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

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 Ca2+-independent conversion of L-arinine to L-Citrulline that can be completely blocked by the specific NOS inhibitor NG-monomethyl-L-arginine. The induced NOS activity exhibits an apparent K. of 16.5 FM 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.

Ionizing radiation induces apoptosis and inhibits neuronal differentiation in rat neural stem cells via the c-Jun NH2-terminal kinase (JNK) pathway

“…Primary astrocyte cultures (RNB cells) were prepared from the cerebral cortex of 1-day-old neonatal rats as described previously (Galea et al, 1992;Kondo et al, 1996). RNB cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA).…”

TGFβ1 and TNFα potentiate nitric oxide production in astrocyte cultures by recruiting distinct subpopulations of cells to express NOS-2

“…Inflammatory mediators induce NOS-2 expression in astrocyte cultures in vitro (Galea et al 1992;Hewett et al 1993;Simmons and Murphy 1992) Paradoxically, transforming growth factor-β1 (TGFβ1), a cytokine with anti-inflammatory properties, enhanced NO production from astrocytes treated with LPS plus IFNγ and this correlated with a 6-fold increase in the fraction of cells that expressed NOS-2 protein . Since TGFβ1 alone did not induce NOS-2, this suggested that TGFβ1 enabled additional astrocytes to respond to LPS plus IFNγ.…”

“…Inflammatory mediators induce NOS-2 expression in astrocyte cultures in vitro (Galea et al 1992;Hewett et al 1993;Simmons and Murphy 1992) and NOS-2 has been detected in astrocytes in several CNS inflammatory conditions in vivo, including cerebral ischemia and Alzheimer and Parkinson diseases (Acarin et al 2002;Endoh et al 1994;Garcion et al 1998;Katsuse et al 2003;Kwak et al 2005;Luth et al 2002). Importantly, NO from glial NOS-2 has been shown to disrupt the blood-brain barrier (Boveri et al 2006) and promote or exacerbate excitotoxic neuronal cell death Hewett et al 1994;Hewett et al 1996;Vidwans and Hewett 2004), an underlying cause of neuropathology in several neurological diseases (Choi 1988;Coyle and Puttfarcken 1993;Meldrum and Garthwaite 1990).…”

Brain nitric oxide production by a proline-rich decapeptide from Bothrops jararaca venom improves baroreflex sensitivity of spontaneously hypertensive rats

“…The expression and activity of the latter are induced by inflammatory stimuli, independent of the cytosolic calcium concentration. 5 In the citrulline-NO cycle, argininosuccinate synthase (ASS) activity is rate-limiting with regard to the supply of L-arginine as substrate for NOS. ASS is an ubiquitous enzyme that is expressed in all tissues, including the brain.…”