Submitted on June 24, 2004
Accepted on August 27, 2004
Cytosolic NADPH May Regulate Differences in Basal Nox Oxidase-Derived Superoxide Generation in Bovine Coronary and Pulmonary Arteries
Sachin A Gupte, Pawel M Kaminski, Beverly Floyd, Ritu Agarwal, Noorjahan Ali, Mansoor Ahmad, John Edwards, and Michael S Wolin*
* To whom correspondence should be addressed. E-mail: mike_wolin{at}nymc.edu.
Since systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox-oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated approximately 60-80% more lucigenin (5 µM) chemiluminescence detectable superoxide than BCA. Apocynin (10 µM), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70 %) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox-2 or Nox-4 mRNA or protein detected by western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus, BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Since both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA.
