ryanodine and diphenyleneiodonium

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.

Topics: Acetophenones; ADP-ribosyl Cyclase; Animals; Calcimycin; Calcium; Calcium Signaling; Catalase; Cattle; Cells, Cultured; Coronary Vessels; Cyclic ADP-Ribose; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Ionophores; Microscopy, Confocal; Muscarinic Agonists; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Niacinamide; Onium Compounds; Oxotremorine; Receptor, Muscarinic M1; RNA Interference; RNA, Small Interfering; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Superoxide Dismutase; Superoxides; Time Factors

NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)-mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120 micromol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart copurified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited >9-fold higher catalytic activity (8.8 micromol/mg protein per minute) in the absence of exogenous mitochondrial complex I (ubiquinone) or complex III (cytochrome c) electron acceptors, but was inhibited by rotenone and pyridaben (IC50=2 to 3 nmol/L), antimycin A (IC50=13 nmol/L), and diphenyleneiodonium (IC50=28 micromol/L). Cardiac junctional SR treated with [3H](trifluoromethyl)diazirinyl-pyridaben specifically labeled a single 23-kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative-feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Calcium; Calcium Signaling; Electron Transport Complex I; Enzyme Inhibitors; Feedback, Physiological; Ion Channel Gating; Ion Transport; Mitochondria, Heart; Molecular Weight; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Onium Compounds; Pyridazines; Rats; Rats, Sprague-Dawley; Rotenone; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Substrate Specificity