Comparative pharmacology of chemically distinct NADPH oxidase inhibitors

Abstract

BACKGROUND AND PURPOSE Oxidative stress [i.e. increased levels of reactive oxygen species (ROS)] has been suggested as a pathomechanism of different diseases, although the disease‐relevant sources of ROS remain to be identified. One of these sources may be NADPH oxidases. However, due to increasing concerns about the specificity of the compounds commonly used as NADPH oxidase inhibitors, data obtained with these compounds may have to be re‐interpreted. EXPERIMENTAL APPROACH We compared the pharmacological profiles of the commonly used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and 4‐(2‐amino‐ethyl)‐benzolsulphonyl‐fluoride (AEBSF), as well as the novel triazolo pyrimidine VAS3947. We used several assays for detecting cellular and tissue ROS, as none of them is specific and artefact free. KEY RESULTS DPI abolished NADPH oxidase‐mediated ROS formation, but also inhibited other flavo‐enzymes such as NO synthase (NOS) and xanthine oxidase (XOD). Apocynin interfered with ROS detection and varied considerably in efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low micromolar concentrations, and interfered neither with ROS detection nor with XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without effect. CONCLUSIONS AND IMPLICATIONS Our data suggest that triazolo pyrimidines such as VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to be a major source of ROS in aortas of SHRs.

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia

“…Pretreatment of platelets with diphenylene iodonium (DPI), an inhibitor of various sources of oxygen radical including NADPH oxidase, 31 resulted in less binding of opsonized platelets to CRP compared with untreated opsonized platelets ( Figure 4E). A similar reduction in binding of platelets to CRP was observed in the presence with Rotenone, a compound more specifically inhibiting the mitochondrial electron transport chain ( Figure 4F).…”

The decrease of NAD(P)H:quinone oxidoreductase 1 activity and increase of ROS production by NADPH oxidases are early biomarkers in doxorubicin cardiotoxicity

“…Moreover, Nox has been identified as the major source for NADPH-dependent production of superoxide anion in cardiac tissue (Nediani et al 2007). As apocynin, the most used Nox inhibitor, interferes with H2O2 detection with Amplex Red in cell-free systems (Heumuller et al 2008) and can behave itself as an antioxidant acting as a scavenger of reaction products of hydrogen peroxide (Wind et al 2010), we have experimentally assessed that apocynin affords a large inhibition of the coupled NADPH oxidase activity in our cardiac homogenates of doxorubicin-treated rats (day 3 post-injection). Our results showed that 250 and 500 μM apocynin inhibited 75±5% and >90%, respectively, of the total NADPH oxidase activity, which was 1.5±0.1 nmoles/min/mg of protein measured as indicated in the Materials and Methods section (average of data obtained with three different homogenates).…”

Taking up the cudgels for the traditional reactive oxygen and nitrogen species detection assays and their use in the cardiovascular system

“…Nrf2/HO-1) [19] or source specific inhibitors (e.g. for different Nox isoforms) [20], or even repair the resulting oxidative damage (e.g. activators and stimulators of oxidized soluble guanylyl cyclase) thus leaving important cellular redox signaling mechanisms intact [13,21].…”