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Glucose-6-phosphate dehydrogenase deficiency promotes endothelial oxidant stress and decreases endothelial nitric oxide bioavailability¹

Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA; and
^* Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02115, USA

²Correspondence: Whitaker CVI, CABR-507, Boston University School of Medicine, 715 Albany St., Boston, MA 02118, USA. E-mail: jane.leopold{at}bmc.org

SPECIFIC AIMS

The vascular endothelium responds to local oxidant stress by increasing the activity of antioxidant enzymes such as glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway and the principal source of cellular NADPH. NADPH is used as an intracellular reducing equivalent to protect against oxidative injury in addition to a cofactor for nitric oxide synthase (NOS) activity. In this study, we examined the role of deficient G6PD activity on cellular oxidant stress and NOS activity in aortic endothelial cells.

PRINCIPAL FINDINGS

1. Inhibition of G6PD activity promotes endothelial cell oxidant stress
Bovine aortic endothelial cells (BAEC) were treated with dehydroepiandrosterone (DHEA) (100 µM), a noncompetitive inhibitor of G6PD. Treatment with DHEA for 24 h significantly decreased G6PD activity (116.6±6.0 vs. 30.1±6.0 units/6 min/mg protein, n=6, P<0.0001), resulting in diminished NADPH stores (0.4±0.03 vs. 0.3±0.003 nmol/mg protein, n=3, P<0.03). Basal levels of reactive oxygen species (ROS) as determined by DCF fluorescence were not significantly different in BAEC with reduced G6PD activity than untreated cells (186.2±10.2 vs. 253.8±23.7 arbitrary fluorescent units (FU), n = 5, p = NS); however, once exposed to hydrogen peroxide (H₂O₂) (100 µM), there was a marked increase in ROS accumulation compared to control cells (617.9±62.9 vs. 1090.3±121.1 AFU, n=6, P<0.0005). After exposure to H₂O₂, cellular GSH levels were significantly decreased in G6PD-deficient cells compared to untreated cells (227.4±3.5 vs. 67.09±1.0 µmol/l/mg protein, n=4, P<1x10^-10).

2. Inhibition of G6PD expression promotes endothelial cell oxidant stress
G6PD expression was decreased by treatment with an antisense oligodeoxynucleotide to G6PD mRNA. After transfection, there was a significant reduction in G6PD expression as determined by Western analysis. Compared to cells treated with a scrambled oligodeoxynucleotide, there was a decrease in G6PD activity (76.0±2.0 vs. 18.2±5.1 units/6 min/mg protein, n=6, P<0.0002) and NADPH levels (0.4±0.01 vs. 0.2±0.001 mmol/mg protein, n=3, P<0.0001). Similar to DHEA-treated cells, ROS accumulation after exposure to H₂O₂ was increased (617.9±62.9 vs. 976.4±17.5 AFU, n=6, P<0.0005). This was accompanied by a significant decrease in cellular GSH levels in G6PD-deficient cells (214.3±3.7 vs. 138.7±4.2 µmol/l/mg protein, n=4, P<1x10^-10) (Fig. 1 ).

View larger version (27K): [in this window] [in a new window]   Figure 1. Antisense phosphorothioate oligodeoxynucleotide to G6PD and G6PD expression, activity, NADPH levels, and ROS production in BAECs. A) BAEC were transfected with an antisense oligodeoxynucleotide to G6PD mRNA (+AS) for 5 h or with the OligofectinTM I vehicle (-AS), and G6PD protein expression was determined by Western analysis. B) G6PD activity (units/6 min/mg protein) was determined in transfected cells (n=6) and data are presented as mean ± SE. *P < 0.0002 vs. -AS. C) Cellular NADPH levels were measured (n=3) as mmol/l/mg protein and reported as mean ± SE. *P < 9 x 10-5 vs. -AS. D) Transfected BAEC were exposed to H2O2 (100 µmol/l) for 4 h and cellular ROS production was determined by DCF fluorescence (n=6). Fluorescence is expressed in arbitrary units and data are reported as mean ± SE. *P < 1 x 10-8 vs. -H2O2.

3. Endothelial nitric oxide synthase (eNOS) augments ROS accumulation in G6PD-deficient cells
To determine the source of increased oxidant stress in endothelial cells exposed to H₂O₂, BAEC were treated with inhibitors of enzymes that generate ROS. Only the addition of diphenylene iodonium, an inhibitor of flavin-containing enzymes, and L-NAME (1 mM) to inhibit NOS decreased ROS accumulation, suggesting that eNOS may be contributing to ROS generation in G6PD-deficient cells. To evaluate this response further, G6PD-deficient BAEC were treated with L-NMMA (100 µM) to inhibit nitric oxide (NO), but not superoxide production by eNOS. DHEA-treated BAEC in the presence of L-NMMA demonstrated increased DCF fluorescence in the absence of H₂O₂ exposure (255.5±27.3 vs. 607.6±30.8 AFU, n=8, P<1x10^-9), as did AS-transfected BAEC (192.6±15.0 vs. 469.2±13.1 AFU, n=4, P<8x10^-6). This response was not observed after treatment with L-NAME (Fig. 2 ).

View larger version (37K): [in this window] [in a new window]   Figure 2. eNOS and cellular oxidant stress in G6PD-deficient endothelial cells. Transfected BAEC were pretreated for 1 h with L-NAME (1 mmol/l) to inhibit NO and superoxide production by eNOS or with L-NMMA (100 µmol/l) to inhibit superoxide production alone. Cellular ROS production was measured by DCF fluorescence (n=4) and expressed in arbitrary units. The data are reported as mean ± SE. *P < 8 x 10-6 vs. + AS, no addition.

4. Decreased NO bioavailability in endothelial cells with deficient G6PD activity
To determine whether deficient G6PD activity influenced NO production and activity, we measured cGMP levels in DHEA-treated and AS-transfected cells. At baseline, cGMP levels were similar between treatments; however, DHEA-treated cells failed to achieve cGMP levels analogous to control cells in response to the agonists A23187 (5 µM) (2.44±0.20 vs. 1.43±0.20 pmol/mg protein, n=3, P<0.05) and bradykinin (5 µM) (3.38±0.36 vs. 1.33±0.21 pmol/mg protein, n=3, P<0.009). A comparable blunted response was observed in AS-transfected cells after stimulation with A23187 (2.44±0.20 vs. 1.31±0.04 pmol/mg protein, n=3, P<0.02) or bradykinin (3.38±0.36 vs. 1.44±0.05 pmol/mg protein, n=3, P<0.006). Similarly, compared with control cells, AS-transfected cells stimulated with bradykinin had diminished nitrate (321.06±16.05 vs. 104.47±3.4 nmol/mg protein, n=6, P<0.001) and nitrite (65.74±0.80 vs. 20.73±0.05 nmol/mg protein, n=6, P<0.001) levels. This abrogated response was not due to differences in eNOS protein levels secondary to DHEA treatment or AS transfection as determined by Western analysis, nor was it due to an inherent difference in eNOS activity in the presence of saturating levels of cofactors. To demonstrate further that deficient G6PD activity was associated with decreased NO production and not depletion, AS-transfected cells were treated with an NO donor. AS-transfected cells exposed to DEA-NONOate (1 µM) achieved cGMP levels similar to control cells (3.92±0.23 vs. 4.25±0.76, pmol/mg protein, n=3, p=NS).

5. Sepiapterin pretreatment decreases oxidant stress in G6PD-deficient endothelial cells
Given that G6PD-deficient endothelial cells demonstrate increased eNOS-mediated ROS accumulation concomitant with decreased bioavailable NO, we hypothesized that decreased G6PD activity adversely influenced eNOS function by decreasing stores of necessary cofactors whose synthesis is dependent on NADPH, such as tetrahydrobiopterin (BH₄). To determine whether a relative BH₄ deficiency contributed to ROS production, G6PD-deficient endothelial cells were treated with the BH₄ precursor sepiapterin (100 µM) for 24 h. In DHEA-treated cells, sepiapterin pretreatment decreased basal ROS levels (246.4±11.4 vs. 135.4±5.7 AFU, n=5, P<5x10^-6) and H₂O₂-mediated increases in ROS accumulation (1270±33.9 vs. 471.0±35.7 AFU, n=4, P<2x10^-8). In AS-transfected BAEC, pretreatment with sepiapterin similarly influenced basal ROS levels (158.8±18.4 vs. 88.0±12.9 AFU, n=8, P<0.007); in the presence of H₂O₂, sepiapterin pretreatment significantly decreased ROS accumulation (927.9±32.2 vs. 465.4±6.9 AFU, n=8, P<2x10^-9).

CONCLUSIONS

A mounting body of evidence suggests that G6PD modulates the vascular endothelial cell response to oxidant stress and that deficient G6PD activity (and associated diminished NADPH stores) are associated with adverse sequelae (Fig. 3 ). In our studies, normal vascular endothelial cells respond to a modest H₂O₂ challenge by increasing G6PD activity to respond to a decrease in cellular GSH levels and enhance glutathione recycling. G6PD-deficient BAEC experience a more pronounced oxidant stress, as shown by the marked increase in cellular ROS accumulation and the corresponding decrease in GSH levels.

View larger version (16K): [in this window] [in a new window]   Figure 3. Central role of NADPH. NADPH serves as a cofactor for the reduction of oxidized glutathione (GSSG) to its reduced form (GSH) by glutathione reductase (GR), synthesis of tetrahydrobiopterin (BH4) from its precursor (BH2) by dihydrofolate reductase (DHFR), conversion of glucose-6-phosphate (G6P) to 6-phosphogluconate (6GP) by glucose-6-phosphate dehydrogenase (G6PD), and the synthesis of NO from L-arginine by eNOS.

The association of G6PD activity with NOS activity and NO generation remains controversial; to date, however, this relationship has only been explored in the setting of markedly elevated concentrations of endogenous NO, generated via the induction of iNOS expression, or exogenous NO from NO donors. We evaluated the association between G6PD and eNOS in endothelial cells and thereby avoided the addition of exogenous mediators to induce iNOS expression with its concomitant nitrosative stress. To characterize the role of G6PD in modulating eNOS activity, we initially made cells G6PD deficient and challenged them with an oxidant stress. Only after the addition of inhibitors of enzymes that generate ROS was it apparent that eNOS, a flavin-containing enzyme, was contributing to cellular oxidant stress. This observation was confirmed by the differential response to the NOS inhibitors L-NAME, which inhibits both NO and superoxide production by NOSs, and L-NMMA, which inhibits only NO production; there was a significant decrease in ROS production only in the presence of L-NAME. This is not entirely surprising, as in the setting of decreased cofactor availability, NOS may preferentially generate superoxide anion. Deficient G6PD activity is associated with a reduction in NADPH stores, which in turn may influence levels of BH₄, another essential cofactor for NOS activity whose synthesis and salvage via dihydrofolate reductase depend on NADPH. Finally, although DCF fluorescence also detects peroxynitrite, the differential response to NOS inhibitors suggests that we are evaluating ROS production.

In our system, decreased G6PD activity did not influence levels of eNOS protein expression as demonstrated by Western analysis; however, it was associated with a reduction in bioavailable NO, as revealed by cGMP and nitrate/nitrite levels after agonist stimulation. There are several possible explanations for this observation. First, treatment with DHEA or an antisense oligodeoxynucleotide may adversely influence enzyme function. To evaluate this possibility, we determined eNOS activity by the conversion of L-arginine to L-citrulline and found there was no inherent difference in enzyme activity (with exogenous cofactor addition in the assay). Second, because there is increased ROS formation and decreased NO synthesis from NOS, bioavailable NO may be rapidly consumed in a 1:1 stoichiometric reaction to form peroxynitrite. DCF fluorescence may also detect peroxynitrite; however, in our system, DCF fluorescence was increased in G6PD-deficient cells treated with L-NMMA, suggesting we were measuring ROS alone and not peroxynitrite in this experiment. Finally, as already suggested, G6PD deficiency is associated with decreased eNOS cofactor availability and, therefore, eNOS may preferentially synthesize ROS instead of NO.

To determine whether replenishing deficient cofactors may reverse eNOS-mediated ROS formation, we pretreated G6PD-deficient BAEC with sepiapterin, a precursor of BH₄. In cells pretreated with sepiapterin, there was a significant reduction in detectable ROS levels after exposure to H₂O₂. In our system, despite reduced NADPH levels, conversion of sepiapterin to BH₄ by dihydrofolate reductase may not be adversely influenced and intracellular BH₄ content may be effectively repleted to scavenge H₂O₂ and decrease cellular ROS accumulation. Alternatively, sepiapterin itself, as well as BH₄, may directly scavenge ROS and thereby confer protection on cells against oxidant stress.

G6PD is increasingly recognized as an important antioxidant enzyme in vascular cells to restore intracellular redox state in the setting of increased oxidant stress. We now show that deficient G6PD activity in vascular endothelial cells promotes ROS accumulation by failing to maintain intracellular reducing equivalents in the form of NADPH. In addition, eNOS, which under basal conditions generates NO to maintain reduced vascular tone, appears to contribute significantly to intracellular ROS formation at the expense of bioavailable NO. By depleting bioavailable NO and increasing cellular ROS, deficient G6PD activity promotes an oxidizing environment and adversely modulates the intracellular milieu and endothelial function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0893fje ; to cite this article, use FASEB J. (June 18, 2001) 10.1096/fj.00-0893fje

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