SINGLET OXYGEN INTERACTION WITH CA2+-ATPASE OF CARDIAC SARCOPLASMIC-RETICULUM

We investigated the role of singlet oxygen (generated from photoactivation of rose bengal) on the calcium transport and Ca2+-ATPase activity of cardiac sarcoplasmic reticulum (SR). Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at 560 nm resulted in significant inhibition of Ca2+ uptake (from 2.27 +/- 0.05 to 0.62 +/- 0.05-mu-mol Ca2+/mg.min [mean +/- SEM], p < 0.01) and Ca2+-ATPase activity (from 2.08 +/- 0.05 to 0.28 +/- 0.04-mu-mol P(i)/min.mg [mean +/- SEM], p < 0.01). The inhibition of calcium uptake and Ca2+-ATPase activity by rose bengal-derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. Singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca2+-ATPase against rose bengal-derived activated oxygen species, but superoxide dismutase and catalase did not attenuate the inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal for up to 14 minutes demonstrated complete loss of the Ca2+-ATPase monomer band, which was significantly protected by histidine. The addition of dithiothreitol (5 mM) had a slight protective effect, showing that new disulfide bond formation was not a major cause of aggregation. The results were also confirmed by high-performance liquid chromatography of the SR exposed to irradiated rose bengal. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide radical (generated from xanthine oxidase action on xanthine) and hydroxyl radical (in the presence of Fe3+-EDTA or 0.5 mM H2O2 plus Fe2+-EDTA) as well as H2O2 (0.25-12 mM) were without any effect on the 97,000-d Ca2+-ATPase band of SR. Generation of radical species (superoxide and hydroxyl radical) from rose bengal was studied by electron paramagnetic resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results showed that irradiation of rose bengal formed a 1:2:2:1 quartet, characteristic of the DMPO-OH adduct, which was scavenged by ethanol but not by superoxide dismutase, catalase, or histidine. No radical species could be detected from irradiated rose bengal or irradiated DMPO under the assay conditions used. Peroxy adducts of DMPO might be produced but would be observed only at very low temperatures. Similarly, we could not detect any measurable . O2- anion from irradiation of rose bengal as indicated by either cytochrome c reduction at 550 nm or nitro blue tetrazolium reduction at 560 nm. These results show that SR is damaged most likely by singlet oxygen derived from rose bengal. This may represent an important mechanism by which the oxidative injury to the myocardium induces both a loss of tension development and arrhythmogenesis.