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NADPH oxidases (Noxs) are a major source of reactive oxygen species (ROS) in vascular cells. ROS are important signalling molecules with diverse actions. Mechanisms underlying differential ROS effects may relate, in part, to subcellular localization and Nox isoform specificity. We investigated the compartmentalization of Noxs and ROS in vascular smooth muscle cells (VSMC) and questioned whether these phenomena are altered in hypertension. VSMCs isolated from mesenteric arteries of Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. Subcellular compartmentalization of Noxs was evaluated by immunoblotting after organelle fractionation. ROS levels were measured by chemiluminescence (O2-) and amplex red (H2O2) in the absence or presence of of ML171 (Nox1 inhibitor), GKT136901 (Nox1/4 inhibitor), mito-tempol (mitochondrial-targeted antioxidant) and 4-PBA (ER stress inhibitor). Protein oxidation was assessed using the fluorescent probe DCP-Rho1 for protein sulfenylation and the oxyblot assay for protein carbonylation. Oxidation of protein tyrosine phosphatases (PTP) was evaluated by immunoblotting and Peroxiredoxin (Prx) oxidation was assessed by one-dimensional isoelectric focusing. Vascular reactivity was assessed by myography ± DTT (reducing agent) and peroxiredoxin inhibitor (Conoidin A). Expression of Nox1, Nox2 and Nox4 was greater in total cell homogenates from SHRSP versus WKY. Nox isoforms were detected in plasma membrane, ER and nucleus in both strains, but not in the mitochondria. Basal ROS generation was increased in SHRSP cells. In WKY only Nox1 inhibition decreased Ang II-induced ROS generation. Inhibition of Nox1 and Nox4 decreased basal and Ang II-induced ROS in SHRSP. Additionally, mito-tempol and 4-PBA reduced basal ROS generation in SHRSP. Analysis of protein oxidation revealed increased protein carbonylation and PTP oxidation in SHRSP. Furthermore, oxidation of the antioxidant enzymes Prxs was increased in SHRSP. Prx2, localised in the cytosol, and mitochondrial Prx3 were more oxidised in SHRSP cells than WKY cells. Noradrenaline-induced vascular contraction was reduced by DTT and Conoidin A. Our data demonstrate that Noxs are expressed in an organelle-specific manner, with Nox1,2,4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHRSP VSMCs Nox expression, ROS generation and protein oxidation are increased. Inhibition of oxidation attenuated vascular reactivity. These findings suggest an important role for Nox1/4 in oxidative stress and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.