Prednisolone augments superoxide formation in porcine pulmonary artery endothelial cells through differential effects on the expression of nitric oxide synthase and NADPH oxidase

Abstract

1 Prednisolone, a potent anti-inflammatory drug, has proved ineffective in treating acute respiratory distress syndrome (ARDS). ARDS is associated with superoxide (O 2 K À ) generation, which negates nitric oxide (NO). NO also downregulates NADPH oxidase and inhibits O 2 K À formation. A possible reason for the lack of effect of prednisolone may due to an inhibition of eNOS expression. In order to test this proposal, the effect of prednisolone on O 2 K À formation and the expression of gp91 phox (catalytic subunit of NADPH oxidase) and eNOS in pig pulmonary artery (PA) segments and PA endothelial cells (PAECs) and PA vascular smooth muscle cells (PAVSMCs) was investigated.2 PA segments and cells were incubated with prednisolone and tumour necrosis factor-alpha (TNF-a) for 16 h. O 2 K À formation was measured spectrophometrically and gp91 phox and eNOS expression by Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride, the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the stable cGMP analogues, 8-bromo cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP). NO release was studied using a fluorescence assay and O 2 formation. Increased O 2 K À release and gp91 phox expression in PAECs elicited by prednisolone was blocked by SIN-1 (3-morpholinosydnonimine hydrochloride), DETA-NONOate, 8-pCPT-cGMP and 8-bromo cGMP. The effects of SIN-1 on gp91 phox expression were reversed by ODQ. Finally, eNOS protein expression was significantly reduced by prednisolone. 4 Prednisolone increases O 2 K À in porcine PAECs through a downregulation of endogenous eNOS expression. Since the NO-cGMP axis inhibits gp91 phox expression, the resultant decrease in endogenous NO formation then augments NADPH oxidase activity, which in turn results in increased O 2 K À formation. Since O 2 K À promotes inflammation, this mechanism may explain why prednisolone is ineffective in treating ARDS. Therapeutically, the coadministration of an NO donor may render prednisolone more effective in treating ARDS.

Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity

“…2C, D and E). We chose to study NOS3 and SERPINE1 expression, as these genes are key endothelial genes regulated by glucocorticoids, and their expression changes also regulate endothelial phenotype (Lou et al 2001, Muzaffar et al 2005, Kimura et al 2009). GRα overexpression correlated with significant decreases in NOS3 (Fig.…”

Reactive oxygen species and erectile dysfunction: possible role of NADPH oxidase

“…102 It was suggested, therefore, that NO may act protectively to prevent NADPH oxidase expression in response to the factors that promote its expression. 103,104 By contrast, the downregulation or impairment of eNOS activity, a possibly key event in ED, would render vascular tissue more susceptible to an increase in NADPH oxidase. Similarly, since NADPH oxidase generates O 2 KÀ which negates NO K bioactivity, then this may constitute a self-perpetuating mechanism that would promote oxidative stress in penile tissue and related vasculature (Figure 2).…”

Adrenal corticosteroids, their receptors and hypertension

“…Consistent with these changes, glucocorticoid-activated GR increases the expression of xanthine oxidase and the catalytic subunit of NADPH oxidase [Muzaffar et al, 2005;Pfeffer et al, 1994], and, as mentioned above, reduces eNOS expression. Apocynin, an inhibitor of NADPH oxidase, was able to prevent and reverse dexamethasone-induced hypertension in rats, suggesting the NADPH oxidase-induced ROS are at least in part responsible for glucocorticoid-induced hypertension [Hu et al, 2006].…”