The Role Of p38 MAP Kinases And Akt On Fluticasone Propionate Induced Inhibition Of iNOS expression And Nitric Oxide Production In Vascular Smooth Muscle Cells

Introduction: The upregulation of the inducible nitric oxide synthase (iNOS) enzyme and the subsequent production of nitric oxide (NO) have been shown to be regulated by glucocorticoids but the underlying mechanisms that mediate the regulation are yet to be fully elucidated. In addition, whilst there has been much focus on the nuclear actions of glucocorticoids, there is relatively little known about their effects on upstream signalling pathways which may be critical for the induction of iNOS and thus NO production. The aim of this study therefore was to investigate the effects of the selective glucocorticoid fluticasone propionate (FP) on iNOS expression and NO production induced by pro-inflammatory mediators in rat cultured aortic smooth muscle cells (RASMCs). In addition, further studies have been carried out to establish the potential mechanism of action of FP by examining whether it regulates the p38 MAP kinases and/or the protein kinase B/Akt pathways which have both been reported to be critical for the induction of iNOS in different cell systems (1, 2).

Methods: All experiments were conducted using primary cultures of RASMCs. The cells were activated with bacterial lipopolysaccharide (LPS; 100 μg ml-1) and interferon-gamma (IFN-γ; 100 U ml-1) for 24 h to induce iNOS and NO. When used FP was added at 0.1 to 30 nM 30 min prior to activation of cells. In parallel studies, cultures were incubated for 1 h with RU-486 (10 μM) prior at the addition of FP. Nitrite levels were quantified by the Greiss assay and changes in the expressions of iNOS, phospho-p38 (P-p38), and phoshop-Akt (P-Akt) were monitored by western blotting. Statistical analysis was carried out using one way ANOVA followed by Dunnett’spost-hoc test using Graph Pad Prism.

Results: Exposure of cells to LPS and IFN-γ resulted in the expected induction of iNOS and NO. These responses were significantly inhibited(p<0.05) by FPwhich exerted its effect in a concentration dependent manner. The inhibitions were however only partial, reaching about 50 % with 3 nM FP. Higher concentrations of the drug were without further effect. The inhibition caused by 3 nM FP was completely reversed by 10μMRU-486 which was able to restore iNOS expression and NO production back to the activated control levels. Interestingly, FP also inhibited the phosphorylation of both p-38 and Akt induced by LPS and IFN-γ significantly (p < .05). These inhibitions were also prevented when RU-486 (10 μM) was incubated with cells for 1 h prior to adding FP.

Conclusions: The data generated has shown novel findings demonstrating that FP is able to attenuate iNOS expression and function via an action on the glucocorticoid receptor which may be linked to the phosphorylation of the p38 MAPK and to Akt. Further studies are now in progress to determine the mechanism(s) by which FP inhibits the phosphorylation of these upstream signalling kinases.

1. Baydoun AR et al. (1999). Biochem J 344: 265–272.

2. Hattori Y et al. (2003). Eur J Pharmacol 481: 153–158.