Regulation of prolyl hydroxylase activity in L-929 cells by mechanisms unrelated to glycolytic metabolism.

Previous studies showing that prolyl hydroxylase is activated by addition of lactate to logarithmic cultures of L-929 and 3T6 mouse cell lines and that the enzyme normally increases in dense cultures which accumulate lactic acid, have led to the hypothesis that regulation is mediated through glycolytic metabolism. We present evidence that the extent of lactate production plays a minor role in controlling the density-dependent activation of prolyl hydroxylase in L-929 cells. The initial rate of glycolysis in stationary phase cells was lower than that in the logarithmic phase so that intracellular levels of glycolytic intermediates would not be related to activation of the enzyme. Nevertheless, because of increased numbers of cells in stationary phase, higher concentrations of lactate occur in the culture medium. Eliminating accumulation of lactate during the growth of L-929 cells by reducing the normal glucose concentration (5.5 mM) to low-glucose (0.5 mM) did not significantly affect the growth rate and still allowed a 10-fold activation of prolyl hydroxylase when cells reached stationary phase. Growth in normal glucose resulted in only an additional 2-fold increase in specific enzyme activity. These results imply that density-dependent activation of prolyl hydroxylase occurs mainly through a mechanism independent of lactate production. The elevated enzyme activity in dense cells was not decreased when lactate was removed and its production prevented by replacement of high-glucose medium with low-glucose medium for 24 hours. This result differed markedly from the dramatic decrease in enzyme activity observed within several hours after replating stationary phase cells in fresh medium, but at a lower cell density. The conclusion that there are at least two mechanisms regulating prolyl hydroxylase activity in L-929 cells was further supported by the observation that the maximal extent of enzyme activation by ascorbate or lactate in logarithmic phase L-929 cells was much less than the extent of density-dependent activation.