Original ContributionHypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through the mitochondrial ROS-PKCɛ signaling axis in pulmonary artery smooth muscle cells
Section snippets
Reagents
5,6-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate and Fura-2/AM were obtained from Molecular Probes (Eugene, OR, USA), PKCɛ translocation peptide inhibitor and chelerythrine from Calbiochem (La Jolla, CA, USA), antibodies against the Nox subunits Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, and p67phox, as well as actin antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and rotenone, myxothiazol, antimycin A, hydrogen peroxide, phorbol 12-myristate 13-acetate (PMA), and
Major NADPH oxidase subunits are equivalently expressed in pulmonary and mesenteric arteries
Previous studies have shown that Nox is highly expressed in both pulmonary and systemic arteries [37]; thus, it is very unlikely that the heterogeneity of hypoxic responses in pulmonary and mesenteric arteries is related to the inherent nature of a distinct expression of Nox in these two different tissues. To exclude this possibility, nevertheless, we investigated the expression of the major phagocytic Nox subunits, including the membrane-bound subunits gp91phox (Nox2) and p22phox, as well as
Discussion
In this study we have for the first time demonstrated: (1) the major Nox subunit Nox1, Nox4, p22phox, p47phox, and p67phox proteins are equally expressed in both pulmonary and mesenteric (systemic) arteries; (2) acute hypoxia stimulates Nox in pulmonary, but not mesenteric, arteries; (3) Nox plays an important role in the hypoxia-induced increases in [ROS]i and [Ca2+]i as well as contraction in PASMCs; and (4) the hypoxia-induced activation of Nox in pulmonary arteries is mediated by the
Acknowledgments
The authors thank Ms. Jodi Heim and Ms. Krista Wardsworth for technical assistance. This work was supported by AHA Scientist Development Grants 0630236N (Y.-M.Z.) and 0730242N (Q.-H.L.), EHS Center Grant P30ES06639 (Y.-S.H.), and AHA Established Investigator Award 0340160N and NIH R01HL064043 and R01HL075190 (Y.-X.W.).
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