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 (O-2(center dot-)) via NAD(P)H oxidase (NOX) in coronary arterial myocytes (CAMs) and to address whether cADPR-RyR/Ca2+ signaling pathway regulates this local O-2(center dot-) production from the SR. Using confocal microscopic imaging analysis in intact single CAMs, a cell-permeable indicator CM-H(2)DCFDA for dynamic changes in intracellular ROS (in green color) and a highly selective ER-Tracker (TM) Red dye for tracking of the SR were found co-localized. A quantitative analysis based on the intensity of different spectra demonstrated a local O-2(center dot-) production derived from the SR. M-1-receptor agonist, oxotremorine (Oxo) and a Ca2+ ionophore, A23187, time-dependently increased this O-2(center dot-) 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 O-2(center dot-), demonstrating an involvement of NOX. This SR-derived O-2(center dot-) production was also abolished by the inhibitors of cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling, such as nicotinamide (Nicot, 6 mM), ryanodine (Rya, 50 mu M) or 8-Br-cADPR (30 mu M). However, IP3 antagonist, 2-APB (50 mu M) had no effect. In CAMs transfected with siRNA of ADP-ribosyl cyclase or RyR, this SR O-2(center dot-) production was attenuated. Electron spin resonance (ESR) spectromic assay in purified SR also demonstrated the production of O-2(center dot-) that was dependent on NOX activity and Ca2+ concentrations. These results provide direct evidence that O-2(center dot-) could be locally produced via NOX on the SR and that this local O-2(center dot-) producing system is controlled by cADPR-RyR/Ca2+ signaling pathway. (C) 2007 Elsevier Inc. All rights reserved.