Induction of argininosuccinate synthetase in rat brain glial cells after striatal microinjection of immunostimulants

The enzyme argininosuccinate synthetase (ASS) initiates the metabolic pathway leading from L-citrulline to L-arginine, the only physiological substrate of all isoforms of nitric oxide synthases. The presence of ASS in glial cells in vivo was investigated by immunohistochemical methods in a model of rat brain inflammation. Phosphate-buffered saline or a mixture of bacterial lipopolysaccharide and interferon-gamma was injected into the left striatum, and animals were killed 24 hours later. Ipsilateral and contralateral sides of brain sections were incubated with an antiserum against ASS or antibodies against cell-specific markers. In the three areas examined, striatum, corpus callosum, and cortex, a strong induction of ASS immunoreactivity was observed in glial cells after injection of immunostimulants. A detailed quantitative analysis of double-stained sections revealed that ASS was almost exclusively expressed in reactive, ED1-positive microglial cells/brain macrophages in immunostimulant- or sham-injected ipsilateral sides of the sections. Furthermore, ASS/ED1 costaining was observed in perivascular cells. Colocalization of ASS with astroglial marker glial fibrillary acidic protein was given only occasionally after immunostimulation. ASS-positive neurons were detected in control and experimental animals; staining intensity was comparable in both cases. The results suggest that neurons express ASS constitutively, whereas the enzyme is induced in glial cells in response to proinflammatory stimuli. This finding is the first demonstration of an induction of a pathway auxiliary to generation of nitric oxide in brain in response to immunostimulants and provides new insight into neural arginine metabolism.