Glutathione redox cycle regulates nitric oxide-mediated
glyceraldehyde-3-phosphate dehydrogenase inhibition.
Padgett, Christine M., and A. Richard Whorton.
Department of Pharmacology, Duke University Medical Center, Durham,
NC 27710
APStracts 3:0257C, 1996.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been identified
as a potential target for nitric oxide (NO)-mediated cellular
toxicity. We have previously shown that NO inhibits GAPDH by S
-nitrosylation of the active site cysteine residue, which is reversed
by low molecular weight thiols. Since endothelial cells contain high
concentrations of low molecular weight thiols, principally
glutathione, we investigated the effect of NO on GAPDH activity in
intact endothelial cells and the influence that cellular glutathione
has on GAPDH inhibition. Our results show that incubation of cells
with an exogenous NO-generating system resulted in inhibition of
GAPDH activity. The mechanism for inhibition appears to involve
reversible modification of GAPDH because addition of thiols to cell
extracts restored activity. Further, cells were able to completely
recover GAPDH activity following removal of the NO-generating system.
Recovery did not require de novo protein synthesis. Depletion of
cellular glutathione levels by treatment of cells with buthionine
sulfoximine resulted in greater NO-mediated GAPDH inhibition as well
as a lesser ability to recover activity. Finally, disruption of the
glutathione redox cycle with the glutathione reductase inhibitor,
1,3-bis(2-chloroethyl)-1-nitrosourea, increased the extent of NO
-mediated GAPDH inhibition and decreased both the rate and degree of
recovery of GAPDH activity. These results suggest that the
glutathione redox cycle plays an important role not only in
regulating the extent of NO-mediated GAPDH inhibition, but also in
the ability of endothelial cells to recover from NO-mediated GAPDH
inhibition.
Received 3 May 1996; accepted in final form 24 July 1996.
APS Manuscript Number C237-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 21 August 1996