1. Local production of O2- by NAD(P)H oxidase in the sarcoplasmic reticulum of coronary arterial myocytes: cADPR-mediated Ca2+ regulation.
- Author
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Zhang F, Jin S, Yi F, Xia M, Dewey WL, and Li PL
- Subjects
- ADP-ribosyl Cyclase antagonists & inhibitors, ADP-ribosyl Cyclase genetics, ADP-ribosyl Cyclase metabolism, Acetophenones pharmacology, Animals, Calcimycin pharmacology, Calcium metabolism, Catalase metabolism, Cattle, Cells, Cultured, Coronary Vessels enzymology, Coronary Vessels metabolism, Cyclic ADP-Ribose analogs & derivatives, Cyclic ADP-Ribose antagonists & inhibitors, Cyclic ADP-Ribose pharmacology, Electron Spin Resonance Spectroscopy, Enzyme Inhibitors pharmacology, Ionophores pharmacology, Microscopy, Confocal, Muscarinic Agonists pharmacology, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular enzymology, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle enzymology, NADPH Oxidases antagonists & inhibitors, NADPH Oxidases genetics, Niacinamide pharmacology, Onium Compounds pharmacology, Oxotremorine pharmacology, RNA Interference, RNA, Small Interfering metabolism, Receptor, Muscarinic M1 agonists, Receptor, Muscarinic M1 metabolism, Ryanodine pharmacology, Ryanodine Receptor Calcium Release Channel genetics, Ryanodine Receptor Calcium Release Channel metabolism, Sarcoplasmic Reticulum drug effects, Sarcoplasmic Reticulum enzymology, Superoxide Dismutase metabolism, Time Factors, Calcium Signaling drug effects, Cyclic ADP-Ribose metabolism, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism, NADPH Oxidases metabolism, Sarcoplasmic Reticulum metabolism, Superoxides metabolism
- Abstract
The present study was designed to determine whether the sarcoplasmic reticulum (SR) could locally produce superoxide (O2-) via NAD(P)H oxidase (NOX) in coronary arterial myocytes (CAMs) and to address whether cADPR-RyR/Ca2+ signaling pathway regulates this local O2- production from the SR. Using confocal microscopic imaging analysis in intact single CAMs, a cell-permeable indicator CM-H2DCFDA for dynamic changes in intracellular ROS (in green color) and a highly selective ER-Tracker Red dye for tracking of the SR were found co-localized. A quantitative analysis based on the intensity of different spectra demonstrated a local O2- production derived from the SR. M(1)-receptor agonist, oxotremorine (Oxo) and a Ca2+ ionophore, A23187, time-dependently increased this O2- production colocalized with the SR. NOX inhibitors, diphenylene iodonium (DPI) and apocynin (Apo), or superoxide dismutase (SOD) and catalase, and Nox4 (a major intracellular NOX subunit) siRNA all substantially blocked this local production of O2-, demonstrating an involvement of NOX. This SR-derived O2- production was also abolished by the inhibitors of cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling, such as nicotinamide (Nicot, 6 mM), ryanodine (Rya, 50 muM) or 8-Br-cADPR (30 microM). However, IP3 antagonist, 2-APB (50 microM) had no effect. In CAMs transfected with siRNA of ADP-ribosyl cyclase or RyR, this SR O2- production was attenuated. Electron spin resonance (ESR) spectromic assay in purified SR also demonstrated the production of O2- that was dependent on NOX activity and Ca2+ concentrations. These results provide direct evidence that O2- could be locally produced via NOX on the SR and that this local O2- producing system is controlled by cADPR-RyR/Ca2+ signaling pathway.
- Published
- 2008
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