NITRIC OXIDE AND HOST DEFENCE 589 DISCUSSION G. Werner-Fe|mayer, E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger and H. Wachter: In rodent cells, synthesis of nitric oxide is easily inducible by interferon-gamma in combination with a second stimulus such as tumour necrosis factor al- pha or lipopolysaccharide. No comparable amount of NO synthesis could be found in human cells in vitro using various strategies in a number of differ- ent laboratories. Nevertheless, increasing evidence suggests that cytokine-inducible NO formation may also occur in humans in vivo. In murine cells, induc- tion of NO formation is counteracted by inhibitory cytokines such as transforming growth factor [31 or macrophage-deactivating factor. Thus, one reason for the different behaviour of human cells in vitro could be a stronger expression of inhibitory cytokines under culture conditions as compared to murine cells. Other reasons for the lack of inducible NO-synthase in human cells, which remain to be investigated, may be found in differences in nitrogen metabolism in different species, e.g. interference of NO formation with arginase, methylation of arginine or different metabolism of the product NO. Recent work shows unambiguously that the con- stitutive form of NO synthase found in cerebellum as well as the cytokine-inducible form in macro- phages require NADPH, flavin and tetrahydrobi- opterin. In contrast to the inducible form of the enzyme, the constitutive form of NO synthase also depends on calmodulin. Inhibition of NO formation by serine protease inhibitors in macrophages and the fact that the molecular weight of the calmodulin- independant macrophage enzyme is about 120,000 kDa as compared to 150,000 kDa for the calmodulin- dependent brain enzyme, could indicate that differ- ent posttranscriptional modifications of the same protein account for different forms of NO synthase. Hopefully, sequences of the different enzyme forms will become available soon to test the validity of this hypothesis. conclusion is justified neither by the data, nor by the interpretation given by BuchmiJller-Rouiller and Mau61 themselves. Furthermore, one should realize that induction and activation of NO synthase are two different processes. Firstly, while we in fact measured the changes in intracellular free Ca 2+ concentration in murine bone-marrow-derived macrophages after stimulation with several different NO-synthase-inducing stimu- li, and failed to observe a correlation with induction of the NO synthase, Buchmfiller-Rouiller and Mau~l provided only indirect evidence for a Ca z+-dependent regulation of macrophage NO syn- thase by studying the effect of EGTA and Ca 2+ ionophore A23187 on intracellular parasite killing and nitrite formation. Unfortunately, these authors did not mention whether EGTA decreased the via- bility of macrophages, which we observed in some preparations, and which could explain the inhibition by EGTA of parasite killing and nitrite formation. We also observed an increase by A23187 in LPS- induced nitrite formation, but like Buchmiiller- Rouiller and Mau~l, we dit not take this as evidence for a direct participation of Ca 2+ in induction and/or " yes, ien of NO synthase activity in these macrophages, in accordance with the conclusion given by those authors, the A23187-induced increase in nitrite formation might be related to membrane perturbation and/or other non-Ca 2+-related effects. Secondly, a direct effect of Ca 2÷ on the activity of inducible NO synthase has never been demonstrat- ed, neither in cell homogenates nor with the purified enzyme (Stuehr et al., 1990), in contrast to the direct Ca 2+ sensitivity of constitutive NO synthase in Ca2 ÷-activatable cells. However, there was one recent report claiming the presence of tiny amounts of an A23187-activatable constitutive NO synthase in the macrophage-derived cell line RAW 264 (Nakane et al., 1991), which did not increase further upon stimulation with LPS. It remains to be clarified whether this enzyme is also present in primary cultures of bone-marrow-derived macrophages since, due to its low activity, it might have escaped from detection so far. A. Miilseh: Regulation of NO synthase activity Werner-Felmayer et al. (this Forum) stated that the data obtained by Buchmiiller-Rouiller and Mauifl (1991) clearly indicated an activation of the macrophage-type NO synthase in intact cells by the intracellular free Ca z+ concentration, in contrast to our findings (Hauschildt et al., 1990). I feel that this Induction of NO synthase in human cells Some contributors addressed the question whether an NO synthase could also be induced in human cells (Werner-Felmayer et ai. ; Billiar et ai. ; Granger), since nitrite/nitrate levels in human serum rise un- der certain conditions associated with bacterial in- fections. Anticipating that macrophages or related cells should be a primary source ef L-arginine-derived NO under these conditions, these investigators, like