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l-Ascorbic Acid Potentiates Endothelial Nitric Oxide Synthesis via a Chemical Stabilization of Tetrahydrobiopterin*

Open AccessPublished:January 05, 2001DOI:https://doi.org/10.1074/jbc.M004392200
      Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 μm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 μm. Accordingly, the effect of ascorbic acid on Ca2+-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001–100 μm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.
      NOS
      nitric-oxide synthase
      eNOS
      endothelial cell NOS
      M199
      medium 199
      FCS
      fetal calf serum
      HSA
      human serum albumin
      TNF-α
      tumor necrosis factor-α
      IFN-γ
      interferon-γ
      l-NAME
      l-nitroarginine methylester
      DAHP
      2,4-diamino-6-hydroxypyrimidine
      LPS
      lipopolysaccharide
      HUVEC
      human umbilical vein endothelial cell(s)
      PBS
      phosphate-buffered saline
      HPLC
      high performance liquid chromatography
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      Endothelium-derived nitric oxide (NO) is a potent signaling molecule in the cardiovascular system participating in many processes such as vascular relaxation, inhibition of platelet aggregation, regulation of endothelial cell adhesivity, and preservation of the normal vessel wall structure (
      • Ignarro L.J.
      • Cirino G.
      • Casini A.
      • Napoli C.
      ). NO is generated from the conversion of l-arginine to l-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS)1 that requires Ca2+/calmodulin, FAD, FMN, and tetrahydrobiopterin as cofactors (
      • Mayer B.
      • Hemmens B.
      ). The endothelial NOS isoform (eNOS) is constitutively expressed and activated upon an increase of intracellular Ca2+ following cell stimulation with agonists such as thrombin and bradykinin or through serine phosphorylation subsequent to cell stimulation with shear stress or insulin (
      • Busse R.
      • Mülsch A.
      ,
      • Fulton D.
      • Gratton J.-P.
      • McCabe T.J.
      • Fontana J.
      • Fujio Y.
      • Walsh K.
      • Franke T.F.
      • Papapetropoulos A.
      • Sessa W.C.
      ).
      Evidence is accumulating that NO determines the antiatherosclerotic properties of the endothelium (
      • Bult H.
      • Herman A.G.
      • Matthys K.E.
      ). All major risk factors for atherosclerosis including hypercholesterolemia, hypertension, and smoking have been associated with impaired vascular NO synthesis (
      • Cooke J.P.
      • Dzau V.J.
      ). The underlying mechanisms are thought to involve reduced formation of NO due to a decrease in NOS expression or a limited availability ofl-arginine, as well as increased degradation of NO by reaction with superoxide anions or oxidized low density lipoproteins (
      • Bult H.
      • Herman A.G.
      • Matthys K.E.
      ,
      • Cooke J.P.
      • Dzau V.J.
      ). Recent studies indicate that under certain pathological conditions, decreased availability of tetrahydrobiopterin may also be responsible for dysfunction of endothelial nitric-oxide synthase. A close link between cellular tetrahydrobiopterin levels and NO synthesis was demonstrated for a number of different cell types including endothelial cells (
      • Schmidt K.
      • Werner E.R.
      • Mayer B.
      • Wachter H.
      • Kukovetz E.R.
      ,
      • Werner-Felmayer G.
      • Werner E.R.
      • Fuchs D.
      • Hausen A.
      • Reibnegger G.
      • Schmidt K.
      • Weiss G.
      • Wachter H.
      ,
      • Schoedon G.
      • Schneemann M.
      • Blau N.
      • Edgell C.-J.S.
      • Schaffner A.
      ,
      • Rosenkranz-Weiss P.
      • Sessa W.C.
      • Milstien S.
      • Kaufman S.
      • Watson C.A.
      • Pober J.S.
      ), suggesting that an optimal concentration of tetrahydrobiopterin is essential for agonist-induced production of NO. Furthermore, tetrahydrobiopterin induced vasodilation in isolated arteries (
      • van Amsterdam J.G.
      • Wemer J.
      ,
      • Tsutsui M.
      • Milstien S.
      • Katusic Z.S.
      ,
      • Rosenblum W.I.
      ) and inhibition of tetrahydrobiopterin biosynthesis impaired endothelium-dependent relaxation in canine basilar artery (
      • Kinoshita H.
      • Milstien S.
      • Wambi C.
      • Katusic Z.S.
      ). Accordingly, tetrahydrobiopterin supplementation was capable of restoring endothelium-dependent vasodilation in experimental diabetes and reperfusion injury as well as in patients with hypercholesterolemia, coronary artery disease and in cigarette smokers (
      • Pieper G.M.
      ,
      • Tiefenbacher C.P.
      • Chilian W.M.
      • Mitchell M.
      • DeFily D.V.
      ,
      • Stroes E.
      • Kastelein J.
      • Cosentino F.
      • Erkelens W.
      • Wever R.
      • Koomans H.
      • Lüscher T.
      • Rabelink T.
      ,
      • Maier W.
      • Cosentino F.
      • Lutolf R.B.
      • Fleisch M.
      • Seiler C.
      • Hess O.M.
      • Meier B.
      • Lüscher T.F.
      ,
      • Heitzer T.
      • Brockhoff C.
      • Mayer B.
      • Warnholtz A.
      • Mollnau H.
      • Henne S.
      • Meinertz T.
      • Münzel T.
      ,
      • Ueda S.
      • Matsuoka H.
      • Miyazaki H.
      • Usui M.
      • Okuda S.
      • Imaizumi T.
      ). Although the reason for a reduced availability of tetrahydrobiopterin is not clear, it might be related to an impaired synthesis, to a decreased affinity of the enzyme for its cofactor or to prolonged oxidative stress. Since tetrahydrobiopterin has profound effects on the structure of NOS including the ability to shift the heme iron to its high spin state, the promotion of arginine binding and the stabilization of the active dimeric form of the enzyme (
      • Gorren A.C.F.
      • Mayer B.
      ), a lack of this cofactor may decrease NOS activity. There is also increasing evidence that NOS-bound tetrahydrobiopterin acts as a redox-active cofactor (
      • Bec N.
      • Gorren A.C.F.
      • Voelker C.
      • Mayer B.
      • Lange R.
      ,
      • Hurshman A.R.
      • Krebs C.
      • Edmondson D.E.
      • Huynh B.H.
      • Marletta M.A.
      ,
      • Witteveen C.F.B.
      • Giovanelli J.
      • Kaufman S.
      ), but, unlike aromatic amino acid hydroxylases where the fully reduced pterin serves as a reductant for oxygen, NOS is not coupled to the dihydropteridine reductase as a tetrahydrobiopterin-regenerating system (
      • Werner E.R.
      • Habisch H.J.
      • Gorren A.C.
      • Schmidt K.
      • Canevari L.
      • Werner-Felmayer G.
      • Mayer B.
      ). Interestingly, a decreased availability of tetrahydrobiopterin may cause a shift in the balance between the production of NO and oxygen free radicals by NOS. Several biochemical studies indicated that activation of purified eNOS in the presence of suboptimal levels of tetrahydrobiopterin results in uncoupling of oxygen reduction and arginine oxidation, thereby generating superoxide anions and subsequently hydrogen peroxide (
      • Vasquez-Vivar J.
      • Kalyanaraman B.
      • Martasek P.
      • Hogg N.
      • Masters B.S.
      • Karoui H.
      • Tordo P.
      • Pritchard K.A.
      ,
      • Xia Y.
      • Tsai A.-L.
      • Berka V.
      • Zweier J.L.
      ,
      • Leber A.
      • Hemmens B.
      • Klösch B.
      • Goessler W.
      • Raber G.
      • Mayer B.
      • Schmidt K.
      ). Thus, deficiency of tetrahydrobiopterin may cause both impaired NO formation and increased oxygen radical formation with the latter leading to increased NO inactivation.
      Recently, we were able to demonstrate that preincubation of human endothelial cells from umbilical veins and coronary arteries with ascorbic acid led to an up to 3-fold increase of agonist-induced NO synthesis (
      • Heller R.
      • Münscher-Paulig F.
      • Gräbner R.
      • Till U.
      ). The ascorbate effect was specific, saturated at 100 μm, and was dependent on cellular uptake. Ascorbic acid induced neither eNOS expression nor l-arginine uptake. Since the potentiating ascorbate effect was minimal, when NOS activity was measured in lysates of ascorbate-pretreated cells in the presence of exogenous tetrahydrobiopterin, we suggested that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for endothelial NOS. The present study was designed to investigate the mechanisms underlying the effect of ascorbic acid on endothelial NO synthesis. We demonstrate that ascorbate treatment of endothelial cells causes an increase of intracellular tetrahydrobiopterin levels and that this effect is based on a chemical stabilization of the fully reduced form of the pterin.

      DISCUSSION

      The present study demonstrates that the ascorbic acid-induced potentiation of endothelial NO synthesis that has been described in a previous paper (
      • Heller R.
      • Münscher-Paulig F.
      • Gräbner R.
      • Till U.
      ) is due to an increase of intracellular tetrahydrobiopterin levels. Since ascorbic acid had only a marginal effect on the tetrahydrobiopterin concentration required for half-maximal stimulation of recombinant eNOS, our data also suggest that ascorbate does not modify the pterin affinity of the enzyme. The effect of ascorbic acid on endothelial tetrahydrobiopterin levels was concentration-dependent in the physiological range and saturated at 100 μm, corresponding to saturation of both ascorbate uptake (
      • Ek A.
      • Ström K.
      • Cotgreave I.A.
      ) and potentiation of NO synthesis (
      • Heller R.
      • Münscher-Paulig F.
      • Gräbner R.
      • Till U.
      ) and suggesting that intracellular tetrahydrobiopterin concentration and thus NO formation are critically dependent on the tissue levels of ascorbate. Tetrahydrobiopterin levels in cultured endothelial cells have already been shown to be insufficient to allow saturation of eNOS with its cofactor and optimal NO synthesis (
      • Werner-Felmayer G.
      • Werner E.R.
      • Fuchs D.
      • Hausen A.
      • Reibnegger G.
      • Schmidt K.
      • Weiss G.
      • Wachter H.
      ,
      • Rosenkranz-Weiss P.
      • Sessa W.C.
      • Milstien S.
      • Kaufman S.
      • Watson C.A.
      • Pober J.S.
      ). This was confirmed in our study since sepiapterin, which is intracellularly converted to tetrahydrobiopterin (
      • Nichol C.A.
      • Smith G.K.
      • Duch D.S.
      ), led to an increase of agonist-induced citrulline and cGMP formation. Additionally, we demonstrated that sepiapterin abolished the potentiating effect of ascorbic acid on NO production in a concentration-dependent manner, suggesting that ascorbate exerts its effect on NO synthesis only under suboptimal intracellular tetrahydrobiopterin concentrations. From the data presented here, we can speculate that tetrahydrobiopterin levels between 1 and 2 pmol/mg of protein provide optimal reaction conditions for NO formation in HUVEC.
      Tetrahydrobiopterin is synthesized de novo from GTP by the sequential action of three enzymes, GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase. GTP cyclohydrolase I has been shown to be the key enzyme of the de novo pathway and to be regulated by cytokines such as TNF-α, IFN-γ, and interleukin-1β in a number of cell types including endothelial cells (
      • Werner-Felmayer G.
      • Werner E.R.
      • Fuchs D.
      • Hausen A.
      • Reibnegger G.
      • Schmidt K.
      • Weiss G.
      • Wachter H.
      ,
      • Rosenkranz-Weiss P.
      • Sessa W.C.
      • Milstien S.
      • Kaufman S.
      • Watson C.A.
      • Pober J.S.
      ,
      • Hattori Y.
      • Nakanishi N.
      • Kasai K.
      • Shimoda S.-I.
      ). The cytokine-induced elevation of GTP cyclohydrolase I activity in HUVEC has been related to an increased transcription rate and an enhanced expression of the mRNA (
      • Katusic Z.S.
      • Stelter A.
      • Milstien S.
      ). Likewise, but to a lower extent, 6-pyruvoyl-tetrahydropterin synthase activity and mRNA abundance have been shown to be regulated by inflammatory cytokines in HUVEC (
      • Linscheid P.
      • Schaffner A.
      • Blau N.
      • Schoedon G.
      ), although other studies have reported a constitutive expression of the enzyme and no significant changes upon treatment with these stimuli (
      • Werner-Felmayer G.
      • Werner E.R.
      • Fuchs D.
      • Hausen A.
      • Reibnegger G.
      • Schmidt K.
      • Weiss G.
      • Wachter H.
      ,
      • Werner E.R.
      • Werner-Felmayer G.
      • Fuchs D.
      • Hausen A.
      • Reibnegger G.
      • Yim J.J.
      • Pfleiderer W.
      • Wachter H.
      ). Our data confirm the low activity and mRNA expression of GTP cyclohydrolase I in control HUVEC underlining its rate-limiting role, and the up-regulation of both parameters by a mixture of cytokines (TNF-α, IFN-γ) and LPS. Ascorbic acid treatment of cells, however, did not alter GTP cyclohydrolase I mRNA levels nor enzyme activity, regardless whether it was added to the culture medium alone or together with cytokines. 6-Pyruvoyl-tetrahydropterin synthase activities in control HUVEC were considerably higher than GTP cyclohydrolase I activities, but were also not changed by preincubation of HUVEC with ascorbic acid. Furthermore, ascorbate did not act as a direct cofactor of GTP cyclohydrolase I since no increase in enzyme activity was measured when the compound was added to the enzyme assay. Taken together, these results suggest that ascorbic acid effects on intracellular tetrahydrobiopterin levels are not due to an increased synthesis of the compound. Accordingly, inhibition of tetrahydrobiopterin formation by DAHP, an inhibitor of GTP cyclohydrolase I (
      • Gal E.M.
      • Nelson J.M.
      • Sherman A.D.
      ), did not prevent the ascorbate-mediated increase of the pteridine although it substantially decreased tetrahydrobiopterin levels in both control and ascorbic acid-treated endothelial cells. Likewise, an inhibition of Ca2+-dependent NO synthesis by DAHP was seen, but the potentiating effect of ascorbate on ionomycin-stimulated citrulline and cGMP formation was maintained.
      Since ascorbic acid did not affect tetrahydrobiopterin synthesis, we speculated that it might act by preventing the degradation of the compound. Ascorbic acid has already been added to biological fluids to increase tetrahydrobiopterin stability and to allow storage of the samples before pteridine measurements (
      • Hyland K.
      ). Accordingly, we found an increase in tetrahydrobiopterin half-life when the compound was dissolved in an aqueous solution in the presence of ascorbate. Our data additionally demonstrate that ascorbic acid stabilizes tetrahydrobiopterin in intact endothelial cells. The increase of tetrahydrobiopterin in cytokine-stimulated cells treated with ascorbate was paralleled by a decrease of 7,8-dihydrobiopterin and biopterin in cells and cell supernatants, suggesting that a chemical stabilization of the fully reduced pterin is the underlying mechanism for its increased intracellular concentration. Since the total amount of tetrahydrobiopterin, dihydrobiopterin, and biopterin remained constant under the different experimental incubations, these data further underline that ascorbate does not influence pterin formation. The stabilizing function of ascorbate is most probably due to a chemical reduction of the quinonoid 6,7-[8H]dihydrobiopterin to tetrahydrobiopterin, which had already been shown for other reducing compounds such as dithioerythritol and NADPH. We suggest that the presence of the latter in the assay solution might also be responsible for the minimal effect of ascorbic acid on the activation of purified eNOS by tetrahydrobiopterin that was seen in our study.
      The results presented here show that the oxidation of tetrahydrobiopterin to the quinonoid 6,7-[8H]dihydrobiopterin with a rearrangement to 7,8-dihydrobiopterin and further oxidation to biopterin is most likely the main pathway of tetrahydrobiopterin degradation in HUVEC. Other pterins generated by the loss of the side chain at C6 could hardly be detected. Interestingly, tetrahydrobiopterin remained intracellular under the different experimental conditions investigated which is in contrast to previous data obtained in human and murine endothelial cell lines (
      • Schoedon G.
      • Schneemann M.
      • Blau N.
      • Edgell C.-J.S.
      • Schaffner A.
      ,
      • Walter R.
      • Schaffner A.
      • Blau N.
      • Kierat L.
      • Schoedon G.
      ). Our data show, however, that up to 91% of the dihydrobiopterin + biopterin formed in HUVEC was released into the medium, thereby preventing an intracellular accumulation of dihydrobiopterin.
      So far, beneficial vascular effects of ascorbic acid have been attributed to its radical scavenging properties, which may lead to a protection of NO from inactivation and may explain the improvement of endothelium-dependent vasodilation in cardiovascular patients by an acute ascorbate application (
      • Ting H.H.
      • Timimi F.K.
      • Boles K.S.
      • Creager S.J.
      • Ganz P.
      • Creager M.A.
      ,
      • Levine G.N.
      • Frei B.
      • Koulouris S.N.
      • Gerhardt M.D.
      • Keaney J.F.
      • Vita J.A.
      ,
      • Heitzer T.
      • Just H.
      • Münzel T.
      ,
      • Solzbach U.
      • Hornig B.
      • Jeserich M.
      • Just H.
      ,
      • Ting H.H.
      • Timimi F.K.
      • Haley E.A.
      • Roddy M.-A.
      • Ganz P.
      • Creager M.A.
      ,
      • Hornig B.
      • Arakawa N.
      • Kohler C.
      • Drexler H.
      ). Long term ascorbic acid administration also reversed endothelial vasomotor dysfunction in patients, although the plasma levels reached might not have been high enough to interfere with the reaction between superoxide anion and NO (
      • Gokce N.
      • Keaney J.F.
      • Frei B.
      • Holbrook M.
      • Olesiak M.
      • Zachariah B.J.
      • Leeuwenburgh C.
      • Heinecke J.W.
      • Vita J.A.
      ). We suggest that the stabilization of the NOS cofactor tetrahydrobiopterin resulting in an increased NO formation may represent an additional mechanism of vascular protection by ascorbate, which may be effective in vivo when plasma levels of ascorbic acid supply saturated intracellular ascorbate concentrations. Interestingly, conditions that are thought to be associated with tetrahydrobiopterin deficiency (i.e. coronary artery disease or smoking) have been characterized by low ascorbic acid levels in plasma or leukocytes (
      • Ramirez J.
      • Flowers N.C.
      ,
      • Vita J.A.
      • Keaney J.F.
      • Raby K.E.
      • Morrow J.D.
      • Freedman J.E.
      • Lynch S.
      • Koulouris S.N.
      • Hankin B.R.
      • Frei B.
      ,
      • Schectman G.
      • Byrd J.C.
      • Gruchow H.W.
      ), suggesting that cellular deficiency of ascorbate may promote tetrahydrobiopterin oxidation and lead to endothelial dysfunction.
      In summary, this study shows that l-ascorbic acid in physiologically relevant concentrations increases intracellular tetrahydrobiopterin levels in endothelial cells in a concentration-dependent and saturable fashion. Ascorbic acid did not affect the synthesis of tetrahydrobiopterin but led to a chemical stabilization of the compound. The results presented in this study suggest that tissue saturation with ascorbic acid may maintain tetrahydrobiopterin levels in endothelial cells in vivo, thus providing optimal reaction conditions for NO synthesis and preventing endothelial dysfunction.

      Acknowledgments

      We thank Bettina Fritz, Gunda Guhr, Renate Kaus, Elke Teuscher, and Anabella Weiskopf-Delucchi for excellent technical assistance.

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