Local production of O2- by NAD(P)H oxidase in the sarcoplasmic reticulum of coronary arterial myocytes: cADPR-mediated Ca2+ regulation.
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.
Zhang
Fan
F
Department of Pharmacology & Toxicology, Medical College of Virginia, Virginia Commonwealth University, VA 23298, United States.
Jin
Si
S
Yi
Fan
F
Xia
Min
M
Dewey
William L
WL
Li
Pin-Lan
PL
D036541:ADP-ribosyl Cyclase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002374:Catalase / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000201:enzymology / Q000378:metabolism; D036563:Cyclic ADP-Ribose / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018613:Microscopy, Confocal; D018721:Muscarinic Agonists / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009536:Niacinamide / Q000494:pharmacology; D009861:Onium Compounds / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors
C493642:8-bromo-cyclic-ADP-ribose; D000098:Acetophenones; D004791:Enzyme Inhibitors; D007476:Ionophores; D018721:Muscarinic Agonists; D009861:Onium Compounds; D034741:RNA, Small Interfering; D043643:Receptor, Muscarinic M1; D019837:Ryanodine Receptor Calcium Release Channel; D013481:Superoxides; D036563:Cyclic ADP-Ribose; D012433:Ryanodine; D009536:Niacinamide; D000001:Calcimycin; D010095:Oxotremorine; C007517:diphenyleneiodonium; C056165:acetovanillone; D002374:Catalase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D036541:ADP-ribosyl Cyclase; D002118:Calcium