Roflumilast N-oxide inhibits NADPH oxidase expression and activity in human pulmonary artery smooth muscle cells

Saima Muzaffar, Nilima Shukla, Gianni Angelini, Jamie Jeremy

Bristol Heart Institute, University of Bristol, Bristol, UK

Chronic obstructive pulmonary disease (COPD) is associated with intrapulmonary superoxide (O2•¯) formation derived from NADPH oxidase (NOX), which augments progression of the disease (Rahman & Adcock, 2006). Prostacyclin (PGI2), through cAMP-PKA signaling, blocks NOX expression and activity in response to inflammatory mediators (Muzaffar et al., 2004). It follows that type 4 phosphodiesterase (PDE4) inhibitors should augment the inhibitory impact of cAMP-PKA on NOX expression and activity. In order to test this proposal, the effect of a PDE4 inhibitor, roflumilast N-oxide (RNO; which is the active metabolite of roflumilast, currently in development for COPD) on O2▪¯ formation, NOX-1 expression (mRNA and protein) and activity was studied in human pulmonary arterial smooth muscle cells (hPASMCs). Rac1 activity was studied since this is an obligatory co-factor for NOX activity (Muzaffar et al., 2008).

hPASMCs were incubated with 10 nM TXA2 analogue, U46619 (±RNO) for 1 h or 16 h and O2•- formation measured and NOX-1 expression assessed using Western blotting and quantitative PCR. Effects on Rac1 activity induced with U46619 were studied using a pull-down assay (Muzaffar et al., 2008).

RNO inhibited the formation of O2•– in a concentration-dependent manner when co-incubated with U46619 for16 h (IC50 RNO: 0.30 nM with 95% CI of 0.14 nM to 0.66 nM, n=8-10). In the acute studies (1 h incubation) following a 16 h pre-incubation with U46619, RNO inhibited O2•– formation at IC50 of 0.20 nM (95% CI of 0.02 nM to 2.01 nM, n=8-10). At 2 nM, RNO completely inhibited NOX-1 mRNA and NOX-1 protein expression induced with U46619. 16 h incubation with U46619 also increased Rac1 activity in hPASMCs which was blocked to basal levels following 1h incubation with 2nM RNO.

The PDE4 inhibitor, roflumilast N-oxide, inhibits the expression and activity of NOX-1 and therefore O2•– formation in hPASMCs, in part, through inhibition of Rac1 activity.

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