Cell type-specific recycling of tetrahydrobiopterin by dihydrofolate reductase explains differential effects of 7,8-dihydrobiopterin on endothelial nitric oxide synthase uncoupling

Abstract

(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) availability regulates nitric oxide and superoxide formation by endothelial nitric oxide synthase (eNOS). At low BH4 or low BH4 to 7,8-dihydrobiopterin (BH2) ratios the enzyme becomes uncoupled and generates superoxide at the expense of NO. We studied the effects of exogenously added BH2 on intracellular BH4/BH2 ratios and eNOS activity in different types of endothelial cells. Incubation of porcine aortic endothelial cells with BH2 increased BH4/BH2 ratios from 8.4 (controls) and 0.5 (BH4-depleted cells) up to ~20, demonstrating efficient reduction of BH2. Uncoupled eNOS activity observed in BH4-depleted cells was prevented by preincubation with BH2. Recycling of BH4 was much less efficient in human endothelial cells isolated from umbilical veins or derived from dermal microvessels (HMEC-1 cells), which exhibited eNOS uncoupling and low BH4/BH2 ratios under basal conditions and responded to exogenous BH2 with only moderate increases in BH4/BH2 ratios. The kinetics of dihydrofolate reductase-catalyzed BH4 recycling in endothelial cytosols showed that the apparent BH2 affinity of the enzyme was 50- to 300-fold higher in porcine than in human cell preparations. Thus, the differential regulation of eNOS uncoupling in different types of endothelial cells may be explained by striking differences in the apparent BH2 affinity of dihydrofolate reductase.

Keywords: Dihydrofolate reductase; Endothelial nitric oxide synthase uncoupling; Human endothelial cells; Porcine endothelial cells; Tetrahydrobiopterin recycling.

Graphical abstract

Fig. 1

Effect of pteridine supplementation on eNOS activity in BH4-depleted PAECs. PAECs were pretreated for 24 h with culture medium in the absence (Ctrl) or the presence of 10 mM DAHP. Where indicated, pteridines were added to the medium 1 h prior to the end of the DAHP treatment. Then, cells were washed and l-arginine to l-citrulline conversion (A) or cGMP formation (B) determined as described in Section 2. Data are mean values ± S.E.M. of 3–5 independent experiments (**p < 0.01, *p < 0.05 compared with untreated control).

Fig. 2

Effect of pteridine supplementation on ROS formation in BH4-depleted PAECs. PAECs grown on glass coverslips were pretreated for 24 h with culture medium in the absence (Ctrl) or the presence of 10 mM DAHP. Where indicated, BH4 or BH2 (final concentration 0.1 mM) was added to the medium 1 h prior to the end of the DAHP treatment. Then, cells were incubated with dihydroethidium (10 μM, 10 min) and stimulated for 30 min with A23187 (1 μM) in the absence and the presence of l-NNA or l-NMA (0.3 mM, each). ROS formation was quantitated by fluorescence imaging as described in Section 2. Data are mean values ± S.E.M. of 3–5 independent experiments (**p < 0.01 compared with untreated control).

Fig. 3

Effect of pteridine supplementation on eNOS activity in PAECs. PAECs were pretreated for 1 h with culture medium in the absence or the presence of BH4 or BH2. Where indicated, aminopterin (final concentration 0.1 mM) was added to the medium 30 min prior to addition of BH2. Then, cells were washed and l-arginine to l-citrulline conversion (A) or cGMP formation (B) determined as described in Section 2. Data are mean values ± S.E.M. of 3–5 independent experiments (**p < 0.01, *p < 0.05 compared with untreated control).

Fig. 4

Effect of pteridine supplementation on eNOS activity in HMEC-1 cells and HUVECs. HMEC-1 cells or HUVECs were pretreated for 1 h with culture medium in the absence or the presence of BH2. Where indicated, aminopterin (final concentration 0.1 mM) was added to the medium 30 min prior to addition of BH2. Then, cells were washed, and l-arginine to l-citrulline conversion was determined as described in Section 2. Data are mean values ± S.E.M. of 3–5 independent experiments (**p < 0.01, *p < 0.05 compared with untreated control).

Fig. 5

Basal and A23187-induced ROS formation in PAECs und HUVECs. Cells grown on glass coverslips were incubated for 4 h in phenol red free culture medium containing 10 μM dihydroethidium. Where indicated, cells were stimulated with A23187 (1 μM) and in the absence or the presence of l-NNA (0.3 mM) during the last hour of incubation. For loading the cells with BH4, BH2 (final concentration 0.1 mM) was added to the culture medium 1 h prior to the addition A23187. ROS formation was quantitated by fluorescence imaging as described in Section 2. Data are mean values ± S.E.M. of 3–5 independent experiments (**p < 0.01, *p < 0.05 compared with untreated control).

Fig. 6

DHFR activity in broken cell preparations. DHFR activity was measured in cell cytosols or PD10 eluates as described in Section 2 using either BH2 (A) od DHF (B) as substrate. The data points shown are from experiments with cytosols and are mean values ± S.E.M. of 4–6 cell preparations. The curves shown were obtained by fitting the mean values to the Michealis–Menten equation. For clarity, results obtained with PD10 eluates (n = 3) are only shown as curve fits without data points.