Elsevier

Neuroscience

Volume 117, Issue 1, 17 March 2003, Pages 37-42
Neuroscience

Research paper
Potentiation by ATP of lipopolysaccharide-stimulated nitric oxide production in cultured astrocytes

https://doi.org/10.1016/S0306-4522(02)00804-7Get rights and content

Abstract

The functional changes of astrocytes are deeply involved in neurodegenerating processes of various CNS diseases. ATP is released during various neuronal damages such as brain ischemia and may control astrocyte functions. We examined the effect of ATP on the production of nitric oxide in the cultured astrocytes from rat embryo. The astrocytes were stimulated by lipopolysaccharide instead of pathological activation in vivo. Nitric oxide production was evaluated by the fluorometric assay of nitrite accumulated in the medium. The expression of inducible nitric oxide synthase was analyzed by Western blotting. Nitric oxide production induced by 1 ng/ml lipopolysaccharide was enhanced by ATP with maximal enhancement of three- to four-fold; a half-effective concentration was about 0.3 mM. In the absence of ATP, half-effective concentration of lipopolysaccharide on nitric oxide production was about 3 ng/ml; however, half-effective concentration shifted to 0.3 ng/ml in the presence of 1.5-mM ATP. Several other P2 receptor agonists (uridine triphosphate, ADP, adenosine monophosphate, 2′- and 3′-O - (4-benzoylbenzoyl)-ATP, and 2-methylthioATP) showed a similar enhancing effect, and an antagonist, ATP-2′,3′-dialdehyde, showed an inhibiting effect. Western blotting analysis revealed that the extent of lipopolysaccharide-induced expression of nitric oxide synthase increased several-fold by the addition of ATP; half-effective concentration was about 0.5 mM.

These results suggest that the extracellular ATP plays an important role as a transmitter and regulates astrocyte functions via a certain P2 receptor and that such a change in astrocyte function is involved in either protection or aggravation in neurodegenerative processes.

Section snippets

Materials

LPS (from Salmonella enteritidis), 2′- and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP), and ATP-2′,3′-dialdehyde (o-ATP; periodate-oxidized ATP) were purchased from Sigma Chemical Co. (St Louis, MO, USA). 2-Methylthioadenosine triphosphate (2-MeSATP) was purchased from Tocris (Bristol, UK). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco BRL (Grand Island, NY, USA). Fetal calf serum (FCS) was obtained from Filtron (Brooklyn, Australia).

Preparation of astrocyte culture

This study was carried out in compliance with the

Enhancement by ATP of LPS-induced NO production from cultured astrocytes

The concentration of NO2 in the medium of cultured astrocytes was measured for more than 30 h (Fig. 1). The addition of 1.5-mM ATP alone showed no effect compared with the background. The addition of 100 ng/ml LPS induced a large amount of NO production after several hours’ lag time. In the presence of 1.5-mM ATP, LPS-induced NO production was remarkably increased. The lag time was not changed but the slope of the increase became about twice. When the cells were treated with 1.0 ng/ml of LPS,

Discussion

One of the most important findings in this study is that ATP enhanced NO production from cultured astrocytes remarkably. We examined the mechanism of this ATP enhancement of NO production. In the time course of NO2 accumulation in the medium, the addition of ATP did not affect the lag time of NO2 accumulation after LPS addition, but increased the slope thereafter. The lag time seems to be the period necessary for induction of iNOS enzyme; therefore, ATP is likely to enhance iNOS activity

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