Glutamine's role as an energetic fuel has been extensively studied in the past using I4C-and 3H-labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N-tracer. We therefore used I5N-labeled glutamine and glutamate to address t h e following questions: 1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and 2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: 1) intracellular [15N]-glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure I5N-glutamine and no glutamate; in contrast, intracellular l5N-gIutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure I5N-glutamate and no glutamine; 2) the cell-free glutamine content was tightly dependent on extracellular glutamine level, while the cell-free glutamate remained steady irrespective of the extracellular glutamate level; 3) the cells took u p glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and 4) when cells were confronted with a glutamine-free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: 1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measurable using stable isotope methodology; 2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and 3) intracellular free glutamate is divided u p between several pools in cultured human fibroblasts.
Glutamine is both a n essential fuel for most cultured stable isotope methodology can provide further insight human cells (e.g., Eagle, 1955; Reitzer et al., 1979; Stan-into intracellular nitrogen pools; they further demonisz et al., 1983; Zielke et al., 1984) and a major nitrogen strate that the fibroblast-free glutamine pool mainly carrier and donor, in vitro (Engstrom and Zetterberg, depends on extracellular glutamine.