Figure 3. A673 neuroepithelioma cells take up extracellular citrulline and convert it to arginine, but do not use this arginine for NO-synthesis.

(A) and (C): Confluent A673 cells were preincubated for 30 min in LS containing 1 mM of each indicated amino acid. The cells were then washed 3 times in ice-cold PBS and amino acids were extracted with 70% ethanol. Intracellular amino acid concentrations were calculated from the amino acid content of the cell lysates measured by HPLC, the cell volume of A673 cells (determined as 94±16 fL), and the cell number (mean ± S.E.M., n = 7–9, one way ANOVA with Bonferroni post hoc test). * significant difference to arginine-incubated cells, #: significant difference to cells incubated with lysine and citrulline (in C without consideration of the arginine-incubated cells). (B) A673 cells were incubated for 30 min at 37°C in LS supplemented with 1 mM lysine, 10.8 µM [¹⁴C]-citrulline, 100 µM L-NAME and with (dashed columns) or without (white columns) 1 mM glutamine as indicated. TLC analyses of supernatants and cell lysates were performed as detailed in the methods section. The [¹⁴C]-arginine spots were quantified using a phosphor-imager. All values were calculated as % of the mean of the values obtained in total from cells incubated without glutamine. Columns represent mean ± S.E.M. (n = 4, unpaired students t-test). (D) NO measurements and data analyses were performed as described in Figure 1. Thirty min preincubations and transfers were performed in LS with the indicated amino acids (single or in combination): 1 mM arginine, 1 mM lysine, 4 mM citrulline, 4 mM aspartic acid. Columns represent mean ± S.E.M. (n = 4–6, one way ANOVA with Bonferroni post hoc test).