ToxSci Advance Access published online on August 9, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm212
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Glutathione-dependent Reduction of Arsenate by Glycogen Phosphorylase – Responsiveness to Endogenous and Xenobiotic Inhibitors
Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School
1 Corresponding author: Zoltán Gregus, M.D., Ph.D., D.Sc., Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary. Tel.: +36-72-536-000; Fax: +36-72-536-218; E-mail: zoltan.gregus{at}aok.pte.hu
Received June 13, 2007; revision received August 2, 2007; accepted August 2, 2007
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Abstract |
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Rabbit muscle glycogen phosphorylase a (GPa) reduces arsenate (As(V)) to the more toxic arsenite (As(III)) in a glutathione (GSH)-dependent fashion. To determine whether reduction of As(V) by GPa is countered by compounds known to inhibit GP-catalyzed glycogenolysis, the effects of thiol reagents, endogenous compounds (glucose, ATP, ADP) as well as non-specific GP inhibitors (GPI; caffeine, quercetin, flavopiridol), and specific GPIs (DAB, BAY U6751, CP320626) were tested on reduction of As(V) by rabbit muscle GPa in presence of glycogen (substrate), AMP (activator) and GSH, and the As(III) formed from As(V) was quantified by HPLC-HG-AFS. The As(V)-reducing activity of GPa was moderately sensitive to thiol reagents. Glucose above 5 mM and ADP or ATP at physiological levels diminished GPa-catalyzed As(V) reduction. All GPIs inhibited As(V) reduction by GPa in a concentration dependent fashion, however, their effects were differentially affected by glucose (10 mM) or AMP (200 µM instead of 25 µM), known modulators of the action of some GPIs on the GP-catalyzed glycogenolysis. Inhibition of As(V) reduction by 1,4-dideoxy-1,4-iminoarabinitol (DAB) and quercetin was not influenced by glucose or AMP. Glucose that potentiates the inhibitory effects of caffeine, BAY U6751 and CP320626 on the glycogenolytic activity of GPa also enhanced the inhibitory effects of these GPIs on GPa-catalyzed As(V) reduction. AMP at high concentration alleviated the inhibition by BAY U6751 and CP320626 (whose antagonistic effect on GP-catalyzed glycogen breakdown is also AMP sensitive), whereas the inhibition in As(V) reduction by flavopiridol or caffeine was little affected by AMP. Thus, GPIs inhibit both the glycogenolytic and As(V)-reducing activities of GP, supporting that the latter is coupled to glycogenolysis. It was also shown that a GPa-rich extract of rat liver contained GSH-dependent As(V)-reducing activity that was inhibited by specific GPIs, suggesting that the liver type GPa can also catalyze reduction of As(V).
Key Words: arsenate; reduction; glycogen phosphorylase; glycogen phosphorylase inhibitors; glutathione.