Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.

TLDR

Exposure of neonatal rat cortical astrocyte cultures to bacterial lipopolysaccharide induces nitric oxide synthase activity, and inducible NOS in glia may contribute to neuronal damage in cerebral ischemia or demyelinating diseases. The induction of NOS, blocked by actinomycin D, is directly related to LPS exposure duration and dose, with a 2‑hour pulse sufficient to induce enzyme activity. LPS‑treated astrocytes generate a Ca²⁺‑independent, NG‑monomethyl‑L‑arginine‑sensitive conversion of L‑arginine to L‑citrulline with a Km of 16.5 µM, dependent on NADPH, FAD, and tetrahydrobiopterin; this inducible NOS is also induced in C6 glioma cells and microglia but not neurons, as confirmed by immunocytochemistry and histochemical staining, indicating that CNS glial cells express an inducible NOS akin to that of macrophages.

Abstract

Exposure of primary cultures of neonatal rat cortical astrocytes to bacterial lipopolysaccharide (LPS) results in the appearance of nitric oxide synthase (NOS) activity. The induction of NOS, which is blocked by actinomycin D, is directly related to the duration of exposure and dose of LPS, and a 2-hr pulse can induce enzyme activity. Cytosol from LPS-treated astrocyte cultures, but not from control cultures, produces a Ca(2+)-independent conversion of L-arginine to L-citrulline that can be completely blocked by the specific NOS inhibitor NG-monomethyl-L-arginine. The induced NOS activity exhibits an apparent Km of 16.5 microM for L-arginine and is dependent on NADPH, FAD, and tetrahydrobiopterin. LPS also induces NOS in C6 glioma cells and microglial cultures but not in cultured cortical neurons. The expression of NOS in astrocytes and microglial cells has been confirmed by immunocytochemical staining using an antibody to the inducible NOS of mouse macrophages and by histochemical staining for NADPH diaphorase activity. We conclude that glial cells of the central nervous system can express an inducible form of NOS similar to the inducible NOS of macrophages. Inducible NOS in glia may, by generating nitric oxide, contribute to the neuronal damage associated with cerebral ischemia and/or demyelinating diseases.