Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells

doi:10.1016/S0014-2999(97)01369-1

European Journal of Pharmacology

Volume 339, Issues 2–3, 27 November 1997, Pages 253-260

https://doi.org/10.1016/S0014-2999(97)01369-1 Get rights and content

Abstract

Lipopolysaccharide is known to stimulate production of nitrite via expression of inducible nitric oxide (NO) synthase in not only macrophages but also glial cells. We found that in glial cell cultures lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation were synergistically enhanced by pretreatment with endothelin, whereas endothelin itself did not induce these responses. Pretreatment with endothelin-1, endothelin-3, and the selective endothelin type B (ET_B) receptor agonist IRL 1620 caused the same effect with similar potencies, suggesting that the synergism was mediated via the endothelin ET_B receptor. A protein kinase C inhibitor, calphostin C, suppressed endothelin-3-enhanced inducible NO synthase expression. Pretreatment with either endothelin-3 or phorbol ester enhanced lipopolysaccharide-induced production of tumor necrosis factor-α (TNF-α). Simultaneous addition of TNF-α increased lipopolysaccharide-stimulated inducible NO synthase expression. These results suggest that the increase in inducible NO synthase expression by endothelin was due to the elevated TNF-α production via protein kinase C. Our findings present the possibility that endothelin is implicated in neurotoxicity via enhancement of inducible NO synthase expression.

Introduction

Nitric oxide (NO) is a molecular mediator that has been implicated in many physiological and pathological processes in various tissues including the brain (for review, see Bredt and Snyder, 1994). NO is synthesized by NO synthase, an enzyme for which several isoforms have been identified (Wong and Billiar, 1995). Two distinct types of Ca²⁺-dependent NO synthase are constitutively expressed, one in some neurons (Bredt and Snyder, 1990) and the other in endothelial cells (Lamas et al., 1992). A Ca²⁺-independent inducible isoform (inducible NO synthase) can be induced in a wide variety of cell types including glial cells by exposure to endotoxins or cytokines (Wong and Billiar, 1995).

There is increasing evidence that inducible NO synthase is induced after focal and global ischemia primarily in reactive astrocytes (Endoh et al., 1994; Iadecola et al., 1995), and that NO release from glial cells appears to be involved in neuronal cell death (Boje and Arora, 1992; Chao et al., 1992; Hewett et al., 1994). In glial cells, increased expression of endothelin has also been observed following focal and global ischemia (for review, see Patel, 1996). However, the involvement of endothelin in inducible NO synthase expression in the nervous system has never been studied in detail.

The endothelins comprise a family of three isopeptides (endothelin-1, -2, and -3), each with 21 amino acids (Yanagisawa and Masaki, 1989). Endothelin has been implicated in a wide variety of physiological functions in various sites including the central nervous system (Masaki et al., 1992). At least two endothelin receptors, which belong to the superfamily of receptors coupled to the heterotrimeric G protein, have been classified via their different affinities for the three endothelin isopeptides. The order of affinity for the endothelin ET_A receptor is endothelin-1≥endothelin-2≫endothelin-3, whereas the endothelin ET_B receptor subtype has equal affinity for all three endothelins (Sakurai et al., 1992). In situ hybridization analysis in the rat brain demonstrated that endothelin ET_B receptor mRNA was expressed in glial cells of many brain regions (Hori et al., 1992).

In the present study, we examined the effect of endothelins on lipopolysaccharide-stimulated inducible NO synthase expression in glial cell cultures prepared from neonatal rat brain. We found that pretreatment with endothelin remarkedly potentiated lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation, whereas endothelins by themselves exerted no effect. We suggest the possible involvement of the endothelin ET_B receptor and the protein kinase C system in the regulation of inducible NO synthase expression by endothelins via production of tumor necrosis factor-α (TNF-α).

Section snippets

Materials

Cyclo (d-Trp-d-Asp–Pro-d-Val–Leu) (BQ-123) was obtained from American Peptide (California), and N-cis-2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine (BQ-788), calphostin C, and phorbol 12-myristate 13-acetate (PMA) were from Sigma (Milwaukee). Endothelin-1, -3, and Suc-[Glu⁹,Ala^11,15]-endothelin-1 (8-21) (IRL 1620) were purchased from Peptide Institute (Osaka). Lipopolysaccharide was purchased from Wako Pure Chemicals (Osaka), and tumor necrosis

Effects of endothelins and IRL 1620 on lipopolysaccharide-stimulated nitrite accumulation

Treatment of glial cells with 100 ng/ml lipopolysaccharide for 24 h led to the accumulation of nitrites 3-fold greater than basal levels in the culture medium (Fig. 1A). Interestingly, 100 ng/ml lipopolysaccharide-stimulated nitrite accumulation was significantly enhanced by pretreatment with 100 nM endothelin-1 for 24 h (p<0.005, n=3). Endothelin-1 by itself did not affect the basal level of nitrite production at concentrations between 1 nM and 10 μM (data not shown).

As shown in Fig. 1B, we

Discussion

In glial cells, 100 ng/ml lipopolysaccharide-stimulated nitrite accumulation and inducible NO synthase expression were enhanced by pretreatment with 100 nM endothelin-1 for 24 h (Fig. 1A, Fig. 2A and B). The synergistic potencies of 100 nM endothelin-1 were similar in cases of both nitrite formation and inducible NO synthase expression induced by 100 ng/ml lipopolysaccharide, indicating that the stimulatory effect of endothelin-1 on lipopolysaccharide-induced nitrite accumulation is due to the

Acknowledgements

This study was supported in part by the Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and from the Research Foundation of Pharmaceutical Research in Japan.

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Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells

Abstract

Introduction

Section snippets

Materials

Effects of endothelins and IRL 1620 on lipopolysaccharide-stimulated nitrite accumulation

Discussion

Acknowledgements

Brain Res.

Brain Res.

Anal. Biochem.

Neuron

Life Sci.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Adv. Pharmacol.

Adv. Pharmacol.

Interleukin-1β induction of TNF-α gene expression: Involvement of protein kinase C

J. Cell. Physiol.

Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme

Proc. Natl. Acad. Sci. USA

Nitric oxide: A physiologic messenger molecule

Annu. Rev. Biochem.

Activated microglia mediate neuronal cell injury via a nitric oxide mechanism

J. Immunol.

Tumor necrosis factor-α production by astrocytes

J. Immunol.

Role of protein kinase C activity in tumor necrosis factor-α gene expression

J. Immunol.