary cultures of adult rat hepatocytes were plated on collagen or Matrigel-precoated dishes, cell number and GSH varied inversely. This density effect on cell GSH occured as early as 2 h after plating, when the media contained 1 mM methionine, but was delayed until 20 h if the media contained only 0.5 mM cystine. The density effect on GSH synthesis occurred in the absence of serum, hormones, changes in cell volume, GSH efflux, ATP levels, and uptake of methionine or cystine and was blocked by cycloheximide or actinomycin D. When methionine was available, the cellular cysteine level was 65% higher at LD than at high density (HD). Gamma-glutamylcysteine synthetase (GCS) activity was 64% higher at LD than at HD. GSH synthetase activity was unaffected by density. Both the increase in cellular cysteine levels and GCS activity were blocked by cycloheximide and actinomycin D. When cells were cocultured using cluster plates and Transwell inserts for 4 h, cell GSH of HD cells was unaffected by the density of cocultured cells; however, LD cells exhibited significantly lower GSH and GCS activity when cocultured with HD cells than when cocultured with LD cells. Cysteine levels were elevated in the LD cells regardless of the density of cocultured cells. Thus the availbility of cysteine appears to be regulated by cell-cell contact, whereas the activity of GCS may be modulated by soluble factor(s). These changes in GSH metabolism may be important during growth and liver regeneration.