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Am J Physiol Heart Circ Physiol 290: H786-H793, 2006. First published September 30, 2005; doi:10.1152/ajpheart.00759.2005
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Increased blood flow causes coordinated upregulation of arterial eNOS and biosynthesis of tetrahydrobiopterin

Chen-Fuh Lam,1,2,4 Timothy E. Peterson,1,2 Darcy M. Richardson,1,2 Anthony J. Croatt,3 Livius V. d'Uscio,1,2 Karl A. Nath,3 and Zvonimir S. Katusic1,2

Departments of 1Anesthesiology and 2Molecular Pharmacology and Experimental Therapeutics and 3Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota; and 4Department of Anesthesiology, National Cheng Kung University Hospital, Tainan, Taiwan

Submitted 18 July 2005 ; accepted in final form 26 September 2005


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Shear stress, imposed on the vascular endothelium by circulating blood, critically sustains vascular synthesis of nitric oxide (NO). Endothelial NO synthase (eNOS) activity is determined by heat shock protein 90 (HSP90), caveolin-1, and the cofactor tetrahydrobiopterin (BH4). To determine whether increased blood flow concomitantly upregulates eNOS and GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme in BH4 biosynthesis), an aortocaval fistula model in the rat was employed wherein aortic blood flow is enhanced proximal but decreased distal to the fistula. Eight weeks after the creation of the aortocaval fistula, the proximal and distal aortic segments were harvested; sham-operated rats served as controls. Vasomotor function was assessed by isometric force recording. Expression of eNOS, HSP90, caveolin-1, Akt, phosphorylated eNOS (eNOS-Ser1177), and GTPCH I were determined by Western blot analysis. Biosynthesis of BH4 and GTPCH-I activity was examined by HPLC. In the aortic segments exposed to increased flow, contractions to KCl and phenylephrine were reduced, whereas endothelium-dependent relaxations were not affected compared with sham-operated or aortic segments with reduced blood flow. Expression of eNOS, caveolin-1, phosphorylated Akt, and eNOS-Ser1177 was enhanced in aortas exposed to increased blood flow. High flow augmented levels of cGMP and BH4 and increased expression of GTPCH I. In aggregate, these findings provide the first demonstration in vivo that coordinated vascular upregulation of eNOS, and GTPCH I accompanies increased blood flow. This induction of GTPCH I increases BH4 production, thereby optimizing the generation of NO by eNOS and thus the adaptive, vasorelaxant response required in sustaining increased blood flow.

guanosine 3',5'-cyclic monophosphate; endothelium; guanosine 5'-triphosphate cyclohydrolase I; nitric oxide; shear stress

VASCULAR ENDOTHELIAL CELLS are constantly exposed to shear stress incurred by circulating blood (4, 19). Shear stress is an important regulator of vascular diameter, vascular wall remodeling, hemostasis, and inflammatory responses (9). Endothelial nitric oxide (NO) synthase (eNOS) is the key molecule in the regulation of vascular function during changes in shear stress or blood flow and exhibits increased expression and activity in endothelial cells that are exposed to high laminar flow (3, 27, 29). Upregulation of eNOS produces higher levels of NO, which relaxes the vascular smooth muscle cells, inhibits smooth muscle cell proliferation, and prevents adhesion of inflammatory cells and platelets (15). Laminar shear stress thus exerts a significant antiatherogenic effect in the cardiovascular system (37).

The biosynthesis of NO by eNOS can be regulated by the protein-protein interaction, phosphorylation of the enzyme, and the availability of the substrate L-arginine, the cofactor tetrahydrobiopterin (BH4), and calcium (12, 16). Two of the most important proteins influencing activity of eNOS are heat shock protein 90 (HSP90) and caveolin-1. Binding of HSP90 enhances the activity of eNOS, whereas binding to the caveolin-1 scaffold domain holds eNOS in a primed yet inactive state (16). Shear stress influences the NO-generating machinery at several steps. For example, the association of eNOS and HSP90 is stimulated when cultured endothelial cells are exposed to shear stress (14). In cultured endothelial cells, shear stress increases the density of caveolae (32), the latter being considered a mechanosensor of shear stress in the plasma membrane of the endothelium (1, 31). eNOS is rapidly phosphorylated at Ser1177 (eNOS-Ser1177) by shear stress and enhances the production of NO via the activation of protein kinase B/Akt pathway (6, 10, 13).

Although these in vitro studies on cultured endothelial cells provide important insights regarding the behavior of vascular endothelium in response to shear stress, the extent to which these findings faithfully predict the behavior of the vasculature exposed to increased blood flow in vivo is a largely unaddressed issue. Vascular responses, including gene and protein expression as shown in a prior study from Nath's laboratory (28), may not accurately reflect responses observed in vivo. Moreover, few, if any, studies have examined whether changes in caveolin-1 and protein kinase B/Akt accompany induction in eNOS in vivo in response to increased blood flow, and none, either in vivo or in vitro, has examined the effect of blood flow on BH4 metabolism. The effect of shear stress on BH4 metabolism cannot be reliably studied by an approach based on an endothelial cell culture system, because levels of BH4 measured in cultured endothelial cells are very low, the latter being incurred by the artefactual loss of BH4 from cultured cells. The assessment of BH4 and the rate-limiting enzyme in the biosynthesis of BH4, GTP cyclohydrolase (GTPCH I), in such in vitro systems is thus problematic (22, 33, 41). An added issue raised by such artefactual reduction in cellular content of BH4 is that eNOS may be uncoupled when BH4 is insufficiently supplied, thereby leading to cellular production of superoxide anions rather than NO (7, 24, 38).

Our present study was thus designed to test the hypothesis that in vivo increased blood flow causes upregulation of GTPCH-I protein expression and enzymatic activity, thereby increasing local concentration of BH4 in arterial wall and thus contributing to the increase in eNOS observed in this setting.


   METHODS
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 METHODS
RESULTS
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Rat model of aortocaval fistula. The aortocaval (AC) fistula model has been described in detail in previous reports (23, 28). In brief, a fistula was created in the abdominal aorta and adjacent inferior vena cava of Sprague-Dawley rats (~250 g) by puncturing the vessels using an 18-gauge needle. The entry point of the needle into aorta was sealed with a drop of cyanoacrylate glue. The sham-operated rats underwent laparotomy, cross-clamping of the aorta and inferior vena cava for 30 s without puncturing, and the placement of a drop of glue at the abdominal aorta. All procedures were approved and performed in accordance with the guidelines of the Animal Care and Use Committee of the Mayo Clinic and Foundation.

Eight weeks after the creation of AC fistula, systolic blood pressure was measured in conscious rats by an automated tail-cuff method (Harvard Apparatus, Kent, UK). Rats were then anesthetized with ketamine (30 mg/kg im) and xylazine (6 mg/kg im) and subjected to a midline abdominal incision, and the aorta was exposed. Blood flow in the abdominal aorta at the level above renal vessels was measured by an ultrasonic flow probe (Transonic System, Ithaca, NY). Rats were euthanized with injection of pentobarbital sodium (250 mg/kg ip) after the hemodynamic measurements, and the abdominal aorta was harvested.

Western blot analysis. Isolated aortic segments were lysed in buffer containing (in mmol/l) 50 NaCl, 50 NaF, 50 sodium pyrophosphate, 5 EDTA, 5 EGTA, and 2 Na3VO4 and 1% Triton X-100, 0.5 mmol/l PMSF, 10 µg/ml leupeptin, and 10 mmol/l HEPES, pH 7.4. Soluble protein extracts (50 µg) were loaded into polyacrylamide gels (9–12%) and transferred onto nitrocellulose membranes. Mouse monoclonal anti-eNOS, anti-eNOS-Ser1177, anti-HSP90, anti-caveolin-1, anti-Akt, and anti-phosphorylated Akt at Ser473 (Akt-Ser473) antibodies were used. After incubation with horseradish peroxidase-linked secondary antibodies was completed, bands were visualized by using enhanced chemiluminescence. Equal loading of proteins was confirmed by Ponceau S staining. Protein levels were quantified by scanning densitometry (Scion Image).

Measurement of cGMP. Aortic segments were incubated in Earle's salts solution containing 0.1% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin with IBMX (10–3 M) for 30 min to inhibit the degradation of cyclic nucleotides by phosphodiesterases. Aortas were then snap-frozen and stored at –80°C until assayed. Aortic segments were homogenized, and tissue concentrations of cGMP were determined by a radioimmunoassay kit (Amersham Pharmacia) (8).

Measurement of vascular reactivity. Rings (~2 mm long) were isolated from aortic segments proximal to the fistula (aortic segments with increased blood flow), from aortic segments distal to the fistula (aortic segments with decreased blood flow), and from the abdominal aorta of sham-operated rats. Aortic rings were mounted in organ chambers containing 25 ml of Krebs solution containing (in mmol/l) 118.6 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.1 NaHCO3, 10.1 glucose, and 0.026 EDTA. The chambers were maintained at 37°C and aerated continuously with 94% O2-6% CO2. Changes in isometric force were recorded continuously using an isometric force-displacement transducer (Grass FT03; Grass Instrument). Each ring was gradually stretched to 2.5 g. After a 45-min equilibration period, the rings were contracted by an addition of KCl (40 mM) and increasing concentrations of phenylephrine (PE, 10–9 to 10–5 M). Concentration-response curves were also obtained by a cumulative addition of acetylcholine and a NO donor diethylaminodiazen-1-ium-1,2-diolate (DEA-NONOate; 10–9 to 10–5 M) during contraction to a median effective concentration (EC50) of PE. Some of the preparations were incubated with a NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10–5 M) for 15 min before contraction with KCl or PE. Papaverine (3 x 10–4 M) was used to induce complete relaxation of the vessels. All experiments were performed in vessels with intact endothelium.

Measurement of tissue biopterin and GTPCH-I activity and expression. Biopterin levels were determined after differential oxidation in acid and base conditions by reverse-phase HPLC (8). GTPCH-I activity was assessed as a function of neopterin production under standard conditions with GTP as a substrate (19). The expression of GTPCH I was also analyzed by Western blot analysis (immunoblotted against rabbit anti-mouse GTPCH-I antibody, 1:50 dilution, custom-made, Invitrogen).

Antibodies and drugs. Unless otherwise specified, all the antibodies used in the present study were purchased from BD Transduction (eNOS, eNOS-S1177, HSP90, and caveolin-1) or Stressgen (Akt and Akt-S473). IBMX, phenylephrine, acetylcholine, DEA-NONOate, and papaverine were obtained from Sigma. All other reagents were of analytical grade.

Statistical analysis. Results are presented as means ± SE. Mean values comparing sham-operated and AC fistula rats were analyzed by an unpaired t-test. Mean values comparing aortic segments proximal from and distal to fistula were analyzed by a paired t-test. ANOVA was used to compare the concentration-dependent curves between groups. Statistical significance was accepted at a level of P < 0.05.


   RESULTS
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Hemodynamic measurements. Eight weeks after surgery, systolic blood pressure was similar between rats that received sham or AC fistula operation (106 ± 5 vs. 103 ± 3 mmHg, sham vs. fistula, respectively; n = 4–5 rats, P = 0.46). When compared with sham-operated animals, rats with the AC fistula had significantly higher aortic blood flow measured at the level of renal vessels (0.12 ± 0.03 vs. 0.22 ± 0.01 ml·min–1·g body wt–1, respectively; n = 3 rats, P = 0.04).

Western blot analysis. When compared with distal aortic segments (lower blood flow), segments proximal to the fistula (higher blood flow) expressed significantly higher levels of eNOS, eNOS-Ser1177, HSP90, and caveolin-1 (Fig. 1, A and B). Expression of these proteins, except HSP90 in aortic segments proximal to the fistula, was also higher than that observed in sham-operated rats (Fig. 1, A and B). Phosphorylation of the Ser473 residue of Akt was increased in vessels exposed to high flow, whereas the total level of Akt remained unchanged (Fig. 2, A and B).



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  		Fig. 1. A: representative Western blot analysis of protein expression in aortas of sham-operated rats and rats with aortocaval fistula 8 wk after operation. Proximal, aortic segments proximal to fistula (high flow); distal, aortic segments distal to fistula (low flow). Proximal and distal aortas in sham-operated rats represent corresponding levels of aortic segments in rats with fistula. eNOS, endothelial nitric oxide synthase; eNOS-S1177, phosphorylated eNOS at Ser1177; HSP90, heat shock protein 90. B: expression of different eNOS-related proteins was quantified by scanning densitometry (Scion Image) and are shown in relative density. In sham-operated group, quantitation of protein expression was undertaken in aortic segments corresponding to proximal aortic segments of rats with aortocaval fistula. *P < 0.05; n = 4–6 different animals for each protein analysis. Unpaired t-test was used to compare sham-operated rats and rats with aortocaval fistula, and paired t-test was used to compare proximal and distal aortic segments in rats with aortocaval fistula.

 


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  		Fig. 2. A: representative Western blot analysis demonstrating expression of phosphorylated Akt at Ser473 (Akt-S473) and Akt in aortas of sham-operated rats and rats with aortocaval fistula. Proximal and distal aortas in sham-operated rats represent corresponding levels of aortic segments in rats with fistula. B: protein levels of Akt-S473 were quantified by scanning densitometry (Scion Image) and are shown in relative density in 3 vessel segments. In sham-operated group, quantitation of protein expression was undertaken in aortic segments corresponding to proximal aortic segments of rats with aortocaval fistula. *P < 0.05; n = 4–5 different animals for each protein analysis. Unpaired t-test was used to compare sham-operated rats and rats with aortocaval fistula, and paired t-test was used to compare proximal and distal aortic segments in rats with aortocaval fistula.

 
Measurement of cGMP. The amount of NO released from the aorta was quantified by the tissue concentrations of cGMP after incubation in IBMX for 30 min. Concentrations of cGMP were significantly elevated in the aortic segments exposed to higher blood flow compared with sham-operated and low-flow aortic segments, whereas there was no difference between aorta isolated from sham-operated animals and aorta distal to fistula (Fig. 3).



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  		Fig. 3. Measurement of tissue concentrations of cGMP in aorta homogenates after 30 min incubation in IBMX (10–3 M). *P < 0.05 analyzed by unpaired t-test (proximal vs. sham) and by paired t-test (proximal vs. distal); n = 6–7 different animals for each analysis group.

 
Vascular reactivity. Contractions to KCl (40 mM) and PE (10–9 to 10–5 M) were significantly reduced in segments isolated from the proximal aorta, as shown in Table 1 and Fig. 4, A and B. EC50 of PE was increased in the aorta proximal to the fistula compared with the distal and sham-operated aortas, but the differences were not statistically significant (Table 1). Treatment with L-NAME significantly potentiated the contraction responses in the proximal aorta, whereas changes in the distal aorta were less affected (Table 1). Endothelium-dependent relaxations to acetylcholine and DEA-NONOate were not significantly different among sham-operated aortas, as well as proximal and distal aortas (Fig. 4, C and D).


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  		Table 1. Contractions to KCl and phenylephrine in aortas of sham- and fistula-operated rats in absence or presence of L-NAME

 


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  		Fig. 4. Measurements of isometric force of aortic segments in sham-operated rats and rats with aortocaval fistula. A: contractions (force in g) to KCl (40 mM) and phenylephrine (PE, 10–5 M). *P < 0.05 compared with proximal (Prox) aorta analyzed by t-tests. B: contraction responses of aortas to cumulative addition of PE (10–9 to 10–5 M). *P < 0.05, distal (Dist) vs. proximal; +P < 0.05, sham vs. proximal analyzed by ANOVA. C: relaxation responses of aortas to cumulative addition of acetylcholine. D: relaxation responses of aortas to cumulative addition of diethylaminodiazen-1-ium-1,2-diolate (DEA-NONOate); n = 5 different animals for all experiments.

 
Expression and enzymatic activity of GTPCH I. When compared with the distal aortic segments with lower blood flow or with aortas obtained from sham-operated rats, both expression and enzymatic activity of GTPCH I were significantly increased in aortic segments proximal to the fistula (Fig. 5, A and B).



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  		Fig. 5. A: representative Western blot analysis demonstrating the expression of GTP cyclohydrolase I (GTPCH I) in aortic segments. Protein levels were quantified by scanning densitometry (Scion Image) and are shown in relative density; n = 4–5 different animals in each group. B: levels of GTPCH-I activity in rat aorta determined as function of neopterin production; n = 9–12 different animals in each group. *P < 0.05 analyzed by unpaired t-test (proximal vs. sham) and by paired t-test (proximal vs. distal).

 
BH4 levels. Total biopterin levels were increased in aortic segments exposed to increased blood flow compared with sham-operated and low-flow aortas (Fig. 6A). This increase was due to the elevated BH4 levels (Fig. 6B). Levels of oxidative products of BH4 (7,8-BH2/biopterin) and the BH4-to-BH2 biopterin ratios were similar among the three vessel segments (Fig. 6, C and D), indicating the absence of increased oxidation of BH4.



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  		Fig. 6. Bar graphs show levels of total biopterin (A), tetrahydrobiopterin (BH4; B), 7,8-BH2/biopterin (C), and BH4-to-BH2 biopterin ratio (BH4/BH2; D) in aortas of sham-operated rats and rats with aortocaval fistula 8 wk after operation. *P < 0.05, analyzed by unpaired t-test (proximal vs. sham) and by paired t-test (proximal vs. distal); n = 6–7 different animals for each analysis group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
With the use of the aortocaval model, the present study demonstrates that increased blood flow causes coordinated upregulation of eNOS and GTPCH I. Elevation of GTPCH-I protein expression is accompanied by increased amounts of BH4, the latter resulting from enhanced enzymatic activity of GTPCH I. We also demonstrate that blood flow does not affect oxidation of BH4, suggesting that increased net biosynthesis of BH4 per se is a major mechanism responsible for increased availability of BH4.

The aortocaval fistula model in rats was developed about 25 yr ago (18). This model predictably raises increased blood flow and shear stress in the proximal aorta, the latter shown to be increased some threefold within 10 min after the creation of the fistula (11). This model has been successfully utilized in studies (27) designed to determine the in vivo effects of blood flow on endothelial function, demonstrating, for example, the enhanced expression (both mRNA and protein) and activity of eNOS in the rat aorta. Such changes in this model occur without any elevation in mean arterial pressure (11, 35, 39). Consistent findings were observed in our studies utilizing this model at 8 wk: systolic blood pressures were unaltered in this model, whereas blood flow was increased almost twofold in the aorta proximal to the fistula compared with aortic blood flow distal to the fistula.

To the best of our knowledge, the present study is the first to demonstrate that increased blood flow stimulates arterial biosynthesis of BH4 and that such increased synthesis results from increased protein expression and enzymatic activity of GTPCH I. As such, our study provides novel insights regarding the molecular basis for increased NO synthesis in the setting of increased blood flow. Increased availability of BH4 (the reduced form of biopterin) has been shown to be vascular protective and can prevent endothelial dysfunction induced by hypercholesterolemia, diabetes, hypertension, and smoking (2, 5, 40). In this regard, it is notable that increased blood flow, and presumably increased laminar shear stress, not only induced the synthesis of BH4 but also preserved it in reduced state. Inspection of the nucleotide sequence of the GTPCH-I gene (20, 21) detected the shear-stress response elements (GAGACC and GGTCTC) (30) in the promoter region of the gene (Table 2), indirectly supporting our findings that GTPCH I may be transcriptionally regulated by shear stress, although this hypothesis requires further investigation. Our data provide the first evidence that biosynthesis of BH4 is governed by hemodynamic forces. This finding has important implications for an understanding of flow-dependent regulation of eNOS. Availability of BH4 is critical for enzymatic activity of eNOS, and the ability of blood flow to upregulate GTPCH I and eNOS in coordinated fashion is most likely designed to optimize the production of NO.


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  		Table 2. Shear-stress responsive elements found in promoter regions of GTP cyclohydrolase I gene

 
In the vessels with increased blood flow, the expression of eNOS and its phosphorylated form (eNOS-Ser1177) were both significantly enhanced. These findings support the concept that both Ca2+/calmodulin-dependent and -independent activation of eNOS is upregulated by prolonged exposure to high flow. High blood flow also increased expression of phosphorylated Akt (Akt-Ser473), whereas the level of total Akt remained unchanged. The upregulation of Akt-Ser473 is consistent with the reported activation of phosphatidylinositol 3-OH kinase/Akt pathway leading to the phosphorylation of eNOS (10, 13). Activation of Akt may also cause the dissociation of eNOS from caveolin-1 and therefore increase the activity of eNOS (26).

Increased level of cGMP, the second messenger of NO, in the aortic segments with increased blood flow, was consistent with a detected increase in the expression of eNOS and phosphorylated eNOS. With the use of Western blot analysis, the expression of heme oxygenase-1, which may also activate soluble guanylyl cyclase via the release of carbon monoxide, was not increased in the aortic segments with increased blood flow (data not shown). Furthermore, contractions to KCl and {alpha}1-adrenergic receptor activation were significantly reduced in the aorta proximal to the fistula. Inhibition of NOS potentiates the contractions to KCl and phenylephrine in the proximal aorta but not in the distal, low-flow aorta, suggesting that high local concentrations of NO and subsequent elevation of cGMP contribute to the reduced reactivity to vasoconstrictors in aorta exposed to high blood flow. These results are consistent with the findings reported by Rudic et al. (34), in which contractions of mouse carotid artery were reduced 7 days after exposure to high blood flow.

In the present study, endothelium-dependent and -independent relaxation responses of aorta exposed to high flow were not significantly different from those in sham-operated aorta or aorta exposed to low flow. Our data are in line with the results reported by Rudic et al. (34) but at variance with the results obtained on arteriovenous fistula in canine femoral vasculature (25). Augmented endothelium-dependent relaxations to acetylcholine were found in the femoral artery exposed to high blood flow for 6 wk (25). Several major differences, including experimental design (6 vs. 8 wk of high flow), different anatomical location of fistula, as well as species differences, may account for differential reactivity to acetylcholine. Furthermore, high local concentrations of NO associated with a significant elevation of cGMP may incur adaptive reduction in the vasodilator effect of NO released from endothelium in response to acetylcholine. This desensitization phenomenon is clearly demonstrated in transgenic mouse overexpressing eNOS wherein endothelium-dependent relaxations to acetylcholine are impaired despite significant elevation of arterial cGMP. Thus increased generation of NO in the vasculature does not necessarily predict enhanced endothelium-dependent and -independent relaxation responses. However, we wish to point out that the major conclusion of our study is in full agreement with prior in vivo studies (25, 27, 36); namely, high arterial blood flow stimulates production of endothelial NO.

With the use of the aortocaval fistula in rats, we demonstrated that a long-term increase in arterial laminar blood flow upregulates the expression of eNOS and its GTPCH I. Activation of Akt/eNOS-Ser1177 pathway is also an important mechanism responsible for stimulation of eNOS enzymatic activity. The results of the present study are the first to show that increased blood flow stimulates vascular biosynthesis of BH4 and to delineate the basis for such increased BH4 synthesis, namely, increased GTPCH-I protein expression and enzymatic activity of GTPCH I. Blood flow does not affect oxidation of BH4, thereby reinforcing the conclusion that increased biosynthesis of BH4 is a major mechanism responsible for increased availability of BH4. Elevation of intracellular BH4 concentration appears to be required for optimal production of NO in arterial endothelium and vasodilatation of arteries exposed to high blood flow.


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This work was supported in part by National Institutes of Health Grants HL-53524, HL-58080, HL-66958, and DK-070124 and by the Mayo Foundation.


   ACKNOWLEDGMENTS
 
The authors thank Janet Beckman for assistance with preparation of this paper and Drs. Sheng Cao and Vijay Shah (Gastrointestinal Research Unit, Dept. of Physiology, Mayo Clinic) for expertise and advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. S. Katusic, Dept. of Anesthesiology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: katusic.zvonimir{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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