Abstract
Quinones undergo redox cycling and/or arylation reactions with key biomolecules involved with cellular Ca2+ regulation. The present study utilizes nanomolar quantities of the fluorogenic maleimide 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) to measure the reactivity of hyperreactive sulfhydryl moieties on sarcoplasmic reticulum (SR) membranes in the presence and absence of quinones by analyzing the kinetics of forming CPM-thioether adducts and localization of fluorescence by SDS-polyacrylamide gel electrophoresis. Doxorubicin, 1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ) are found to selectively and dose-dependently interact with a class of hyperreactive sulfhydryl groups localized on ryanodine-sensitive Ca2+ channels [ryanodine receptor (RyR)], and its associated protein, triadin, of skeletal type channels. NQ and BQ are the most potent compounds tested for reducing the rate of CPM labeling of hyperreactive SR thiols (IC50 = 0.3 and 1.8 μM, respectively) localized on RyR and associated protein. The reduced forms of quinone, tert-butylhydroquinone, and 5-imino-daunorubicin do not alter significantly the pattern or kinetics of CPM labeling up to 100 μM, demonstrating that the quinone group is essential for modulating the state of hyperreactive SR thiols. Nanomolar NQ is shown to enhance the association of [3H]ryanodine for its high-affinity binding site and directly enhance channel-open probability in bilayer lipid membrane in a reversible manner. By contrast, micromolar NQ produces a time-dependent biphasic action on channel function, leading to irreversible channel inactivation. These results provide evidence that nanomolar quinone selectively and reversibly alters the redox state of hyperreactive sulfhydryls localized in the RyR/Ca2+ channel complex, resulting in enhanced channel activation. The Ca2+-dependent cytotoxicities observed with reactive quinones formed at the microsomal surface by oxidative metabolism may be related to their ability to selectively modify hyperreactive thiols regulating normal functioning of microsomal Ca2+ release channels.
Footnotes
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Send reprint requests to: Dr. Isaac N. Pessah, Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616. E-mail:inpessah{at}ucdavis.edu
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This work was supported by Grant ES05002, National Institute of Environmental Health Sciences Center for Environmental Health Sciences Grant ES05707 from the National Institutes of Health (to I.N.P.), and Oregon Affiliate of the American Heart Association (to J.J.A.).
- Abbreviations:
- BLM
- bilayer lipid membrane
- BQ
- 1,4-benzoquinone
- CE
- coupling enzyme
- CPM
- 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin
- DXR
- doxorubicin
- IDAU
- 5-iminodaunorubicin
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- NQ
- 1,4-naphthoquinone
- NQS
- 1,2-naphthoquione-4-sulfonic acid
- NEM
- N-ethylmaleimide
- PAGE
- polyacrylamide gel electrophoresis
- RR
- ruthenium red
- RyR1
- skeletal isoform of ryanodine receptor
- RyR2
- cardiac form of ryanodine receptor
- SERCA
- SR/ER Ca2+ ATPase
- SR
- sarcoplasmic reticulum, TG, thapsigargin
- THQ
- tert-butylhydroquinone
- Received July 29, 1998.
- Accepted February 12, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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