Glutathione redox cycle regulates nitric oxide-mediated glyceraldehyde-3-phosphate dehydrogenase inhibition.
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. Because 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. Furthermore, cells were able to completely recover GAPDH activity after 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.
Padgett
C M
CM
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Whorton
A R
AR
D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D010084:Oxidation-Reduction
D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005980:Glutathione Reductase; D005978:Glutathione; D004229:Dithiothreitol; D002330:Carmustine