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Cardiovascular Research 2002 55(4):838-849; doi:10.1016/S0008-6363(02)00460-1
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Copyright © 2002, European Society of Cardiology

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation

Shijie Caia, Nicholas J Alpa, Denise McDonalda, Ian Smithb, Jonathan Kayb, Laura Canevaric, Simon Healesc and Keith M Channona,*

aDepartment of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, UK
bDepartment of Clinical Biochemistry, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK
cDepartment of Neurochemistry, Institute of Neurology, Queens Square, London, UK

keith.channon{at}cardiov.ox.ac.uk

* Corresponding author. Tel.: +44-1865-851-085; fax: +44-1865-222-077

Received 13 February 2002; accepted 29 April 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Experimental
 3. Results
 4. Discussion
 References
 
Objectives: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. Methods: We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. Results: GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS–GFP fusion protein were significantly increased following GTPCH gene transfer. Conclusions: These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

KEYWORDS Endothelial function; Gene therapy; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Experimental
 3. Results
 4. Discussion
 References
 
Nitric oxide (NO) is generated in the vascular wall by endothelial nitric oxide synthase (eNOS) that oxidises L-arginine to L-citrulline using molecular oxygen. NO plays central roles in maintaining vascular homeostasis, by effects on endothelial cells, smooth muscle cells, leucocytes and platelets. Impaired NO bioactivity, resulting in endothelial dysfunction is a characteristic feature of vascular disease states, including hypercholesterolemia, atherosclerosis, diabetes, hypertension and cigarette smoking [1]. Although NO bioactivity is decreased in dysfunctional endothelium, levels of eNOS mRNA and protein are maintained or even enhanced, but associated with increased NOS-dependent superoxide formation, due to enzymatic ‘uncoupling’ of eNOS [2,3].

Tetrahydrobiopterin (BH4) is an essential cofactor for activity of all NOS enzymes [2,4]. Intracellular BH4 levels are regulated by the activity of the de novo biosynthetic pathway. GTP cyclohydrolase I (GTPCH; EC 3.5.4.1 [EC] 6) catalyses GTP to dihydroneopterin triphosphate. BH4 is generated by further steps catalysed by 6-pyruvoltetrahydropterin synthase (PTPS) and sepiapterin reductase [5]. In most cells, the rate limiting enzyme appears to be GTPCH, although some data suggest that in human endothelial cells, PTPS may also exert a regulatory role [6].

The exact role of BH4 in NOS catalysis is still unclear, but it appears to facilitate electron transfer from the eNOS reductase domain and maintains the heme prosthetic group in its redox active form [4,7,8]. Moreover, BH4 is required for promoting and possibly stabilising NOS protein monomers into the active homodimeric form of the enzyme [9,10]. However, previous studies that have directly investigated the molecular mechanisms of eNOS regulation by BH4 have focused principally on purified recombinant proteins analysed in cell-free systems [11,12]. The role and mechanisms of BH4 in regulating eNOS activity in intact endothelial cells are less well defined.

Recent studies suggest that reduced BH4 levels may mediate eNOS uncoupling and that loss of BH4 availability in vascular disease states may be an important contributor to the loss of NO production leading to endothelial dysfunction [2,13]. Canine basilar artery rings incubated ex vivo with BH4 show augmented NOS-dependent relaxations. Conversely, rings incubated with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) show reduced NO-dependent relaxations that can be restored by sepiapterin [14]. Similarly, endothelial dysfunction is improved by supplementation of BH4 in vessel rings from animals with atherosclerosis [15], diabetes [16,17] or hypertension [18]. In humans, BH4 administration appears to augment NO-mediated effects on forearm blood flow in smokers [19] and in patients with diabetes [3,20] or elevated cholesterol [21]. However, the effects of BH4 on NO bioactivity observed in these studies may not be entirely mediated through direct modulation of eNOS activity, because high extracellular BH4 concentrations may result in nonspecific antioxidant effects that indirectly increase NO bioactivity by superoxide scavenging.

We sought to investigate the potential utility of a novel approach to modulate intracellular BH4 levels in endothelial cells without recourse to pharmacological supplementation using high biopterin concentrations. We used adenoviral gene transfer of GTPCH to increase the activity of the BH4 de novo biosynthetic pathway, and used this approach to investigate how intracellular BH4 levels regulate eNOS activity, protein levels and dimerisation in intact human endothelial cells.


   2. Experimental
Top
 Abstract
 1. Introduction
 2. Experimental
3. Results
 4. Discussion
 References
 
2.1 Cell cultures
293 cells, the human endothelial cell lines EAhy926 and hMEC (human dermal microvascular endothelial cells), and NIH/3T3 murine fibroblasts were grown in a 5% CO2 atmosphere in Dulbecco's modified Eagles’ medium (DMEM) supplemented with 10% foetal calf serum (FCS) (Sigma), 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine. For hMEC, medium was also supplemented with 30 ng/ml endothelial growth factor and 4 mg/ml hydrocortisone. For adenoviral infection, cells were grown to 70–80% confluence. The monolayers were inoculated with virus, diluted in DMEM supplemented with 2% FCS for 1 h, then replaced into DMEM containing 10% FCS for 72 h.

2.2 Construction of adenovirus vectors
A 1.2-kb human GTPCH-1 cDNA (kindly provided by Dr. H Ichinose, Japan) [22] was modified by PCR and subcloned to incorporate a haemagglutinin (HA) epitope tag at the 5' end. The HA-tagged GTPCH cDNA was cloned into the plasmid pShuttleCMV (kindly provided by Dr. Bert Vogelstein) [23] and used to generate a recombinant adenovirus, AdGCH, encoding HA–GTPCH under the control of the cytomegalovirus immediate-early promoter, by transfection in 293 cells using the AdEasy system [23]. A recombinant adenovirus, AdeNOS–GFP, encoding a C-terminus GFP fusion protein of human eNOS [24] was generated using the same system, and a recombinant adenovirus encoding β-galactosidase (AdβGal) was used as a control for viral infection [25]. Viruses were isolated by three rounds of plaque purification, amplified in 293 cells, and purified using CsCl gradient ultracentrifugation, as previously described [25].

2.3 Western blot analysis
Cells were lysed in 50 mM Tris–HCl, pH 8, containing 0.2% Nonidet P-40, 180 mM NaCl, 0.5 mM EDTA, 100 mM phenylmethylsulforyl fluoride, 1 M DTT and protease inhibitors (Boerhinger Complete). Equal amounts of cellular proteins were resolved by 6 or 12% SDS–PAGE and transferred to PVDF membranes. To investigate eNOS homodimer formation in endothelial cells, low-temperature SDS–PAGE was performed as described previously [12]. Membranes were incubated with either a 1:1000 dilution of rat anti-HA high affinity monoclonal antibody (Roche), a 1:400 dilution of chicken anti-human GTPCH polyclonal antibody (a generous gift of Dr. M Gutlich, Germany), or a 1:2000 dilution of mouse anti-eNOS monoclonal antibody (Transduction Laboratories). Bands were visualised using chemiluminescence, and quantified using NIH IMAGE software. The amount of eNOS–GFP fusion protein in cell lysates was measured by fluorescence as described [24], calculated from a standard curve of purified GFP (Clontech).

2.4 Determination of biopterins in cell lysates and culture medium
Two different HPLC methods were used to determine biopterin levels in separate experiments: acid–base oxidation followed by fluorometric detection, and direct coulometric detection. For fluorometric detection, measurements of BH4 and dihydrobiopterin (BH2) were performed by HPLC analysis, after iodine oxidation in acidic or alkaline conditions, as previously described [26]. Briefly, cell pellets were lysed in cold extract buffer (50 mM Tris–HCl, pH 7.4, 1 mM DTT, 1 mM EDTA, containing 0.1 µM neopterin as an internal recovery standard). Protein concentration was measured using the Bio-Rad protein assay. Proteins were removed by adding 10 µl of a 1:1 mixture of 1.5 M HClO4 and 2 M H3PO4 to 90 µl of extracts, followed by centrifugation. To determine total biopterins (BH4, BH2, biopterin) by acid oxidation 10 µl of 1% iodine in 2% KI solution was added to 90 µl protein-free supernatant. To determine BH2 and biopterin by alkali oxidation 10 µl of 1 M NaOH was added to 80 µl of extract, then 10 µl of iodine/KI solution. Samples were incubated at room temperature for 1 h in the dark. Alkaline-oxidation samples were then acidified with 20 µl of 1 M H3PO4. Iodine was reduced by adding 5 µl of fresh ascorbic acid (20 mg/ml). HPLC was performed using a Spherisorb ODS-1 column (Waters, Elstree, UK) with a methanol–water (5:95, v/v) mobile phase run at 1 ml/min. Fluorescence detection (350 nm excitation, 450 nm emission) was performed using an FP-1520 detector (Jasco, Essex, UK). BH4 concentration, expressed as pmol/mg protein, was calculated by subtracting BH2+biopterin from total biopterins. In some experiments, biopterins were also measured by HPLC using direct coulometric detection, as described previously [27]. Cells or medium were treated in 0.2 M perchloric acid containing dithiothreitol (6.5 mM) and 2.5 mM diethylenetriaminepentaacetic acid. Following centrifugation (15 000 g for 5 min) 100 µl of supernatant were analysed by HPLC, and biopterins were detected electrochemically using an ESA Coulochem detector. The sensitivity of the assays was approximately 1 pmol/mg protein. All samples were determined in triplicate, and HPLC results were calculated blind to the samples’ identity.

2.5 NOS activity assays by nitrite determination
Cells were passaged into 12-well plates. After 48 h incubation, with or without viral infection, the cell monolayers were washed three times with Krebs–HEPES buffer (KHB) (NaCl 99 mM, KCl 4.7 mM, MgSO4 1.2 mM, KH2PO4 1.0 mM, CaCl2 1.9 mM, NaHCO3 25 mM, glucose 11.1 mM, HEPES 20 mM) and were incubated for 2 h with KHB containing calcium ionophore (A23187 [GenBank] ; 1 µM) and L-arginine (100 µM) in the presence or absence of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA; 1 mM), as previously described [28]. We determined NOS activity by measuring nitrite accumulation in the buffer, in triplicate wells, using a microamperometric assay (NOMkII, World Precision Instruments), expressed as nmol nitrite/h/106 cells.

2.6 Immunostaining
Cells, grown on glass coverslips, were washed with PBS, fixed for 10 min with 3% paraformaldehyde containing 2% glucose, permeablised for 5 min with 0.2% Triton X-100, then blocked for 15 min with 3% BSA. Primary anti-HA epitope monoclonal antibody was bound for 1 h. Following washes, secondary FITC-conjugated antibody was added for 15 min. After additional washes, cells were counterstained with propidium iodide and imaged using a Bio-Rad MRC1024 confocal microscope.

2.7 Statistics
Results are expressed as mean±S.D. Statistical significance of differences between means was assessed using Students’ t-test. A P value <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Experimental
 3. Results
4. Discussion
 References
 
3.1 Effects of GTPCH gene transfer on GTPCH expression and intracellular biopterin production
We first evaluated the effects of GTPCH gene transfer in human EAhy926 endothelial cells, infected with AdGCH, or with AdβGal as control, in a series of increasing titres. Cell lysates, harvested after 48 h, were analysed by immunoblotting for recombinant GTPCH protein and by HPLC determination of cellular biopterins.

Recombinant GTPCH protein, detected by either anti-human GTPCH or anti-HA epitope antibodies, increased in a dose-dependent fashion after AdGCH infection but was not detectable in control or AdβGal-transduced cells (Fig. 1). Levels of total biopterin and BH4 in nontransduced EAhy926 were very low. In contrast, cells transduced with AdGCH greatly augmented both total biopterin and BH4, also in a dose-dependent manner, with a 50-fold increase in total biopterin levels at a viral multiplicity of infection (MOI) of 100 pfu/cell.


Figure 1
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  		Fig. 1 GTP cyclohydrolase gene transfer augments intracellular biopterin levels in human endothelial cells. EAhy926 cells were harvested 48 h after infection with either AdGCH or AdβGal at a series of increasing titres (multiplicity of infection, MOI). Cell lysates were fractionated by SDS–PAGE and immunoblotted with antibodies to HA tag epitope, GTPCH, or eNOS (A). Biopterin content and BH4 levels were measured in cell pellets by differential oxidation in acid and base followed by the reverse phase HPLC (B). Data are shown as pmol/mg of cell protein (mean±S.D. of triplicate determinations).

 
At very low AdGCH viral titres (<10 MOI) GTPCH expression was below of the detection limit of Western blotting. However, AdGCH at these levels still generated a 6-fold increase in biopterin levels compared with nontransduced cells, suggesting that even very low-level GTPCH overexpression is sufficient to significantly augment intracellular biopterin levels.

We further determined total cellular biopterins, BH4 and neopterin levels in endothelial cell lysates infected with AdGCH or AdβGal (MOI 50), compared with cells incubated with the synthetic pterin sepiapterin, that elevates intracellular BH4 indirectly via the pterin salvage pathway. We determined biopterin levels in separate experiments using two different HPLC methods: acid–base oxidation followed by fluorometric detection, and by direct coulometric detection (Table 1). Biopterin measurements showed similar differences when determined using either method. Control cells or cells after AdβGal infection had very low levels of total biopterin, BH2 and BH4 that were increased more than 10-fold by AdGCH infection. Sepiapterin (10 µM) increased BH4 to levels similar to AdGCH, but total biopterins were even more greatly elevated, such that BH4 was a much smaller proportion of total biopterins (AdGCH 30% vs. sepiapterin <10%). Changes in biopterins in cell culture media showed changes that corresponded with the changes in cellular levels (Table 1). In contrast to the striking elevations of BH4 levels following GTPCH gene transfer, the levels of neopterin, produced from the biopterin intermediate between GTPCH and PTPS, remained low, suggesting that PTPS activity was not significantly limiting for BH4 biosynthesis despite the greatly increased GTPCH activity.


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  		Table 1 Effect of GTPCH gene transfer on cellular biopterin production in EAhy926 cells

 
Taken together, these data show that GTPCH overexpression by adenoviral gene transfer is highly effective in augmenting endothelial cell BH4 levels in a dose-dependent fashion. Endothelial cell biosynthesis of BH4 under these conditions does not appear to be limited by the activities of the ‘downstream’ enzymes PTPS and sepiapterin reductase. Furthermore, GTPCH gene transfer results in a more specific elevation of BH4 in relation to other cellular biopterins compared with sepiapterin supplementation.

3.2 Effects of GTPCH gene transfer on eNOS activity in endothelial cells
To investigate the effects of GTPCH overexpression on eNOS activity, we measured nitrite production by calcium ionophore (A23187 [GenBank] )-stimulated endothelial cells. Western blot analysis demonstrated that all EAhy926 cells expressed endogenous eNOS protein (Fig. 1). However, AdGCH infection greatly augmented eNOS activity (>15 fold) in EAhy926 cells in a dose-dependent fashion (Fig. 2). In contrast, eNOS activity in AdβGal transduced cells remained at basal levels. Thus, recombinant GTPCH expression in endothelial cells by adenoviral gene transfer results in parallel increases in both intracellular biopterin levels and eNOS activity.


Figure 2
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  		Fig. 2 GTP cyclohydrolase gene transfer increases eNOS activity in human endothelial cells. EAhy926 cells were infected with either AdGCH or AdβGal at a series of increasing virus titres (multiplicity of infection, MOI). After 48 h incubation, cell monolayers were stimulated with A23187 (1 µM) for 2 h. eNOS activity was determined by electrochemical measurement of nitrite accumulation. Data are shown as pmol nitrite/h/106 cells (mean±S.D. of triplicate determinations).

 
To further investigate the mechanisms of increased eNOS activity following GTPCH gene transfer, we measured nitrite production by EAhy296 cells under different conditions. Increased eNOS activity was abolished by DAHP (3.0 µM), an inhibitor of GTPCH, and by L-NMMA, an inhibitor of eNOS, despite the presence of similar levels of recombinant GTPCH protein and eNOS protein (Fig. 3). As expected, extracellular sepiapterin supplementation (10 µM) also increased eNOS activity, independent of GTPCH overexpression. However, the magnitude of eNOS activation was not as great as with GTPCH gene transfer despite the very large increase in total biopterins, possibly reflecting the greater proportion of oxidised biopterins produced under these conditions.


Figure 3
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  		Fig. 3 Enhanced NO production after GTPCH gene transfer is dependent on GTPCH and eNOS activities. EAhy926 cells were incubated with media alone, with sepiapterin (10 µM), with AdGCH (50 MOI), plus either DAHP (3 µM) or L-NMMA (1 mM), or with AdβGal (50 MOI) as control. After 48 h incubation, proteins of cell lysates were fractionated by SDS–PAGE and immunoblotted with antibodies to HA tag epitope, or to eNOS (A). Cell monolayers were stimulated with A23187 (1 µM) for 2 h. eNOS activity was determined electrochemically by measurement of nitrite concentration in the cell supernatants (B). Data are shown as mean±S.D. of triplicates determination, expressed as pmol/h/106 cells.

 
We next investigated the association between GTPCH overexpression, NO production and eNOS protein, by comparison of AdGCH gene transfer in EAhy926, hMEC and NIH/3T3 cells, that express eNOS at different levels. Fluorescent immunostaining revealed abundant recombinant GTPCH localised in the cytoplasm of all three cell lines after AdGCH infection, but not in control cells (Fig. 4). Correspondingly, HPLC analysis of cell lysates showed that AdGCH gene transfer greatly elevated intracellular biopterin production in all of these cell lines (Table 2). In contrast, nitrite production by AdGCH-transduced hMEC was less than one third that in EAhy926 cells, and in NIH/3T3 cells there was no increase in nitrite production after AdGCH gene transfer (Fig. 5). Western blot analysis showed that these variations in nitrite production were related to the levels of endogenous eNOS protein present in the three cell lines (Fig. 5); EAhy926 cells have abundant eNOS, NIH/3T3 cells do not express eNOS, and hMEC cells have low eNOS levels. These data show that GTPCH gene transfer augments NO production in a GTPCH- and eNOS-specific manner, and in direct relation to the levels of endogenous eNOS present in the target cell.


Figure 4
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  		Fig. 4 Detection of recombinant GTPCH by immunofluorescence. EAhy926, hMEC and NIH/3T3 cells were fixed 48 h after infection with either AdGCH (50 MOI) or AdβGal (50 MOI), or uninfected, then immunostained with a primary anti-HA tag epitope antibody, secondary FITC-conjugated antibody (green fluorescence) and counterstained with propidium iodide (red).

 

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  		Table 2 Effect of recombinant GTPCH expression on intracellular biopterin production in EAhy926, hMEC and NIH/3T3 cells

 

Figure 5
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  		Fig. 5 Enhanced eNOS activity by recombinant GTPCH depends on eNOS protein levels. EAhy926 or hMEC cells were harvested after 48 h incubation with media alone, with either AdGCH (50 MOI) or AdβGal (50 MOI), respectively, with NIH/3T3 cells as control. Proteins from cell lysates were fractioned by SDS–PAGE and immunoblotted with antibody to eNOS (A). Cell monolayers were stimulated with A23187 (1 µM) for 2 h. eNOS activity was determined electrochemically by measurement of nitrite concentration in the cell supernatants (B). Data are shown as mean±S.D. of triplicates determination, expressed as pmol nitrite/h/106 cells.

 
3.3 GTPCH gene transfer promotes eNOS dimerisation in endothelial cells
We next used GTPCH gene transfer to investigate the role of BH4 in modulating eNOS dimerisation in living endothelial cells, and to determine the relationships between eNOS activity, eNOS dimerisation and the total levels of eNOS protein. We used low temperature SDS–PAGE and immunoblotting to investigate eNOS dimerisation in EAhy926 cells, following gene transfer of GTPCH (Fig. 6). In cells incubated with medium alone, or infected with AdβGal, eNOS was present predominantly as the 135-kDa monomer, in keeping with the low eNOS activity previously shown under these conditions (Fig. 6A). In contrast, eNOS protein in cells transduced with AdGCH, was predominantly in the active homodimeric form. Densitometric quantification of blots from three individual experiments revealed that in AdGCH transduced cells, the total quantity of homodimeric eNOS was increased 3-fold and the ratio of dimer:monomer band intensities was more than two-fold higher than in control cells (Fig. 6B and C).


Figure 6
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  		Fig. 6 GTPCH gene transfer promotes eNOS dimerisation and increases eNOS protein levels. EAhy926 cells were harvested 48 h after mock infection (cells alone), infection with 50 MOI AdGCH, AdβGal, or AdβGal plus sepiapterin (10 µM, +Sep). Nonboiled cell lysates (equalised for protein), and a boiled bovine endothelial cell lysates as a positive control (+C), were fractionated by low temperature SDS–PAGE and immunoblotted with a monoclonal antibody to eNOS (A). (B) Quantification of eNOS dimer bands from n = 3 separate experiments. *, P<0.01 vs. cells alone or AdβGal. (C) Ratio of eNOS dimer:monomer band intensities from n = 3 separate experiments. *, P<0.01 vs. cells alone or AdβGal. (D) Quantification of total eNOS protein band intensity (dimer+monomer) from n = 3 separate experiments. *, P<0.05 vs. cells alone.

 
3.4 GTPCH gene transfer increases total eNOS protein levels in endothelial cells
In addition to the striking changes in eNOS dimerisation following GTPCH gene transfer, the total amount of eNOS protein, quantified by immunoblotting (i.e. sum of monomer+dimer band intensities) was also significantly increased in AdGCH-infected cells compared with control cells (Fig. 6D). In view of the potential limitations of immunoblotting for quantification of differences in protein quantity, we also used adenoviral gene transfer to express an eNOS–GFP fusion protein in endothelial cells and determined the molar quantities of recombinant eNOS–GFP protein in cells under different conditions by direct measurement of GFP fluorescence in cell lysates, rather than relying on semiquantitative analysis by immunoblotting (Table 3). In hMEC expressing recombinant eNOS–GFP, GTPCH gene transfer greatly increased eNOS–GFP dimerisation, and augmented eNOS–GFP activity, as observed for native eNOS in EAhy296 cells (data not shown). In EAhy296 cells, GTPCH gene transfer increased the total amount of recombinant eNOS–GFP protein by approximately one quarter, when quantified directly by GFP fluorescence. Taken together, these findings suggest that adenoviral gene transfer of GTPCH greatly augments BH4 levels and eNOS activity in human endothelial cells. This effect is mediated principally by increased enzyme activation and eNOS homodimerisation, but is also associated with a significant increase in total eNOS protein quantity.


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  		Table 3 Effect of GTPCH gene transfer on recombinant eNOS–GFP protein levels in EAhy926 endothelial cells

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Experimental
 3. Results
 4. Discussion
References
 
In this study we have used GTPCH gene transfer to investigate how eNOS is regulated by changes in intracellular BH4 biosynthesis in human endothelial cells. Our findings establish the utility of GTPCH gene transfer as a novel and specific technique to augment intracellular BH4 levels, and provide novel insights into the mechanisms of intracellular eNOS regulation in intact endothelial cells. Furthermore, these approaches are important in establishing proof of principle for future strategies aimed at regulating eNOS activity and function through modulation of intracellular BH4 biosynthesis.

Several previous studies have established that BH4 is a required cofactor for NOS activity. BH4-free NOS is inactive for NO formation; activity is restored by reconstituting BH4 [9]. Structural studies suggest a role for BH4 in heme catalysis and NOS dimerisation [29,30]. However, most previous functional studies of the role of BH4 in eNOS function have relied on purified recombinant proteins in reconstituted cell-free systems. Bovine eNOS expressed in E. coli suggested that BH4 was required for eNOS activation by modulation of heme redox function, although the effect on eNOS homdimerisation appeared to be minimal [11]. In another study of recombinant eNOS purified from a baculovirus system, exogenously added BH4 increased both eNOS activity and dimerisation [31]. However, in none of these studies was it possible to address the functional importance of BH4 in intracellular eNOS regulation, where eNOS is subject to a host of other regulatory mechanisms including membrane association and subcellular trafficking, phosphorylation and protein–protein interactions [32]. Our data now suggest that in intact human endothelial cells eNOS dimerisation is an important regulator of BH4-mediated eNOS activation. Since we used human endothelial cell lines, further studies are needed in primary cell cultures and in endothelial cells isolated from disease states, where absolute levels of BH4 and eNOS protein may be different. However, our experiments with both native eNOS and recombinant eNOS–GFP fusion proteins allowed us to investigate the relative contribution of these factors to the overall increase in eNOS activity in this model system. GTPCH gene transfer increased total eNOS activity in EAhy296 10-fold; the quantity of dimerised eNOS was increased 3-fold and total eNOS protein levels were increased by 25%. These observations suggest that the principal action of BH4 in intact endothelial cells is to increase the enzymatic specific activity of eNOS. This is accompanied by eNOS homodimerisation, and by a modest but significant increase in total eNOS protein levels.

Our observation of increased eNOS protein levels in response to increased BH4 levels in endothelial cells suggests a novel aspect of intracellular eNOS regulation by BH4. This observation may reflect increased stability of the eNOS homodimer compared with the BH4-deficient monomer. Indeed, recent studies suggest that the neuronal NOS (nNOS) homodimer is more stable than free monomers, resulting in proteolytic degradation of monomers [32,33]. Our findings now raise the possibility that an important action of BH4 in regulating eNOS in endothelial cells is to maintain eNOS homodimerisation that in turn stabilises the protein and leads to an increase in steady-state protein levels.

Our study has important implications for therapeutic strategies aimed at augmenting or normalizing eNOS activity by increasing intracellular BH4 levels. eNOS uncoupling in diseased endothelium appears to be an important aspect of endothelial dysfunction in vascular disease states, suggesting that increasing BH4 synthesis could restore eNOS function in conditions such as diabetes [17] and atherosclerosis [15]. Indeed, GTPCH overexpression in combination with inducible NOS (iNOS) gene transfer to vein grafts appeared to increase NOS activity [34], although it was not clear whether recombinant GTPCH led to the expected increase in BH4 levels, or whether other mechanism(s) may have contributed—for example, increased iNOS expression due to the effects of viral coinfection. Our studies in human endothelial cells now provide more definitive proof-of-principle that strategies targeting the intracellular BH4 biosynthetic pathway are a rational and effective approach to augment BH4 levels in endothelial cells, and that this has specific effects on eNOS activity and regulation. This approach avoids the potential nonspecific effects of high-level extracellular biopterin supplementation, that may increase NO bioactivity, but do so by simple removal of superoxide anions rather than any specific effect on NOS activity or regulation. Furthermore, these effects of BH4 supplementation are unpredictable, as high concentrations of this redox-active compound can be pro-oxidant, leading directly to superoxide generation that reduces NO bioactivity [35,36].

GTPCH gene transfer results in a proportionately greater increase in the BH4 fraction compared with the elevation in total biopterins, including the oxidised form BH2, than results from pharmacologic supplementation using sepiapterin. This observation has potentially important implications for studies of eNOS regulation, as BH2 is inactive for eNOS activation and may compete with BH4 for eNOS binding and worsen eNOS uncoupling [15,17]. We measured biopterins using two separate assay systems: differential acid–base oxidation with fluorometric detection and by direct coulometric detection [26,27]. Both assays gave very similar results, although quantification of the BH2 fraction in conditions of very high pharmacologic biopterin supplementation, using sepiapterin, tended to be underestimated by the differential oxidation method. This is likely due to the known limitations of this method in the efficiency of oxidation [5,26]. Similarly, the absolute levels of the BH2/BH4 subfractions in cell culture media are likely to have been affected by ambient oxidation during the experimental time course. Overall, the BH2:BH4 ratio, determined by either method, tended to be low in these endothelial cells in culture, suggesting relatively high oxidative stress and/or a relative deficiency in recycling of BH4 from quinonoid BH2 catalysed by dihydropteridine reductase (DHPR) [5]. Nevertheless, our results clearly show that GTPCH overexpression alone is sufficient to greatly increase BH4 levels in endothelial cells, suggesting that the other ‘downstream’ enzymes in the de novo synthetic pathway do not become significantly rate-limiting even when GTPCH activity is greatly increased [6]. Thus, endothelial GTPCH overexpression in experimental models should provide a powerful and specific approach to investigate the effects of BH4 on eNOS activity and regulation, and could potentially provide a therapeutic strategy in vascular disease states where reduced BH4 availability results in endothelial dysfunction.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by grants from the Garfield Weston Trust and the British Heart Foundation. NJA is a Wellcome Trust Clinical Research Fellow.


   References
Top
 Abstract
 1. Introduction
 2. Experimental
 3. Results
 4. Discussion
 References
 

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