Citrulline immunohistochemistry for demonstration of NOS activity in vivo and in vitro

Nitric oxide (NO), a biomolecule with major cytotoxic potency, is generated by NO synthases (NOS) utilizing L-arginine as substrate and citrulline is formed as a "side product." In brain tissue, citrulline is considered to be produced exclusively by NOS, due to the incomplete urea cycle in the brain. We aimed to characterize NOS activity by citrulline immunostaining in different cell types of the brain under in situ conditions and in slice and culture experiments, NOS-positive neurons and activated microglial cells were the most prominent citrulline-positive structures, Lack of citrulline immunoreaction in neurons of nNOS knockout mice emphasizes the dependency of citrulline positivity on NOS activity, and likewise there was no citrulline staining after application of the NOS inhibitors 7-nitroindazole and NIL, Interestingly, only a portion of NOS-containing neurons costained for citrulline, The inhibition of argininosuccinate synthetase by alpha -methyl-DL-aspartate increased the number of citrulline-positive cells, apparently due to reduction of the turnover rate of citrulline, Cells positive for NOS but negative for citrulline may indicate that the enzyme is either not activated or inhibited by cellular control mechanisms. The fact that not all citrulline-positive cells were NOS positive may be explained by an insufficient detection sensitivity or by disparate sites of citrulline production and recycling, The present results show that citrulline immunocytochemistry offers a viable and convenient means for studying NOS activity at the single-cell level to elicit its posttranslational control under physiological and pathophysiological conditions. (C) 2000 Academic Press.