Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle.
It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is 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 on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.
Yi
Xiu-Yu
XY
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, USA.
Li
Victoria X
VX
Zhang
Fan
F
Yi
Fan
F
Matson
Daniel R
DR
Jiang
Ming Tao
MT
Li
Pin-Lan
PL
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000201:enzymology*; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D019255:NADPH Oxidases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D019837:Ryanodine Receptor Calcium Release Channel; D019255:NADPH Oxidases; D010100:Oxygen