Superoxide Anion Radical-Triggered Ca2+ Release from Cardiac Sarcoplasmic Reticulum through Ryanodine Receptor Ca2+ Channel
- Department of Pharmacology and ESR Laboratory, Kanagawa Dental College, Yokosuka, Kanagawa 238, Japan
Abstract
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.
Footnotes
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Send reprint requests to: Dr. Eiichiro Okabe, D.D.S., Ph.D., Professor & Chairman, Department of Pharmacology and ESR Laboratory, Kanagawa Dental College, 82 Inaoka-Cho, Yokosuka, Kanagawa 238, Japan. E-mail: okabe{at}kdcnet.ac.jp
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This work was supported by Grants 09877356 (E.O.) and 07557119 (E.O.) from Scientific Research Fund of The Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Research Fund of JEOL Ltd., Tokyo, Japan.
- Abbreviations:
- SR
- sarcoplasmic reticulum
- Cai
- intravesicular free Ca2+
- O2⨪
- superoxide anion radical
- RyRC
- ryanodine receptor Ca2+-release channel(s)
- Po
- open probability
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- DMPO
- 5,5-dimethyl-1-ryrroline-N-oxide
- MnO
- manganese oxide marker
- SOD
- superoxide dismutase
- DMSO
- dimethylsulfoxide
- HO⋅
- hydroxyl radical
- Ca2+-ATPase
- Ca2+-stimulated, Mg2+- dependent ATPase
- SH
- sulfhydryl
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- Received July 14, 1997.
- Accepted October 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
