Abstract 1151: Glucocorticoids Modulate Glucose-6-Phosphate Dehydrogenase Activity to Preserve Bioavailable Nitric Oxide in Vascular Endothelial Cells
Glucocorticoids (GC) have been shown to impair vascular endothelial cell function, in part, by stimulating the production of reactive oxygen species (ROS). Glucose-6-phosphate dehydrogenase (G6PD) plays a central role in modulating endothelial cell oxidant stress by serving as the principal intracellular source of NADPH, which provides reducing equivalents to the cell, and is a cofactor for the endothelial isoform of nitric oxide synthase (eNOS) to produce nitric oxide (NO•). When G6PD activity is deficient, endothelial cells demonstrate increased oxidant stress and decreased levels of bioavailable NO•. We, therefore, hypothesized that G6PD regulates the response of endothelial cells to GC. To test this, bovine aortic endothelial cells (BAEC) were exposed to dexamethasone (DEX) (10−7, 10−6, or 10−5 mol/L) for 24 h and confirmed an increase in ROS formation (at 10−5 mol/L; 125 ± 14 % control, p<0.05). Compared to untreated cells, DEX (10−5 mol/L) increased G6PD protein (119 ± 5.1 % control, p<0.001) and mRNA expression (138 % control), which was associated with a concomitant increase in G6PD activity (139 ± 4.9 vs. 220 ± 43.7 units/6 min/mg protein, p<0.05) to maintain NADPH levels similar to that observed in untreated cells. This increase in G6PD expression was mediated by the GC receptor as co-incubation with RU-486 (10 μmol/L), a GC receptor antagonist, abolished this effect. In DEX-treated cells, with increased G6PD activity, there was an increase in bioavailable NO• as determined by nitrite levels (at 10−5 mol/L; 0.00302 ± 0.001 vs. 0.0044 ± 0.001 mmol/L/mg protein, p<0.05). Next, to confirm the protective role of G6PD in endothelial cells exposed to DEX, we transfected cells with an siRNA to G6PD for 48 h and demonstrated a 53 % decrease in G6PD protein expression and a 41% decrease in G6PD activity. In G6PD-deficient cells exposed to DEX (10−5 mol/L), ROS formation was increased as compared to control cells exposed to DEX (115.9 ± 4.9 vs. 161.9 ± 9.8 % control, p<0.01) and nitrite levels were decreased by 21% (p<0.04). Taken together, these data suggest that GC increases G6PD expression and activity in vascular endothelial cells to limit cellular oxidant stress and preserve bioavailable NO• when G6PD activity is deficient, endothelial dysfunction occurs.