The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O2- was increased simultaneously. NOS inhibitor, inhibited O2- release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process.     

Tetrahydrobiopterin Recycling, a Key Determinant of Endothelial Nitric-oxide Synthase-dependent Signaling Pathways in Cultured Vascular Endothelial Cells

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated “knockdown” GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.Regulation of endothelial nitric oxide (NO)² production represents a critical mechanism for the modulation of vascular homeostasis. NO is released by endothelial cells in response to diverse humoral, neural, and mechanical stimuli (1–4). Endothelial cell-derived NO activates guanylate cyclase in vascular smooth muscle cells, leading to increased levels of cGMP and to smooth muscle relaxation. Blood platelets represent another key target for the actions of endothelium-derived NO (5): platelet aggregation is inhibited by NO-induced guanylate cyclase activation. Many other effects of NO have been identified in cultured vascular cells and in vascular tissues, including the regulation of apoptosis, cell adhesion, angiogenesis, thrombosis, vascular smooth muscle proliferation, and atherogenesis, among other cellular responses and (patho)physiological processes.The endothelial isoform of NO synthase (eNOS) is a membrane-associated homodimeric 135-kDa protein that is robustly expressed in endothelial cells (2, 4, 6, 7). Similar to all the mammalian NOS isoforms, eNOS functions as an obligate homodimer that includes a cysteine-complex Zn²⁺ (zinc-tetrathiolate) at the dimer interface (8–10). eNOS is a Ca²⁺/calmodulin-dependent enzyme that is activated in response to the stimulation of a variety of Ca²⁺-mobilizing cell surface receptors in vascular endothelium and in cardiac myocytes. The activity of eNOS is also regulated by phosphorylation at multiple sites (11) that are differentially modulated following the activation of cell surface receptors by agonists such as insulin and vascular endothelial growth factor (VEGF) (12). The phosphorylation of eNOS at Ser-1179 activates eNOS, but phosphorylation at Thr-497 or Ser-116 is associated with inhibition of eNOS activity (13–17). eNOS is reversibly targeted to plasmalemmal caveolae as a consequence of the protein''s N-myristoylation and thiopalmitoylation. The generation of NO by eNOS requires several redox-active cofactors, including nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, and tetrahydrobiopterin (BH4), which have key roles in the electron flow required for eNOS catalysis. If the flow of electrons within eNOS is disrupted, the enzyme is uncoupled from NO production and other redox-active products are generated, including hydrogen peroxide and superoxide anion radical (18, 19).In vascular disease states such as diabetes, endothelial dysfunction is characterized by a decrease in NO bioactivity and by a concomitant increase in superoxide formation, while eNOS mRNA and protein levels are maintained or even increased. “Uncoupled” eNOS generates reactive oxygen species (ROS), shifting the nitroso-redox balance and having adverse consequences in the vascular wall (20). Several enzymes expressed in vascular tissues contribute to the production and efficient degradation of ROS, and an enhanced activity of oxidant enzymes and/or reduced activity of antioxidant enzymes may cause oxidative stress. Various agonists, pathological conditions, and therapeutic interventions lead to modulated expression and function of oxidant and antioxidant enzymes. However, the intimate relationship between intracellular redox state, eNOS regulation, and NO bioavailability remains incompletely characterized.BH4 is a key redox-active cofactor for activity of all NOS enzymes (21). The exact role of BH4 in NOS catalysis is not yet completely defined, but this cofactor appears to facilitate electron transfer from the eNOS reductase domain and maintains the heme prosthetic group of the enzyme in its redox-active form (18, 22, 23). Moreover, BH4 promotes formation of active NOS homodimers (24) and inhibits the formation of hydrogen peroxide or superoxide by uncoupled eNOS (18, 19). It has been reported that the endothelial dysfunction associated with diabetes is accompanied a decrease in the abundance of bioactive BH4. Supplementation with BH4 has been shown to improve endothelial function in the models of diabetes and hypertension (25, 26, 27). Moreover, BH4 oxidation is seen in vascular cells in the setting of oxidative stress associated with diabetes (28) and hypertension (29).BH4 can be formed either by a de novo biosynthetic pathway or by a salvage pathway. Guanosine triphosphate cyclohydrolase-1 (GTPCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate. BH4 is generated by further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (30). GTPCH1 appears to be the rate-limiting enzyme in BH4 biosynthesis; overexpression of GTPCH1 is sufficient to augment BH4 levels in cultured endothelial cells (31). On the other hand, dihydrofolate reductase (DHFR) catalyzes the regeneration of BH4 from its oxidized form, 7,8-dihydrobiopterin (BH2), in several cell types (30, 32). DHFR is mainly involved in folate metabolism and converts inactive BH2 back to BH4 and plays an important role in the metabolism of exogenously administered BH4. However, the relative contributions of endothelial GTPCH1 and DHFR to the modulation of eNOS-dependent pathways are incompletely understood.In these studies, we have used siRNA-mediated “knockdown” of GTPCH1 and DHFR to explore the relative roles of BH4 synthesis and recycling in the modulation of eNOS bioactivity, as well as in the regulation of NO-dependent signaling pathways in endothelial cells.     

Manganese Supplementation Reduces High Glucose-induced Monocyte Adhesion to Endothelial Cells and Endothelial Dysfunction in Zucker Diabetic Fatty Rats

Endothelial dysfunction is a hallmark of increased vascular inflammation, dyslipidemia, and the development of atherosclerosis in diabetes. Previous studies have reported lower levels of Mn²⁺ in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. This study examines the hypothesis that Mn²⁺ supplementation can reduce the markers/risk factors of endothelial dysfunction in type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured with or without Mn²⁺ supplementation and then exposed to high glucose (HG, 25 mm) to mimic diabetic conditions. Mn²⁺ supplementation caused a reduction in monocyte adhesion to HUVECs treated with HG or MCP-1. Mn²⁺ also inhibited ROS levels, MCP-1 secretion, and ICAM-1 up-regulation in HUVECs treated with HG. Silencing studies using siRNA against MnSOD showed that similar results were observed in MnSOD knockdown HUVECs following Mn²⁺ supplementation, suggesting that the effect of manganese on monocyte adhesion to endothelial cells is mediated by ROS and ICAM-1, but not MnSOD. To validate the relevance of our findings in vivo, Zucker diabetic fatty rats were gavaged daily with water (placebo) or MnCl₂ (16 mg/kg of body weight) for 7 weeks. When compared with placebo, Mn²⁺-supplemented rats showed lower blood levels of ICAM-1 (17%, p < 0.04), cholesterol (25%, p < 0.05), and MCP-1 (28%, p = 0.25). These in vitro and in vivo studies demonstrate that Mn²⁺ supplementation can down-regulate ICAM-1 expression and ROS independently of MnSOD, leading to a decrease in monocyte adhesion to endothelial cells, and therefore can lower the risk of endothelial dysfunction in diabetes.     

In vivo expression and function of recombinant GTPCH I in the rabbit carotid artery

Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 10(9) plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or beta-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10(-5) to 10(-9) M), endothelium-dependent relaxation to acetylcholine (10(-5) to 10(-9) M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.     

Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H(2)O(2) to ECs markedly increased the BH4 level, but not its oxidized forms. The H(2)O(2)-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H(2)O(2) induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H(2)O(2)-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H(2)O(2). Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H(2)O(2), and the H(2)O(2)-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H(2)O(2) induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H(2)O(2) appears to be one of the important signaling molecules to regulate the BH4-NOS system.     

Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling: RELATIVE IMPORTANCE OF THE DE NOVO BIOPTERIN SYNTHESIS VERSUS SALVAGE PATHWAYS*

Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.In vascular disease states such as atherosclerosis and diabetes, endothelial nitric oxide (NO) bioactivity is reduced, and oxidative stress is increased, resulting in endothelial dysfunction. It has become apparent that enzymatic “coupling” of endothelial NO synthase by its cofactor tetrahydrobiopterin (BH4)² plays a key role in maintaining endothelial function. Indeed, the balance between NO and superoxide production by eNOS appears to be determined by the availability of BH4 versus the abundance of 7,8-dihydrobiopterin (BH2, that is inactive for NOS cofactor function and may compete with BH4 for NOS binding (1). Intracellular biopterin levels are regulated principally by the activity of the de novo biosynthetic pathway (Fig. 1). Guanosine triphosphate cyclohydrolase I (GTPCH; EC 3.5.4.16) catalyzes the formation of dihydroneopterin triphosphate from GTP, and BH4 is generated by two further steps through 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. GTPCH appears to be the rate-limiting enzyme in BH4 biosynthesis, and overexpression of GTPCH is sufficient to augment BH4 levels in cultured endothelial cells (2). Electron paramagnetic resonance spectroscopy studies have shown that BH4 both stabilizes and donates electrons to the ferrous-dioxygen complex in the oxygenase domain, as the initiating step of l-arginine oxidation (3–5). In this reaction BH4 forms the protonated trihydrobiopterin cation radical, which is subsequently reduced by electron transfer from NOS flavins. When BH4 availability is limiting, electron transfer from NOS flavins becomes uncoupled from l-arginine oxidation, eNOS generates superoxide rather than NO, BH4 becomes oxidized to catalytically incompetent BH2, and a futile feed-forward cascade of BH4 destruction proceeds (1). Recent studies reveal that BH4 and BH2 bind eNOS with equal affinity and that BH2 can efficiently replace eNOS-bound BH4, resulting in eNOS uncoupling (6). Indeed, we have previously shown that the relative abundance of eNOS versus BH4 together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling (7), a hypothesis supported by a recent publication where BH2 levels are elevated after exposure of bovine aortic endothelial cells to DHFR-specific siRNA (8). Thus, mechanisms that regulate the BH4:BH2 ratio independently of overall biopterin levels may play an equally important role in regulating eNOS coupling as the well established role of GTCPH, which regulates de novo BH4 biosynthesis. In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR; EC 1.5.1.3) can reduce BH2, thus regenerating BH4 (9, 10). It is, therefore, likely that net BH4 bioavailability within the endothelium reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. In human liver extracts DHFR has been shown to reduce BH2 back to BH4 as part of the salvage pathway for biopterin synthesis (11). However, the role of this pathway and the extent to which it regulates intracellular BH4 levels in vivo remains unknown. Recent work by Chalupsky and Cai (2) investigated the functionality of endothelial DHFR in cultured bovine aortic endothelial cells. Exposure to angiotensin II down-regulated DHFR expression, decreased BH4 levels, and increased eNOS uncoupling, which was restored by overexpression of DHFR (2). A recent study also suggests that perturbation of BH4 metabolism differentially affects eNOS phosphorylation sites. Knockdown of DHFR by siRNA inhibits vascular endothelial growth factor-induced dephosphorylation of eNOS at Ser-116, an effect that is completely recovered by the addition of exogenous BH4 (8). However, the requirement for DHFR in regulating intracellular BH4 homeostasis and the quantitative relationships that relate BH4 de novo synthesis versus BH4 recycling to eNOS coupling remain uncertain. Accordingly, we sought to address these questions using both pharmacologic and genetic manipulation of DHFR activity and related these interventions to effects on eNOS coupling. We manipulated DHFR in both endothelial cells and in novel cell lines that stably express an eNOS-GFP fusion protein and where expression of human GTPCH can be regulated by doxycycline in order to test the effects of variations in intracellular BH4 biosynthesis (7). We report that although GTPCH is the key regulator of the total amount of intracellular biopterins, DHFR is critical to eNOS function by determining BH4:BH2 ratio and, thus, in maintaining eNOS coupling. In particular, DHFR is important in preventing “self-propagated” eNOS uncoupling in conditions of low total biopterin levels, when eNOS-dependent oxidation of BH4 that would further exacerbate eNOS uncoupling can be rescued by DHFR.Open in a separate windowFIGURE 1.Schematic representation of the BH4 recycling pathway and eNOS coupling. BH4 is synthesized from GTP via a series of reactions involving GTPCH, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase (SR) and DHFR. DHFR activity can be inhibited by MTX. GFRP, GTP cyclohydrolase feedback regulatory protein. PTPS, 6-pyruvoyl-tetrahydropterin synthase.     

l-Cysteine supplementation reduces high-glucose and ketone-induced adhesion of monocytes to endothelial cells by inhibiting ROS

Type 1 diabetic (T1D) patients are hyperglycemic and also show elevated blood levels of ketone bodies, particularly acetoacetate (AA) and β-hydroxybutyrate (BHB). T1D patients have a greater risk of developing endothelial dysfunction and cardiovascular disease (CVD). Supplementation with cysteine-rich milk proteins has been shown to be beneficial in improving various biomarkers of endothelial dysfunction and CVD. This study examines whether l-cysteine (LC) per se prevents monocyte adhesion to endothelial cells, a critical step in endothelial dysfunction. Human umbilical vein endothelial cells and THP-1 monocytes were pretreated with and without LC (500 μM) for 2 h and then exposed to ketones (AA or BHB, 0–4 mM) and/or high glucose (HG) (25 mM) for 24 h. This study shows that LC reduces HG and ketone-induced ROS production, ICAM-1 expression, and the adhesion of monocytes to endothelial cells. This study provides a biochemical mechanism by which milk protein supplementation can be beneficial in preventing the excess endothelial dysfunction and CVD seen in diabetic patients.     

Activation of promoter activity of the catalytic subunit of γ-glutamylcysteine ligase (GCL) in brain endothelial cells by insulin requires antioxidant response element 4 and altered glycemic status: implication for GCL expression and GSH synthesis

Our recent finding that insulin increased the expression of the glutamate-cysteine ligase catalytic subunit (GCLc) with coincident increases in GCL activity and cellular glutathione (GSH) in human brain microvascular endothelial cells (IHECs) suggests a role for insulin in vascular GSH maintenance. Here, using IHECs stably transfected with promoter-luciferase reporter vectors, we found that insulin increased GCLc promoter activity, which required a prerequisite increase or decrease in medium glucose. An intact antioxidant response element-4 was essential for promoter activation, which was attenuated by inhibitors of PI3-kinase/Akt/mTOR signaling. Interestingly, only under low-glucose conditions did promoter activation correlate with increased GCLc expression and GSH synthesis. Low tert-butylhydroperoxide (tBH) concentrations similarly mediated promoter activation, but the maximal activation dose was decreased 10-fold by insulin. Insulin-tBH coadministration abrogated the low or high glucose requirement for promoter activation, suggesting possible ROS involvement. ROS production was elevated at low glucose without or with insulin; however, GSH increases were not inhibited by tempol, suggesting that ROS did not achieve the threshold for driving GCLc promoter activation and de novo GSH synthesis. The minor effect of pyruvate also ruled out a major role for hypoglycemia (± insulin)-induced metabolic stress on GSH induction under these conditions.     

Early determinants of H2O2-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.     

AT1-receptor blockade by telmisartan upregulates GTP-cyclohydrolase I and protects eNOS in diabetic rats

Several enzymatic sources of reactive oxygen species (ROS) were described as potential reasons of eNOS uncoupling in diabetes mellitus. In the present study, we investigated the effects of AT(1)-receptor blockade with chronic telmisartan (25 mg/kg/day, 6.5 weeks) therapy on expression of the BH(4)-synthesizing enzyme GTP-cyclohydrolase I (GCH-I), eNOS uncoupling, and endothelial dysfunction in streptozotocin (STZ, 60 mg/kg iv, 7 weeks)-induced diabetes mellitus (type I). Telmisartan therapy did not modify blood glucose and body weight. Aortas from diabetic animals had vascular dysfunction as revealed by isometric tension studies (acetylcholine and nitroglycerin potency). Vascular and cardiac ROS produced by NADPH oxidase, mitochondria, eNOS, and xanthine oxidase were increased in the diabetic group as was the expression of NADPH oxidase subunits at the protein level. The expression of GCH-I and the phosphorylation of eNOS at Ser1177 was decreased by STZ treatment. Therapy with telmisartan normalized these parameters. The present study demonstrates for the first time that AT(1)-receptor blockade by telmisartan prevents downregulation of the BH(4) synthase GCH-I and thereby eNOS uncoupling in experimental diabetes. In addition, telmisartan inhibits activation of superoxide sources like NADPH oxidase, mitochondria, and xanthine oxidase. These effects may explain the beneficial effects of telmisartan on endothelial dysfunction in diabetes.     

Divergence in regulation of nitric-oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-alpha. Ceramide potentiates nitric oxide synthesis without affecting GTP cyclohydrolase I activity

Synthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a required cofactor for inducible nitric-oxide synthase (iNOS) activity, is usually coordinately regulated with iNOS expression. In C6 glioma cells, tumor necrosis factor-alpha (TNF-alpha) concomitantly potentiated the stimulation of nitric oxide (NO) and BH(4) production induced by IFN-gamma and interleukin-1beta. Expression of both iNOS and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the BH(4) biosynthetic pathway, was also markedly increased, as were their activities and protein levels. Ceramide, a sphingolipid metabolite, may mediate some of the actions of TNF-alpha. Indeed, we found that bacterial sphingomyelinase, which hydrolyzes sphingomyelin and increases endogenous ceramide, or the cell permeable ceramide analogue, C(2)-ceramide, but not C(2)-dihydroceramide (N-acetylsphinganine), significantly mimicked the effects of TNF-alpha on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF-alpha increased BH(4) synthesis and GTPCH activity, neither BH(4) nor GTPCH expression was affected by C(2)-ceramide or sphingomyelinase in IFN-gamma- and interleukin-1beta-stimulated cells. It is likely that increased BH(4) levels results from increased GTPCH protein and activity in vivo rather than from reduced turnover of BH(4), because the GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, blocked cytokine-stimulated BH(4) accumulation. Moreover, expression of the GTPCH feedback regulatory protein, which if decreased might increase GTPCH activity, was not affected by TNF-alpha or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-kappaB and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-kappaB to the nucleus, inhibited TNF-alpha-dependent iNOS mRNA expression without affecting GTPCH mRNA levels. This is the first demonstration that cytokine-stimulated iNOS and GTPCH expression, and therefore NO and BH(4) biosynthesis, may be regulated by discrete pathways. As BH(4) is also a cofactor for the aromatic amino acid hydroxylases, discovery of distinct mechanisms for regulation of BH(4) and NO has important implications for its specific functions.     

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.     

Non-Covalent Interaction between Polyubiquitin and GTP Cyclohydrolase 1 Dictates Its Degradation

GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4). GTPCH1 protein degradation has been reported in animal models of several diseases, including diabetes mellitus and hypertension. However, the molecular mechanisms by which GTPCH1 is degraded remain uncharacterized. Here we report a novel non-covalent interaction between polyubiquitin and GTPCH1 in vitro and in vivo. The non-covalent binding of GTPCH1 to polyubiquitin via an ubiquitin-binding domain (UBD) results in ubiquitination and degradation. Ectopic expression of ubiquitin in cultured cells accelerated GTPCH1 degradation. In cultured cells and in vitro assays, Lys48-linked ubiquitin chains, but not Lys63-linked chains, interacted with GTPCH1 and targeted it for degradation. Consistently, proteasome inhibition attenuated GTPCH1 degradation. Finally, direct mutagenesis of an isoleucine (Ile131) in the hydrophobic patch of the GTPCH1 UBD affected its ubiquitin binding and the enzyme stability. Taken together, we conclude that GTPCH1 non-covalently interacts with polyubiquitin via an ubiquitin-binding domain. The polyubiquitin binding directs GTPCH1 ubiquitination and proteasome degradation.     

Involvement of sphingosine kinase in TNF-alpha-stimulated tetrahydrobiopterin biosynthesis in C6 glioma cells

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a cofactor required for iNOS activity. TNF-alpha also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH(4) biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-alpha on GTPCH expression and activity and BH(4) biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-alpha. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-alpha, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH(4) biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH(4) levels.     

2,4-Diamino-6-hydroxypyrimidine (DAHP) suppresses cytokine-induced VCAM-1 expression on the cell surface of human umbilical vein endothelial cells in a BH(4)-independent manner

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH(4) biosynthesis and is widely used in order to elucidate the possible biological function of BH(4) in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH(4)) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH(4) might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH(4) and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH(4) level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH(4) level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH(4) production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH(4)-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappaB (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappaB level in the nucleus of HUVEC.     

Erythropoietin increases bioavailability of tetrahydrobiopterin and protects cerebral microvasculature against oxidative stress induced by eNOS uncoupling

This study was designed to determine whether treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Wild‐type and GTP cyclohydrolase I (GTPCH‐I)‐deficient hph1 mice were administered EPO (1000 U/kg/day, s.c., 3 days). Cerebral microvessels of hph1 mice demonstrated reduced tetrahydrobiopterin (BH4) bioavailability, increased production of superoxide anions and impaired endothelial NO signaling. Treatment of hph1 mice with EPO attenuated the levels of 7,8‐dihydrobiopterin, the oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8‐dihydrobiopterin. Moreover, EPO decreased the levels of superoxide anions and increased NO bioavailability in cerebral microvessels of hph1 mice. Attenuated oxidation of BH4 and inhibition of endothelial NO synthase uncoupling were explained by the increased expression of antioxidant proteins, manganese superoxide dismutase, and catalase. The protective effects of EPO observed in cerebral microvessels of hph1 mice were also observed in GTPCH‐I siRNA‐treated human brain microvascular endothelial cells exposed to EPO (1 U/mL or 10 U/mL; 3 days). Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of BH4 and endothelial NO in the cerebral microvascular endothelium.

     

Dopa-responsive dystonia induced by a recessive GTP cyclohydrolase I mutation

GTP cyclohydrolase I (GTPCH) catalyzes the rate-limiting step of tetrahydrobiopterin (BH4) biosynthesis. GTPCH has been associated with two clinically distinct human diseases: the recessive hyperphenylalaninemia (HPA) and the dominant dopa-responsive dystonia (DRD). We found a recessive GTPCH mutation (R249S, 747C-->G in a dystonia patient. Her PHA-stimulated mononuclear blood cells had a normal amount of GTPCH mRNA, but low GTPCH activity. Arginine 249 is located at the C-terminus of GTPCH, outside the catalytic site. E. coli expressed recombinant R249S mutant protein possessed normal enzyme activity and kinetics. However, in transfected eukaryotic cells, R249S mutant protein expression level was lower than the wild-type protein. Therefore, this is suspected to be a destabilizing mutation. Our data suggest that DRD could be either dominantly or recessively inherited, and the inheritance might be determined by the mechanism of mutation.