Differential regulation of nitric oxide production by increase of intracellular cAMP in murine primary fibroblasts and L929 fibrosarcoma cell line

doi:10.1016/S0165-2478(99)00178-9

Immunology Letters

Volume 71, Issue 3, 1 March 2000, Pages 149-155

https://doi.org/10.1016/S0165-2478(99)00178-9 Get rights and content

Abstract

The effect of intracellular cAMP rise on nitric oxide (NO) production was compared in murine primary fibroblasts isolated from the spleens of CBA mice, and L929 fibrosarcoma cell line. Treatment of confluent L929 cells with cAMP analogues -dibutyryl-cAMP (db-cAMP) or 8-Cl-cAMP caused dose-dependent augmentation of inducible NO synthase (iNOS)-mediated NO production, which has been abrogated by inhibition of protein synthesis with cycloheximide or addition of selective iNOS inhibitor aminoguanidine. In contrast, under the same cultivating conditions, cAMP analogues were not able to upregulate NO synthesis in primary fibroblasts. Treatment with cAMP analogues or non-selective phosphodiesterase (PDE) inhibitor pentoxifylline affected IFNγ-induced NO synthesis in both cell types, but in the opposite manner-enhancing in L929 cells and suppressive in primary fibroblasts. The induction of iNOS, but not its catalytic activity, was impaired in cAMP-treated primary fibroblasts. Finally, PDE type IV inhibitor rolipram enhanced IFN-γ-triggered NO synthesis in L929 cells, but was unable to mimic cAMP analogue or PTX-mediated suppression of NO synthesis in spleen fibroblasts. These results suggest that, in contrast to L929 fibrosarcoma cell line, intracellular cAMP rise might have a role in downregulation of NO production in murine primary fibroblasts.

Introduction

Nitric oxide (NO), a gaseous free radical produced by inducible NO synthase (iNOS)-mediated oxidation of l-arginine is involved in antimicrobial and antitumour defence, regulation of T cell and macrophage function, and tissue destruction during inflammation (reviewed in [1] and [2]). Fibroblasts, secreting various cytokines and expressing co-stimulatory molecules involved in T cell activation, are active participants in the immune response [3], [4]. Release of high amounts of NO from cytokine-stimulated rodent and human fibroblasts has been implicated in the killing of infectious agents [5], joint erosion during rheumatoid arthritis [6], neurotoxicity [7], as well as in inflammatory pathology of heart, lung and skin [8], [9], [10]. A complex, both positive and negative role of endogenous NO in controlling fibrosis during inflammation resolution has been recently revealed [11], [12], [13]. Although previous study of NO release regulation has focused on macrophages, due to their role in host immune defence [1], control of NO synthesis in fibroblasts seems worthy of attention as a potential target in inflammatory diseases.

Anti-inflammatory properties of agents that elevate intracellular levels of cyclic adenosine monophosphate (cAMP) have been well described (reviewed in [14]). Augmentation of intracellular cAMP inhibits fibroblast proliferation and collagen synthesis [15], [16], [17], both shown to be influenced also by fibroblast-derived NO. Therefore, establishing the connection between augmentation of intracellular cAMP and fibroblast NO release might contribute to better understanding of mechanisms responsible for anti-inflammatory and antifibrotic action of cAMP-elevating drugs. It appears that cAMP elevation stimulates NO synthesis in activated rodent macrophages [18], [19], [20], while acting in an opposite manner in astrocytes [20]. Recent studies, showing induction of NO synthesis in 3T3 cells, or augmentation of IFN-γ-triggered NO production in L929 cells by cAMP-raising agents, have stressed important role of intracellular cAMP rise for iNOS activation in murine fibroblast-like tumour cell lines [21], [22]. However, these results can not automatically apply to primary fibroblasts, since their response to the same stimuli can differ from that of transformed cell lines.

In this report we present evidence for a different role of cAMP signalling pathway in iNOS activation in murine primary spleen fibroblasts and L929 fibrosarcoma cell line, acting to inhibit or enhance, respectively, IFN-γ-induced NO synthesis in these cells.

Section snippets

Cells

Murine L929 fibrosarcoma cell line was obtained from European Collection of Animal Cell Cultures (Salisbury, UK). Plastic-adherent fibroblast-like short-term cell lines (splenic ‘primary’ fibroblasts-SPF) were derived from spleens of CBA mice (animal facility of Institute for Biological Research, Belgrade, Yugoslavia), as previously described [4]. After the mice were killed, spleens were removed, minced into small pieces, and digested for 10 min at 37°C in 2 mg/ml collagenase (Sigma, St Louis,

Effect of cAMP analogues on NO production in murine primary fibroblasts and L929 cells

A recent report by Kleinert et al. presented evidence that elevation of intracellular cAMP induces iNOS expression and subsequent NO production in 3T3 fibroblast [21]. To examine whether cAMP has similar effect on NO synthesis in L929 murine fibrosarcoma cell line and murine primary fibroblasts, cells were treated with cAMP analogues db-cAMP (4–1000 μM) and 8-Cl-cAMP (0.8–200 μM). Treatment of L929 cells with either db-cAMP or 8-Cl-cAMP increased nitrite accumulation in culture supernatants in

Discussion

In this study we showed that elevation of intracellular cAMP level has opposite effects on NO production in murine primary fibroblasts compared to L929 fibrosarcoma cell line. In L929 cells, cAMP analogues induced NO production when applied alone, as well as potentiated IFN-γ-triggered iNOS activation. In contrast, in primary fibroblasts augmentation of intracellular cAMP alone was not sufficient for iNOS induction and even inhibited IFN-γ-induced NO synthesis. While cAMP-mediated upregulation

Acknowledgements

This work was supported by grants from Ministry of Science and Technology, Republic of Serbia, Yugoslavia.

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Background. – As oxidative stress contributes to both progression and pathologic complications of diabetes and effective therapeutic strategies to prevent or delay the damage remain limited, the aim of the present study was to assess the efficacy of pentoxifylline in reducing of oxidative stress. Since there is a relationship between nitric oxide (NO), epidermal growth factor (EGF) and oxidative stress, we measured the effect of this drug on these parameters in comparison to placebo.
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Effects of phosphodiesterase inhibitors on l-arginine-dependent pathways in rat alveolar macrophages, inducible nitric oxide (NO) synthase (iNOS) and arginase, were studied. Culture of rat alveolar macrophages in the presence of lipopolysaccharides (20 h) caused an increase of arginase activity (by 135%) and nitrite concentration (fourfold). The nonselective phosphodiesterase inhibitor IBMX (2-isobutyl-1-methylxanthine) enhanced arginase activity by 35% and nitrite accumulation by 130%. IBMX caused a clear increase in iNOS protein levels and a relatively smaller increase in iNOS mRNA. The effect of IBMX on nitrite accumulation was largely attenuated by the protein kinase A inhibitor K 5720. The phosphodiesterase 4 inhibitor rolipram enhanced nitrite accumulation more effectively than the phosphodiesterase 3 inhibitor siguadozan (about 50% and 20% of IBMX effect, respectively), whereas the phosphodiesterase 3/4 inhibitor benzafendrine was as effective as IBMX. In conclusion, in rat alveolar macrophages, phosphodiesterase 4 and, to a smaller extent, phosphodiesterase 3 play a role in the control of iNOS-mediated NO synthesis.
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Inducible nitric oxide synthase (iNOS) plays a significant role in the pathology of central nervous system diseases. Inducible NOS expression is regulated by intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) signaling, and astrocytes contain both iNOS and adenylate cyclase-coupled neurotransmitter receptors. The data obtained from the present study indicated that acetylcholine, λ-amino-n-butyric acid, glutamate, quinolinic acid, N-methyl-d-aspartate and aspartate have no effect on NO₂⁻ production in C6 glioma cells stimulated by lipopolysaccharide and interferon-gamma. However, dopamine (DA) caused inhibition of NO₂⁻ production and iNOS transcription. The effects of DA were not due to homovanillic acid/3,4-dihydroxyphenylacetic acid, the autoxidative products superoxide (O₂⁻)/hydrogen peroxide (H₂O₂) or direct reactions with NO₂⁻. Forskolin, adenylate cyclase activator, dose-dependently reduced NO₂⁻. Meanwhile, (±) SKF-38393 D₁ receptor agonist attenuated iNOS in a similar fashion to DA. In addition, the results indicated that DA attenuation of iNOS was significantly impeded by the adenylate cyclase inhibitor MDL-12,330A, the D₁ antagonist SCH-23390, the β2 adrenergic receptor antagonist ICI-118,551 and the β1 adrenergic receptor antagonist atenolol. In conclusion, it appears that DA attenuates iNOS through a D₁, β1 and β2 adrenergic receptor-linked adenylate cyclase-mediated cAMP cascade.
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Mycophenolic acid (MPA) and A77 1726, the active components of the immunosuppressants mycophenolate mophetil and leflunomide, respectively, in a dose-dependent manner inhibited interferon (IFN)-γ/LPS-induced interleukin (IL)-6 release in confluent cultures of mouse L929 fibrosarcoma cells. In addition, both drugs markedly reduced the production of the free radical gas nitric oxide (NO), without affecting the viability of L929 cells. The inhibitors of NO synthase, aminoguanidine and L-NMMA, but not L-NMMA inactive counterpart D-NMMA, mimicked the effects of A77 1726 and MPA on IL-6 generation in L929 fibroblasts. Furthermore, NO-releasing substance SNP completely reverted IL-6 accumulation in L929 cultures treated with A77 1726, while only partial recovery of IL-6 production was observed in the presence of MPA. MPA, but not A77 1726, significantly suppressed NO-independent IL-6 release triggered by cAMP-elevating agent rolipram. Thus, while A77 1726 effect on IL-6 production was mediated through concomitant reduction of NO synthesis, MPA action was mainly independent of the interference with NO generation. Finally, both agents inhibited IFN-γ/LPS-triggered IL-6 production in mouse primary fibroblasts, but not in mouse peritoneal macrophages, indicating cell-specificity of this novel anti-inflammatory action of A77 1726 and MPA.
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Differential regulation of nitric oxide production by increase of intracellular cAMP in murine primary fibroblasts and L929 fibrosarcoma cell line

Abstract

Introduction

Section snippets

Cells

Effect of cAMP analogues on NO production in murine primary fibroblasts and L929 cells

Discussion

Acknowledgements

Curr. Opin. Immunol.

Clin. Immunol. Immunopathol.

J. Invest. Dermatol.

J. Invest. Dermatol.

Biochem. Biophys. Res. Commun.

Gen. Pharmacol.

Biochem. Pharmacol.

Int. J. Immunopharmacol.

J. Biol. Chem.

J. Biol. Chem.

Cell. Immunol.

Eur. J. Pharmacol.

Cell. Immunol.

J. Biol. Chem.

J. Biol. Chem.

Immunopharmacology

Annu. Rev. Immunol.

Am. J. Pathol.

J. Immunol.