Regulation of Induced Nitric Oxide Synthase in Vascular Smooth Muscle Cells by Glucocorticoids
The upregulation of the inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production have been implicated in inflammatory pathologies. Although research has revealed that glucocorticoids (GCs) such as dexamethasone and hydrocortisone inhibit iNOS expression and NO production, it remains unclear how these compounds attenuate iNOS expression and function. In response, this thesis has compared the effects of nonselective GCs (i.e., dexamethasone and hydrocortisone) with a selective GC namely, fluticasone propionate (fluticasone) to identify the precise GC actions that regulate the iNOS pathway. Additional investigations were performed to distinguish the GC and non-GC actions using receptor antagonists. Since the effects of GCs on upstream signalling pathways remain vague, further studies were conducted to investigate whether fluticasone regulates the p38 mitogen-activated protein kinases or protein kinase B (Akt) pathways, both of which have been reported to be critical for the induction of iNOS. All experiments were conducted using primary cultures of rat aortic smooth muscle cells (RASMCs). The cells were activated with bacterial LPS (100 μg/mL) and interferon-gamma (IFN-γ, 100 U/mL) to induce iNOS and NO. Nitrite levels in cellular supernatants were quantified by the Griess assay, and expressions of iNOS, phospho-p38 (P-p38), and phospho-Akt (P-Akt) were investigated by western blotting. Dexamethasone (0.1–10.0 μM) inhibited iNOS expression and NO production in a concentration dependent manner that was significant at higher concentrations (0.3–10.0 μM). Hydrocortisone (0.01–10.0 μM) also inhibited iNOS expression and NO production in a concentration dependent manner which was significant at the higher concentrations (0.1–10.0 µM). By contrast, fluticasone (0.1 nM–3.0 µM) inhibited NO production and iNOS expression only partially (~50%), and the effects were significant at 1 nM–3 µM. RU-486 (10 μM), a GC receptor (GCR) blocker, was able to reverse the inhibitions caused by dexamethasone, hydrocortisone, and fluticasone, though eplerenone (0.1–10.0 µM), the mineralocortocoid receptor blocker, had no effect. Fluticasone also inhibited the phosphorylation of p38 and Akt in activated RASMCs. The inhibitions were reversed upon incubation with RU-486 (10 μM) for 1 h prior to the addition of fluticasone. The partial inhibition of iNOS and NO by fluticasone suggests that the actions of dexamethasone and hydrocortisone were not restricted solely to GCR and that other receptors or pathways, if not both, might regulate iNOS and NO in RASMCs. In conclusion, the nonselective GCs (i.e., dexamethasone and hydrocortisone) showed a full inhibition of iNOS expression and function, whereas fluticasone only partially inhibited both processes. The inhibitions were reversed by RU-486, but not eplerenone, which strongly suggests a GC-mediated response to all three compounds investigated. Regarding fluticasone, mechanistic studies revealed that the GC can regulate key signalling pathways associated with the induction of iNOS. More specifically, fluticasone reduced the phosphorylation of p38 and Akt, thereby suggesting that its actions can be mediated by suppressing these kinase pathways, which are widely reported to critically regulate iNOS expression and function.