Tetrahydrobiopterin protects soluble guanylate cyclase against oxidative inactivation

Introduction

(6R)-5,6,7,8-Tetrahydro-l-biopterin (BH4) plays an important role in the vascular system. As an essential cofactor of endothelial nitric oxide synthase (eNOS), BH4 promotes NO synthesis by facilitating the transfer of electrons from the reductase domain to the heme group for reductive activation of molecular O₂, which is required for oxidation of the amino acid substrate l-arginine. At subsaturating concentrations of BH4, the enzymatic reduction of O₂ uncouples from substrate oxidation and results in the generation of superoxide, which rapidly combines with NO form peroxynitrite NO [1].

Superoxide/peroxynitrite formation by uncoupled eNOS has been implicated in endothelial dysfunction associated with a variety of cardiovascular diseases including atherosclerosis, hypertension and diabetes. In accordance with the role of BH4 as “coupling switch”, a causal link between BH4 deficiency and endothelial dysfunction was found in many animal models of vascular diseases. Moreover, administration of BH4 has been shown to restore endothelial function in patients with hypertension, diabetes mellitus, hypercholesterolemia, atherosclerosis, coronary artery disease, chronic heart failure, and in chronic smokers, suggesting that BH4 deficiency, resulting in eNOS uncoupling and limited NO bioavailability, may play a prominent role in the pathogenesis of cardiovascular diseases [2–5].

In addition to preventing eNOS uncoupling, BH4 is also a powerful antioxidant, reacting with superoxide, H₂O₂, and peroxynitrite. Accordingly, the beneficial effects of BH4 could also be ascribed to the antioxidant properties of the pteridine. Some studies have focused on this issue by using (6RS)-5,6,7,8-tetrahydro-d-neopterin (NH4), a compound that exhibits similar antioxidative properties as BH4 but does not bind to eNOS, and the results are ambiguous. For example, in human studies performed with chronic smokers, endothelial-dependent vasodilation measured as forearm blood flow response to acetylcholine, was improved upon intra-arterial infusion of BH4 but not NH4 [6]. However, when endothelial dysfunction was induced by ischemia-reperfusion injury in the forearm of healthy human subjects, both BH4 and NH4 exerted protective effects [7]. These data demonstrate that at least under certain conditions of oxidative stress, the beneficial effect of BH4 supplementation is not explained by the reversal of eNOS uncoupling.

In a previous study aimed at clarifying whether exposure of vascular tissue to nitroglycerin (GTN) results in oxidative stress and BH4 depletion, we observed that long-term treatment of cultured endothelial cells with GTN markedly diminished NO-induced cGMP formation [8]. The apparent dysfunction of soluble guanylate cyclase (sGC) resulted from GTN-triggered oxidation of enzyme-bound heme and was prevented by supplementation of cells with BH4, revealing a novel protective mechanism that might contribute to the beneficial vascular effects of BH4 [9]. In this article, we summarize our work on this hitherto unrecognized action of BH4.

Soluble guanylate cyclase and oxidative stress

The prosthetic heme group of sGC represents the intracellular receptor for NO. Upon binding to the ferrous heme, NO induces a change in heme geometry resulting in enzyme activation and enhanced conversion of GTP to cGMP [10–12]. Oxidation of the heme iron to its ferric form prevents NO binding and renders the enzyme insensitive to activation by NO. Moreover, heme oxidation promotes dissociation of the heme from sGC [13] and induces degradation of the protein by the ubiquitin-proteasome pathway [14, 15]. Preclinical and clinical data suggest that the relative amount of heme-free vascular sGC is increased in cardiovascular disorders such as coronary artery disease, pulmonary hypertension and chronic heart failure [16–18]. In view of the essential role of oxidative stress in most cardiovascular diseases [19], this is probably due to oxidation by reactive oxygen species and subsequent dissociation of sGC-bound heme. Partial dysfunction of sGC may cause NO resistance of the diseased blood vessels, resulting in increased blood pressure and atherosclerosis.

Tetrahydrobiopterin and oxidative inactivation of sGC

Based on functional and spectroscopic data showing that GTN and ODQ oxidize the ferrous heme iron of sGC [8, 20–23], we used both drugs as model compound to study the effect of BH4 on oxidative inactivation of sGC [9]. The results revealed that NO-induced cGMP formation was markedly diminished in GTN-treated endothelial cells and restored upon coincubation of the cells with the BH4 precursors sepiapterin and BH2 (7,8-dihydro-l-biopterin; Figure 1). As BH4 is formed from sepiapterin by the consecutive action of sepiapterin reductase and dihydrofolate reductase, which catalyze the reduction of sepiapterin to BH2 and further reduction of BH2 to BH4, respectively, treatment of the cells with sepiapterin or BH2 results in an increase of both BH2 and BH4. To see whether sGC protection requires the fully reduced biopterin, we blocked BH2-to-BH4 conversion by inhibiting dihydrofolate reductase with methotrexate (MTX), which led to accumulation of BH2 that was accompanied by a decrease in BH4 levels close to that of untreated cells. Under these conditions, the effects of sepiapterin and BH2 on cGMP formation induced by DEA/NO (1,1-diethyl-2-hydroxy-2-nitroso-hydrazine) were virtually abolished, indicating that sGC is protected against oxidative inactivation by BH4 but not by BH2. In contrast to supplementation of the cells with BH4, scavenging of superoxide and/or peroxynitrite with Tiron (Figure 1), superoxide dismutase (SOD), Mn(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) or urate did not prevent the effect of GTN on DEA/NO-induced cGMP accumulation, excluding scavenging of reactive oxygen species as the underlying mechanism.

Figure 1

Effects of pteridines on oxidative inactivation of sGC induced by GTN.

Endothelial cells were pretreated for 24 h in culture medium in the absence and presence of 0.1 mM GTN, sepiapterin (Sep), BH2, MTX (final concentration 0.1 mM, each) or Tiron (10 mM). Following pretreatment, cells were washed, stimulated for 4 min with 1 μM DEA/NO, and cGMP formation was determined by radioimmunoassay. Data are from [9] and are mean values ± SEM of 3–5 independent experiments.

The data obtained with GTN were confirmed with ODQ, a potent sGC inhibitor that acts through heme oxidation [21]. As shown in Figure 2, pretreatment of endothelial cells with sepiapterin or BH2 markedly diminished the inhibitory effect of ODQ on DEA/NO-induced cGMP formation, evident as a more than 20-fold rightward shift of the ODQ concentration-response curve. A similar effect was obtained when endothelial cells were directly preincubated with BH4. To see whether the observed effects reflect a specific feature of endothelial cells, we repeated the experiments with RFL-6 fibroblast and obtained identical results.

Figure 2

Effects of pteridines on oxidative inactivation of sGC induced by ODQ.

Endothelial cells were pretreated in culture medium for 24 h with 0.1 mM sepiapterin or for 1 h with 0.1 mM BH2. Then, cells were washed, preincubated for 15 min with increasing concentrations of ODQ, for 4 min 1 μM DEA/NO, and cGMP formation was determined by radioimmunoassay. Data are from [9] and are mean values ± SEM of 3–5 independent experiments.

Supplementation of cells with tetrahydrobiopterin

The BH4 precursor sepiapterin proved to be ideal for long-term incubations. The compound is fairly stable in aqueous solution under aerobic conditions [24] and efficiently taken up by endothelial cells [25]. Intracellular BH4 levels are rapidly increased and remain constantly high for up to 2 days after addition of sepiapterin to the cell culture medium, In contrast to sepiapterin, BH2 and BH4 are prone to autoxidation [24, 26, 27] and according to our own data, 10%–20% of BH2 and ∬99% of BH4 are lost within 1 h of incubation in culture medium. Consequently, the increases in intracellular BH4 observed upon exogenous addition of BH2 or BH4 were transient and less pronounced than those observed with sepiapterin. Accordingly, we used BH2/BH4 supplementation only in short-term experiments or – in the case of long-term studies – re-added BH2 to the culture medium every 8 h.

Role of tetrahydroneopterin

Using the same experimental protocol that was established for supplementation of cells with reduced biopterins, we investigated whether NH4 mimics the effect of BH4. The results revealed that incubation of endothelial cells or fibroblasts with NH4 protects sGC against oxidative inactivation to a similar extent as observed with BH4. However, in contrast to incubation with BH2, 7,8-diydro-d-neopterin (NH2) had no effect, indicating inefficient uptake and/or reduction to the corresponding tetrahydro derivative by dihydrofolate reductase. The mechanisms of cellular uptake and metabolism of reduced neopterins are poorly understood, mainly due to the lack of analytical techniques for reliable quantification of these compounds and their oxidation products in tissues. Nonetheless, the data obtained with NH4 clearly suggest that protection of sGC against oxidative inactivation is not a peculiarity of BH4 and reflects a general feature of tetrahydropteridines.

Concluding remarks

In addition to eNOS uncoupling and limited NO bioavailability, inactivation of sGC caused by oxidative stress contributes to vascular dysfunction in cardiovascular diseases. Based on our in vitro data showing that BH4 protects sGC against oxidative inactivation, we suggest that this hitherto unrecognized action of the reduced pteridine might contribute to the beneficial vascular effects of BH4 supplementation.