The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by 
angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells 
(VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats 
(SHR) was studied. Expression of Nox1, Nox2, and Nox4 (gene and protein) and 
NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity 
was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and 
by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in 
WKY, only Nox1 was influenced in SHR. Ang II-induced NAD(P)H activity was 
inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox 
expression only in WKY and increased NAD(P)H oxidase activity, an effect 
inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with Nox4, 
implicating association between Nox1 and Nox2 but not between Nox1 and Nox4. 
These data highlight the complexity of Nox biology in VSMCs, emphasising that 
more than one Nox member, alone or in association, may be involved in NAD(P)H 
oxidase-mediated •O(2)(-) production. Nox1 regulation by Ang II, but not by 
ET-1, may be important in •O(2)(-) formation in VSMCs from SHR.