SKELETAL SARCOPLASMIC-RETICULUM DYSFUNCTION INDUCED BY REACTIVE OXYGEN INTERMEDIATES DERIVED FROM PHOTOACTIVATED ROSE-BENGAL

We investigated the role of reactive oxygen intermediates generated fr om photoactivation of xanthene dye rose bengal on skeletal sarcoplasmi c reticulum (SR) function, which plays a major role in the regulation of intracellular Ca++ and thereby in the generation of force. We used SR microsomes of canine masseter muscle as a model system in which to explore the effect of oxidation by determining oxalate-supported Ca+uptake, Ca++, Mg++-adenosine triphosphatase (Ca++-ATPase) activity and Ca++ permeability of the SR vesicles. Skeletal SR vesicles exposed to rose bengal (50 nM) illuminated at 560 nm resulted in significant inh ibition of Ca++ uptake velocity and Ca++-ATPase activity and in stimul ation of Ca++ permeability. The observed effect afforded by illuminate d rose bengal was dependent on intensity of light. Most reactive oxyge n species scavengers tested had no protective effect; histidine (a pow erful quenching agent for singlet oxygen), however, significantly prot ected the effect of illuminated rose bengal on Ca++ uptake velocity an d Ca++-ATPase activity. The illumination of rose bengal also caused hi stidine- inhibitable loss of total sulfhydryl groups of SR. The increa sed Ca++ permeability elicited by illuminated rose bengal was blunted by a cocktail of histidine-catalase, but not by histidine alone. Gener ation of reactive oxygen species (singlet oxygen, superoxide and hydro xyl radical) from photoactivation of rose bengal was studied by electr on spin resonance spectroscopy by use of the spin trap 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP). We found that illumination of rose bengal formed a 1:2:2:1 quartet, chara cteristic of the hydroxyl radical-DMPO spin adduct, which was effectiv ely blunted by hydroxyl radical scavenger, dimethyl sulfoxide, and by superoxide scavenger, superoxide dismutase, The results of electron sp in resonance study also showed that singlet oxygen was produced by pho toactivation of rose bengal and was detected as singlet oxygen-TEMP pr oduct (TEMPO; 2,2,6,6-tetramethylpiperidine-N-oxyl). The formation of TEMPO signal was strongly inhibited by histidine. Similarly, we could detect hydrogen peroxide production from illuminated rose bengal. It i s suggested that photoactivation of rose bengal generates singlet oxyg en, superoxide, hydrogen peroxide and hydroxyl radical, and the data o btained from the present study indicate that singlet oxygen, rather th an superoxide, hydrogen peroxide and hydroxyl radical, to be the activ e agent in the Ca++ transport system of SR; the observed effect of sin glet oxygen may be due to sulfhydryl group oxidation. Our results are also consistent with the view that singlet oxygen does not appear to b e an exclusive species that increases Ca++ permeability of SR vesicles , but the increased Ca++ permeability may be caused in part by hydroge n peroxide as well as singlet oxygen.