The ryanodine receptor Ca2+ channel (RyRC) constitutes the Ca2+-release pathway in sarcoplasmic reticulum (SR) of cardiac muscle. A direct mechanical and a Ca2+-triggered mechanism (Ca2+-induced Ca2+ release) have been proposed to explain the in situ activation of Ca2+ release in cardiac muscle. A variety of chemical oxidants have been shown to activate RyRC; however, the role of modification induced by oxygen-derived free radicals in pathological states of the muscle remains to be elucidated. It has been hypothesized that oxygen-derived free radicals initiate Ca2+-mediated functional changes in or damage to cardiac muscle by acting on the SR and promoting an increase in Ca2+ release. We confirmed that superoxide anion radical (O2-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles; hypoxanthine-xanthine oxidase also decreases Ca2+ free within the intravesicular space of the SR with no effect on Ca2+-ATPase activity. Current fluctuations through single Ca2+-release channels have been monitored after incorporation into planar phospholipid bilayers. We demonstrate that activation of the channel by O2- is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O2--triggered Ca2+ release through the RyRC. For the first time, we show that O2- stimulates Ca2+ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2-. The decreased calmodulin content induced by oxygen-derived free radicals, especially O2-, is a likely mechanism of accumulation of cytosolic Ca2+ (due to increased Ca2+ release from SR) after reperfusion of the ischemic heart.

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In the temporomandibular joint (TMJ), it has been hypothesized that mechanical stresses lead to the oxidative stress of articular tissues. It has also been postulated that cells pertinent to arthritis-including endothelial cells and synovial cells-when stimulated by mechanical stresses and/or pro-inflammatory cytokines, promote oxidative damage. To determine the involvement of reactive oxygen species (ROS) in the diseased joint, we studied the generation of ROS in synovial fluid (SF) from interleukin-1alpha (IL-1alpha)-induced TMJ arthritis by electron spin resonance (ESR) spectroscopy, using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The TMJ arthritis was experimentally induced in rats by the injection of human recombinant IL-1alpha into the TMJ; control rats were treated with normal saline solution. We found that the detected radicals in the collected SF were identified as a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct. The ESR signal intensity of the hydroxyl radical-DMPO spin adduct in the SF from IL-1-treated rats was significantly higher than that from the control rats (P < 0.01). The results of ESR study also showed that hydroxyl radical (HO*) was increased in a time-dependent fashion in the presence of superoxide anion radical (O2*-) scavenger superoxide dismutase (SOD); the formation of DMPO-HO* was strongly inhibited by the iron chelater deferoxamine. We could measure higher levels of free iron (Fe2- and Fe3-) in the SF from TMJ arthritis than in that from controls (P < 0.05). Analysis of the data obtained from the present study suggests that the HO* radical detected in SF from IL-1-induced TMJ arthritis is generated via a modified Haber-Weiss reaction (biological Fenton reaction) in which O2*- can subsequently result in the production of H2O2 through dismutation reaction by SOD. Thus, HO* may be generated from the reaction of resultant H2O2 with free iron ions. The results presented here provide the first evidence of involvement of ROS in IL-1-induced TMJ arthritis.

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