Figure 1. Individual NOS isoforms differ in their dependency on extracellular arginine.

NO production was measured indirectly by cGMP formation in RFL-6 reporter cells exposed to supernatants of confluent human A673 neuroepithelioma (A), human EA.hy926 endothelial (B) or LPS-stimulated murine J774A.1 macrophage cells (C). Confluent NO-producing cells were pre-incubated for 2 hours in LS containing either 1 mM arginine (dark columns) or 1 mM lysine (white and grey columns) at 37°C. They were then incubated consecutively two times for 2 min each in LS containing the same amino acids and in the case of A673 and EA.hy926 cells also 10 µM calcium ionophore A23187. The cells represented in the grey columns were exposed to 1 mM lysine and 1 mM arginine, respectively, in the 1^st and 2^nd incubation. Supernatants of each incubation were singularly transferred to RFL-6 reporter cells, left for 2-minutes and then the cGMP content of the reporter cells was determined by radioimmunoassay. (Here, only values of the 2^nd incubations are shown). Values obtained were calculated as % of the mean of the values obtained from the corresponding control cells incubated in 1 mM arginine. The basal cGMP content of the RFL-6 cells was subtracted. Columns represent mean ± S.E.M. (n = 3–9, one way ANOVA with Bonferroni post hoc test).