AJP - Cell Physiology, Vol 263, Issue 6 C1181-C1189, Copyright © 1992 by American Physiological Society
Loss of suppression of GSH synthesis at low cell density in primary cultures of rat hepatocytes
S. C. Lu and J. L. Ge
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033.
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 occurred
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.(ABSTRACT TRUNCATED AT
250 WORDS)
