Lack of effect of NH 4Cl on protein synthesis in cultured fibroblasts

Abstract

In experiments with isolated hepatocytes, Seglen [1] has shown that in the combined presence of NH 4Cl and high concentrations of valine, incorporation of this amino acid into cell protein is inhibited. He has proposed that NH 4Cl, in addition to inhibiting protein degradation in lysosomes, inhibits protein synthesis in these cells as part of a general toxic effect. To determine if NH 4Cl inhibits protein synthesis in cultured cells we incubated rat embryo fibroblasts, prelabeled with [ 14C]leucine, in the presence of 10 mM NH 4Cl and 15 mM leucine in both growth and serum-free media. We did not detect any effect of NH 4 + on protein synthesis or cell growth over a 3-day period. A partial inhibition of protein degradation was observed, particularly during the first 24 h of the experiment. In pulse-labeling experiments, NH 4Cl had no effect on the incorporation of [ 3H]leucine in the media. High concentrations of leucine, however, reduced re-utilization of endogenously derived leucine and inhibited the transport of valine into the cellular acid-soluble pool. These experiments show that at least in cultured fibroblasts 10 mM NH 4Cl shows no significant toxicity beyond an inhibition of lysosomal function. In addition these data suggest the possibility that high chase concentrations of one amino acid in the medium may be saturating a common transport mechanism, in effect reducing the transport of other amino acids utilizing this mechanism. A combined blockade by both NH 4Cl and a high concentration of a single amino acid may in certain sensitive cells result in a significant reduction in protein synthesis.