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1 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA
2 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA
3 Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA
* To whom correspondence should be addressed. E-mail: pli{at}vcu.edu.
Non-mitochondrial NAD(P)H oxidases have been reported to importantly contribute to the intracellular levels of superoxide (O2.-) in vascular tissues, thereby to the regulation of vascular function. However, topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2.- into cytosol. It is now imperative to clarify the source of intracellular O2.- associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase exists on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle (CASM), which regulates the activity of SR ryandoine receptors (RyRs) by producing O2.- locally. Using Western blot analysis, NAD(P)H oxidase subunits were detected in the purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of the SR with NADH time-dependently produced O2.-, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors including diphenylene iodonium (DPI) and apocynin (Apo), and by superoxide dismutase (SOD) or its mimetic, Tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of the conversion rate of NADH into NAD+. In the experiments of lipid bilayer channel reconstitution, addition of NADH in the cis- solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM with a maximal increase in NPo from 0.0044 ± 0.0005 to 0.0213 ± 0.0018, and this effect of NADH was markedly blocked in the presence of SOD or Tiron or NAD(P)H oxidase inhibitors, DPI, N-vanillylnonanamide (NVN) or Apo. These results suggest that there is a local NAD(P)H oxidase system on the SR from CASM, which regulates the RyR/Ca2+ channel activity and Ca2+ release from the SR by producing O2.-.
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