Karen Bedard and Karl-Heinz Krause

260 KAREN BEDARD AND KARL-HEINZ KRAUSE
upon binding to p67 phox . p40 phox is involved in the regulation
of NOX2. One study suggests that p40 phox might
inhibit NOX2 function (778); however, most evidence indicates
that it enhances oxidase function (173, 493, 888).
Unlike p47 phox and p67 phox , p40 phox is dispensable for
NOX2 activity and no CGD patients with p40phox deficiency
have been described (585). Presently available
data also suggest that p40 phox is specific for NOX2. However,
as NOX1 and NOX3 could potentially be activated by
the p47 phox /p67 phox complex, a role of p40 phox in this
scenario cannot be excluded.
5. Rac GTPases
There are three highly homologous Rac proteins in
mammals: Rac1, ubiquitously distributed; Rac2, mostly
expressed in myeloid cells; and Rac3, predominantly
found in the central nervous system. Rac proteins are
clearly involved in the regulation of the phagocyte
NADPH oxidase NOX2. However, Rac proteins are not
NOX subunits in the strict sense because they regulate
other cellular functions, in particular the cytoskeleton.
Thus we do not discuss Rac GTPases in detail in this
review and refer readers to specific articles (92, 93, 197,
215, 776).
Of relevance for this review is our present knowledge
on the importance of Rac proteins in the activation of
different NOX isoforms. The involvement of Rac in NOX2
and NOX1 activation is established beyond doubt. The
involvement of Rac in NOX3 activation is a matter of
debate (615, 891). NOX4 activity appears to be Rac independent
upon heterologous expression (584); however,
some data from cells endogenously expressing NOX4 provide
arguments compatible with a role of Rac in NOX4
activation (311, 410). Presently there are no data in favor
of a role for Rac proteins in NOX5 activation. Finally,
DUOX-dependent ROS generation in thyroid cells does
not involve Rac1 activation (270).
While there is no doubt that Rac GTPases are important
activators of NOX2 and NOX1 and possibly some
other NOX isoforms, it should be kept in mind that they
may also be involved in regulating ROS generation from
other sources, particularly mitochondria (724, 948). Thus
the numerous studies suggesting the involvement of a
NOX NADPH oxidase in biological processes based on
inhibition by dominant negative Rac should be taken with
caution.
C. NOX Inhibitors
At present, no specific NOX inhibitors exist. Yet,
many studies are based on the use of inhibitors (for a
recent review, see Ref. 155). We therefore briefly describe
some of the most commonly used compounds.
1. Diphenylene iodonium
The most commonly used NOX inhibitor is the iodonium-derivative
diphenylene iodonium (DPI). The compound
acts by abstracting an electron from an electron
transporter and forming a radical, which then inhibits the
respective electron transporter through a covalent binding
step (661). In the case of NOX enzymes, it is not clear
whether the iodonium radical formation occurs through
interaction with the flavin group (661) or the heme group
(224). As suggested by the mechanism of action, DPI is a
nonspecific inhibitor of many different electron transporters.
It inhibits not only all of the NOX isoforms, but also
nitric oxide synthase (835), xanthine oxidase (225), mitochondrial
complex I (544), and cytochrome P-450 reductase
(713). To some extent, the lack of specificity of DPI
can be addressed by using specific inhibitors of other
sources of ROS.
2. Apocynin
The plant phenol 4-hydroxy-3-methoxyacetophenone,
referred to as apocynin, was described around 1990
as a low-affinity inhibitor (IC 50 �10 �M) of the phagocyte
respiratory burst (817). Studies on the effect of the phagocyte
NADPH oxidase suggested that apocynin 1) must be
metabolized by peroxidases to generate the inhibitory
compound and 2) acts by inhibiting the translocation of
cytoplasmic subunits (833). According to statement by
Lafeber et al. (502), the compound has been used in
patients without signs of toxicity. While these initial studies
on apocynin appear solid, a low-affinity prodrug is not
an ideal drug candidate, nor is it a powerful tool for
research. Nevertheless, apocynin is now used indiscriminately
as a bona fide NOX inhibitor. Apocynin at extremely
high concentrations has been used as a NOX4
inhibitor (e.g., Refs. 247, 758) and as a NOX5 inhibitor
(274), although there is presently no evidence for a subunit
dependence of these isoforms. Apocynin is also
widely used in cell types where there is no evidence for
the presence of a peroxidase. A final complicating factor
in the use of apocynin comes from recent studies which
suggest that apocynin may actually stimulate ROS production
in nonphagocytic cells (747, 913).
3. AEBSF
Physiol Rev VOL 87 JANUARY 2007 www.prv.org
4-(2-Aminoethyl)benzenesulfonylfluoride (AEBSF) is
a serine protease inhibitor that inhibits NOX enzymes by
interfering with the association of the cytoplasmic subunit
p47 phox (213). It is not clear whether AEBSF really
inhibits NOX enzymes or rather acts on signaling steps
towards p47 phox activation. AEBSF is also likely to have
NOX-unrelated effects due to serine protease inhibition
(155).
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