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Hypoxia appears to promote contraction [hypoxic pulmonary vasoconstriction (HPV)] of bovine pulmonary arteries (BPA) through removal of a peroxide-mediated relaxation. This study examines the roles of BPA Nox oxidases and mitochondria in the HPV response. Inhibitors of Nox2 (0.1 mM apocynin and 50 muM gp91-dstat) and mitochondrial electron transport (10 muM antimycin and rotenone) decreased superoxide generation in BPA without affecting contraction to 25 mM KCl or the HPV response. Transfection of BPA with small inhibitory RNA (siRNA) for Nox2 and Nox4 decreased Nox2 and Nox4 protein expression, respectively, associated with an attenuation of superoxide detection, without affecting 25 mM KCl contraction. However, Nox4 siRNA, but not Nox2, attenuated HPV in BPA. A Nox4 inhibitor plumbagin (10 muM) increased basal force, decreased superoxide detection and peroxide release, and caused BPA to relax under hypoxia. Although acute removal of peroxide with 0.1 mM ebselen increased 25 mM KCl contraction and decreased hypoxic contraction, prolonged treatment with ebselen only decreased hypoxic contraction without affecting 25 mM KCl contraction, suggesting basal peroxide levels also maintain a contractile mechanism not removed by acute hypoxia. Organ culture of BPA with transforming growth factor (TGF)-beta1 (4 nM) increased Nox4 expression, superoxide, peroxide, and the HPV response. Thus Nox2 and mitochondria are sources for superoxide generation in BPA, which do not appear to influence the HPV response. However, peroxide derived from superoxide generated by Nox4 appears to maintain a basal relaxation in BPA under normoxic conditions, which is removed under hypoxia leading to HPV. Peroxide generated by Nox4 may also function to maintain a contractile mechanism, which is not reversed by acute hypoxia.

Roles for Nox4 in the contractile response of bovine pulmonary arteries to hypoxia