AJP - Heart AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H2448-H2453, 2004. First published August 19, 2004; doi:10.1152/ajpheart.00248.2004
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/6/H2448    most recent
00248.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blackwell, K. A.
Right arrow Articles by Katusic, Z. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blackwell, K. A.
Right arrow Articles by Katusic, Z. S.

Mechanisms of aging-induced impairment of endothelium-dependent relaxation: role of tetrahydrobiopterin

Katherine A. Blackwell,1 Joseph P. Sorenson,1 Darcy M. Richardson,1 Leslie A. Smith,1 Osamu Suda,1 Karl Nath,2 and Zvonimir S. Katusic1

1Departments of Anesthesia Research and Molecular Pharmacology and Experimental Therapeutics and 2Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Submitted 19 March 2004 ; accepted in final form 29 June 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidative stress has been implicated as an important mechanism of vascular endothelial dysfunction induced by aging. Previous studies suggested that tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase, could be a molecular target for oxidation. We tested the hypothesis that oxidative stress, in particular oxidation of BH4, may contribute to attenuation of endothelium-dependent relaxation in aged mice. Vasomotor function of isolated carotid arteries was studied using a video dimension analyzer. Vascular levels of BH4 and its oxidation products were measured via HPLC. In aged mice (age, 95 ± 2 wk), endothelium-dependent relaxation to ACh (10–5 to 10–9 M) as well as endothelium-independent relaxation to the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10–5 to 10–9 M) were significantly reduced compared with relaxation detected in young mice (age, 23 ± 0.5 wk). Incubation of aged mouse carotid arteries with the cell-permeable SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride normalized relaxation to ACh and DEA-NONOate. Furthermore, production of superoxide anion in aorta and serum levels of amyloid P component, which is the murine analog of C-reactive protein, was increased in old mice. In aorta, neither the concentration of BH4 nor the ratio of reduced BH4 to the oxidation products were different between young and aged mice. Our results demonstrate that in mice, aging impairs relaxation mediated by NO most likely by increased formation of superoxide anion. Oxidation of BH4 does not appear to be an important mechanism underlying vasomotor dysfunction in aged mouse arteries.

endothelial dysfunction; nitric oxide; superoxide anion; reactive oxygen species; C-reactive protein

AGING IS A RISK FACTOR for vascular disease; however, the role of aging, either as a process or as the result of longer exposure to other risks, is not well defined (16, 17). In murine vascular tissue, the age-dependent changes in vasomotor function have not been characterized. Studies on rats have shown impairment of endothelium-dependent relaxation due to increased production of superoxide anions, but the source of the superoxide anions has also not been characterized (16, 30). Reactive oxygen species (ROS) have been implicated in endothelial dysfunction associated with aging, hypertension, hypercholesteremia, diabetes, and cigarette smoking (3). ROS can interfere with endothelium-dependent relaxation particularly by the scavenging of NO by superoxide anion (O2–·; Refs. 1, 3, 34). Additionally, the product of this reaction, peroxynitrite, is a potent but selective oxidant in vitro and in vivo and can cause cytotoxic damage, which could contribute to the pathology of vascular disease (1, 28).

One possible target for oxidation by peroxynitrite is tetrahydrobiopterin (BH4), which is an essential cofactor of all NO synthase (NOS) isoforms. During biosynthesis of NO, BH4 prevents the uncoupling of the electron transfer from NADPH to L-arginine and the subsequent production of O2–· (4, 32, 35, 36). Recently BH4 was shown to inhibit O2–· production by endothelial NOS (eNOS), whereas the oxidized analog 7,8-dihydrobioterin (7,8-BH2) potentiated O2–· production by eNOS (33). In addition, BH4 can autoxidize in a radical chain reaction; this could result in the reduction of available cofactors and the contribution of more oxidants to the milieu (14). Finally, peroxynitrite can oxidize BH4 (18, 21, 22). Therefore, reduced BH4 levels can reflect increased oxidative stress as well as contribute to generation of ROS. In this study, we tested the hypothesis that oxidation of BH4 is responsible for aging-induced endothelial dysfunction.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Male C57BL/6 mice were used for all experiments. Young animals were 20–27 wk, and aged animals were 84–114 wk of age. All mice were fed regular pelleted diet and housed in facilities with a 12:12-h light-dark cycle. Experimental protocols and housing facilities were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic.

The mice were killed with a 60 mg/kg ip injection of pentobarbital. Blood samples were obtained via puncture of the right ventricle. The blood was mixed with heparin and centrifuged at 4°C for 10 min at 2,000 rpm. The plasma was aspirated and stored at –80°C. Cholesterol levels were determined using a colorimetric-based assay on a Cobras Mira system. The aorta, carotid arteries, and kidney were removed and placed immediately in ice-cold modified Krebs-Ringer solution that contained (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4 (monobasic), 25 NaHCO3, 11.1 dextrose, and 0.028 calcium disodium versenate. Carotid arteries were dissected, and connective tissue was removed under a microscope (Carl Zeiss; Oberkochen, Germany). Aorta and kidneys were prepared similarly using a lighted magnifying glass. All of the assays were performed using tissue from the same animal.

Vasomotor reactivity. Carotid arteries were studied individually using a microcannula technique that has been previously described (8). Briefly, each artery was sutured to two microcannulas and placed in a vessel chamber (Living Systems Instrumentation; Burlington, VT) filled with aerated (94% O2-6% CO2) Krebs-Ringer solution at 37°C, which flowed from a 250-ml reservoir to the vessel chamber at a rate of 50 ml/min. A pressure of 50 mmHg was maintained in the artery through the microcannulas. The arteries were equilibrated 45 min before each experiment. The arteries were submaximally contracted with the thromboxane analog 9,11-dideoxy-11{alpha},9{alpha}-epoxy-methanoprostaglandin F2{alpha} (U-46619, 10–7 to 10–6 M), and endothelium-dependent relaxation was then obtained using ACh (10–9 to 10–5 M). After washout, equilibration, and submaximal contraction of the arteries with U-46619, endothelium-independent relations were determined using diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10–9 to 10–5 M). Relaxation values were determined as percents of relaxation to a high concentration of papaverine (3 x 10–4 M). In a separate protocol, arteries were preincubated with the SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride (MnTBAP, 10–5 M) 15 min before contraction (8).

Biopterin measurements. Fresh aortas were used for analysis. Aortas were homogenized in extraction buffer that contained (in mM) 50 Tris·HCl (pH 7.4), 1 EDTA, and 10 1,4-dithio-(DL)-threitol using a glass mortar and pestle (Kontes; Vineland, NJ; Ref. 7). The homogenate was centrifuged at 4°C and 10,000 rpm for 10 min, and the resulting supernatant was used in the assay. Amounts of reduced BH4 and total oxidized biopterins including 7,8-BH2 levels were measured after oxidation in acid and base conditions using reverse-phase HPLC (7, 9).

Detection of vascular O2–· production. O2–· production was measured by lucigenin-enhanced chemiluminescence as previously described (7). Briefly, aortas were opened lengthwise and equilibrated for 30 min at 37°C in modified Krebs-HEPES buffer (pH 7.4). Scintillation vials that contained 2 ml of Krebs-HEPES buffer with 5 µM lucigenin were placed into a scintillation counter (LS 5000; Beckman Instruments) that was switched to the out-of-coincidence mode. Background signals were recorded, and vascular segments were then added to each vial. The results were expressed as counts per minute per milligram of dry weight.

Measurement of GTP cyclohydrolase I activity. GTP cyclohydrolase I (GTPCH-I) activity was determined by standardized enzymatic reaction followed by oxidation as previously described with small modifications (31). Concisely, 100 µl of tissue supernatant homogenate, prepared as in the biopterin assay, was filtered using a Sephadex G25M column (Amersham; Piscataway, NJ) to remove endogenous neopterin, BH4, and phenylalanine. The resulting supernatant was then incubated at 37°C for 2 h in a mix that contained 7.14 mM Tris·HCl, 21.4 mM KCl, 0.179 mM EDTA (disodium, dihydrate), 96.9 mM glycerol, 0.714 µg of bovine serum albumin, 7.14 µM PMSF (in isopropanol, 933 mM), and 0.819 mM GTP. Once the reaction was terminated with the addition of 10 µl of 1 M HCl on ice, the resulting reaction product was oxidized with 10 µl of an iodine reagent (1% I2, 2% KI) for 1 h in the dark. This reaction was stopped with 10 µl of 20% ascorbate solution. The neopterin triphosphate was converted to neopterin for analysis by adding 20 µl of 1 M NaOH and 10 µl of 2,500 U/ml alkaline phosphatase in 37.5 mM MgCl2 and reacting at 37°C for 1 h. The product was analyzed as the biopterin using reverse-phase HPLC. All results were standardized to the protein concentration and assayed using DC Protein Assay (Bio-Rad; Hercules, CA) based on the Lowry method (19).

Measurement of serum amyloid P component. The serum amyloid P component (SAP) values were measured using an ELISA method (2). Briefly, SAP protein in plasma from mice was captured onto a 96-well microplate that was previously coated with a sheep anti-mouse polyclonal antibody (catalog no. 565194; Calbiochem; La Jolla, CA). For detection of captured SAP protein, a rabbit anti-mouse SAP antibody (catalog no. 565192; Calbiochem) was employed as a primary antibody followed by a horseradish peroxidase-conjugated, goat anti-rabbit IgG antibody (catalog no. DC03L, Calbiochem) as a secondary antibody and tetramethylbenzidine as the peroxidase substrate (catalog no. 555214; BD Pharmingen; San Diego, CA). Developed color was detected at a 450-nm wavelength using an absorbance plate reader and was quantified against a reference curve constructed using a mouse SAP standard (catalog no. 565192; Calbiochem).

Determination of senescence. Bisected kidneys were embedded in optimal cutting temperature compound (Sakura; Torrance, CA) and frozen at –80°C until sectioning. The tissue was cut in 5-µm sections, and senescence was determined using the senescence {beta}-Galactosidase Staining Kit (Cell Signaling Technology; Beverly, MA).

Drugs. ACh hydrochloride was obtained from Sigma Chemical (St. Louis, MO). DEA-NONOate and U-46619 were purchased from Cayman Chemical (Ann Arbor, MI), and MnTBAP was obtained from BIOMOL Laboratories (Plymouth Meeting, PA).

Statistical analysis. All data are reported as means ± SE. SigmaStat was used to perform all statistical analyses, which were preceded by a test for normality. For comparison between two groups, Mann-Whitney tests or t-tests were performed where needed. Dose-response curves were compared using two-way ANOVA for repeated measures. Comparisons of multiple groups were made using a one-way ANOVA. All ANOVAs were followed with Bonferroni's correction. P < 0.05 was considered significant. Because all arteries did not relax to at least 50%, EC25 values were used.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal characteristics. There were no differences observed between the animals with regard to plasma concentrations of glucose, cholesterol, high-density lipoprotein, or triglycerides (Table 1). Additionally, there were no changes in body weight or in basal diameter and wall thickness of the carotid artery. Senescence-associated {beta}-galactosidase staining of kidney was present in the aged animals (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of different parameters in young and aged mice

 
Vascular reactivity. There was no statistical difference between young and aged animals in the submaximal contractions of carotid arteries to U-46619 (Table 1). Maximum endothelium-dependent relaxations to ACh were reduced in aged mouse carotid arteries compared with young animals. Preincubation with MnTBAP significantly improved relaxation such that the maximum values for both the young and the aged mouse carotids were no longer different (Table 2 and Fig. 1). There was no difference between young mouse carotids treated with and without MnTBAP (Fig. 1A). Similar results were obtained for the endothelium-independent relaxations to DEA-NONOate (Fig. 2).


View this table:
[in this window]
[in a new window]
  		Table 2. Maximum relaxation of carotid arteries from young and aged C57BL/6 mice in presence and absence of MnTBAP

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. Effects of superoxide anion (O2–·) scavenging on endothelium-dependent relaxation in response to ACh in the carotid arteries of young and aged C57BL/6 mice. A: treatment with Mn(III)tetra(4-benzoic acid)porphyrin chloride (MnTBAP) on young mouse arteries did not affect relaxation in response to ACh. B: treatment with MnTBAP improved relaxation in aged mouse arteries. Results are means ± SE and are expressed as a percent of maximal relaxation to papaverine (3 x 10–4 M); P < 0.05; two-way repeated-measures ANOVA and Bonferroni's test.

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2. Effects of O2–· scavenging on endothelium-independent relaxation in response to the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) in the carotid arteries of young and aged C57BL/6 mice. A: in young mouse arteries, there was no difference in relaxation in the presence of MnTBAP compared with control. B: treatment of aged mouse arteries with MnTBAP improved relaxation. P < 0.05.

 
The EC25 values of aged mouse arteries in response to both ACh and DEA-NONOate were significantly increased (Table 3). There was no difference observed between the young and the aged mouse arteries with the addition of MnTBAP.


View this table:
[in this window]
[in a new window]
  		Table 3. Comparison of EC25 values for carotid arteries from young and aged C57BL/6 mice in the presence and absence of MnTBAP

 
Vascular O2–· production. Formation of O2–· was increased approximately fivefold in aged mouse aortas (young, 11,208 ± 4,373; aged, 60,239 ± 16,515 counts·min–1·mg of dry weight–1; n = 4; P < 0.05).

Biopterin levels and GTPCH-I activity. There were no significant differences between young and aged mouse aortas in BH4 concentrations (Fig. 3A) or in combined 7,8-BH2 and biopterin concentrations (Fig. 3B). The ratios of BH4 to 7,8-BH2 and biopterin were unchanged (Fig. 3C). There was also no change in GTPCH-I activity in the aorta of aged mice (Fig. 4).



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Concentrations of biopterin derivatives in young and aged C57BL/6 mouse aortas. There were no differences in levels of derivatives shown. A: tetrahydrobiopterin (BH4). B: 7,8-dihydrobioterin (7,8-BH2) and biopterin. C: ratio of BH4 to 7,8-BH2 and biopterin.

 


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 4. GTP cyclohydrolase I (GTPCH) activity, measured as production of neopterin under standard conditions, in young and aged C57BL/6 mouse aortas. There was no difference in the activity of this enzyme between young and aged mice.

 
Serum concentration of SAP. Levels of circulating SAP were significantly higher in aged compared with young mice (307 ± 167 vs. 16 ± 7 µg/ml, respectively; n = 6 mice; P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to examine the effects of aging on endothelium-dependent relaxation in mice. Aging significantly reduced relaxation to ACh mediated by release of NO from endothelial cells. Furthermore, relaxation in response to the NO donor DEA-NONOate was also reduced in aged mouse arteries, which indicates that impaired reactivity of smooth muscle cells to NO is an important component of endothelial dysfunction of aged mouse arteries. The SOD mimetic MnTBAP normalized vasomotor function of carotid arteries, which suggests that chemical antagonism between O2–· and NO is responsible for impairment of relaxation in response to ACh and DEA-NONOate. We also provide evidence that in a murine model of aging, alterations of BH4 metabolism do not contribute to impaired vasomotor function.

Vascular endothelium plays an essential role in cardiovascular homeostasis. Consistent with the results of the present experiments, numerous previous studies demonstrated that endothelium becomes dysfunctional in aged mouse arteries, mostly due to loss of NO biological activity and/or biosynthesis (10, 12, 13, 15, 20, 23, 30). Pharmacological analysis of impaired endothelium-dependent relaxation in response to ACh in aged mouse arteries demonstrated that reactivity of smooth muscle cells to NO is also reduced. This reduction is an important mechanism that underlies impairment of relaxation induced by the release of NO from endothelium. The ability of the SOD mimetic MnTBAP to normalize relaxation in response to ACh and DEA-NONOate in aged mouse arteries strongly suggests that increased production of O2–· and subsequent chemical inactivation of NO are critical mechanisms of vasomotor dysfunction in aged arteries. Indeed, measurements of O2–· production demonstrated increased formation of this free radical in aortas of old mice. The selectivity of MnTBAP was indicated by the fact that it did not affect relaxation in response to ACh or DEA-NONOate in young arteries. Consistent with our findings, van der Loo et al. (30) previously demonstrated that endothelial dysfunction in aortas of aged rats was due to increased production of O2–·. Interestingly, in aged rats, increased production of O2–· was detected in endothelial cells but not in smooth muscle cells. In contrast, in the present study, MnTBAP normalized endothelium-independent relaxation induced by NO. This finding suggests that in the carotid arteries of aged mice, O2–· is present not only in the endothelium but also in the media. This conclusion is consistent with the fact that in carotid arteries of Cu,Zn-SOD-knockout mice, which have increased O2–· throughout the vascular wall, both endothelium-dependent relaxation in response to ACh and endothelium-independent relaxation in response to NO are impaired (6).

Examination of various parameters, including plasma glucose level and lipid profile, demonstrated that there were no differences between young and aged animals, which rules out a possible contribution of age-induced metabolic changes to vasomotor dysfunction. Furthermore, measurements of basal diameter and wall thickness of the carotid artery did not differ between young and aged animals, which excludes major age-induced remodeling of the arterial wall as an explanation for the observed differences in vasomotor function. In aged animals, we detected senescence-associated {beta}-galactosidase staining in the kidneys, which indicates that the mice used in the present study were of an age at which cells were senescent. Interestingly, serum SAP levels were significantly increased in aged mice. SAP is the murine analog of C-reactive protein (33), which is an important proinflammatory marker in humans. This observation is consistent with the concept that aging is associated with increased production of proinflammatory mediators, including O2–·.

Several in vitro studies demonstrated that BH4 can be inactivated by peroxynitrite-induced oxidation (18, 21, 22). Simultaneous production of NO and O2–· in aged arteries provides favorable conditions for biosynthesis of peroxynitrite. Indeed, existing evidence suggests that increased production of peroxynitrite is an important mechanism of age-induced oxidative stress (30). If the cellular concentration of BH4 is reduced to a level suboptimal to that required for enzymatic activity of NOS, this may have inhibitory effect on NO production. Furthermore, uncoupling of NO synthesis from consumption of NADPH may lead to NOS-mediated reduction of oxygen and formation of O2–· (4, 32, 36, 37). This has been proposed as an important mechanism underlying endothelial dysfunction caused by hypercholesterolemia, hypertension, diabetes, and smoking (5, 11, 24–27, 29, 37). Based on these considerations, we hypothesized that BH4 oxidation may be an important component of endothelial dysfunction that is developed as a result of aging. In contrast with our expectations, we did not detect any major change in BH4 metabolism in aged mouse vascular tissue. Levels of both BH4 and its oxidation products were not different between young and aged mouse tissues. These findings strongly suggest that BH4 is not a molecular target for oxidation by an age-induced increase in oxidative stress. Our conclusion was reinforced by the fact that enzymatic activity of GTPCH-I, which is a rate-limiting enzyme in BH4 biosynthesis, was not affected by aging.

The results of the present study suggest that in mouse carotid arteries, aging-induced impairment of reactivity to NO is due to increased formation of O2–·. Aging apparently does not affect BH4 metabolism in vascular tissue. Therefore, oxidation of BH4 appears to be an unlikely mechanism responsible for vascular dysfunction of aged carotid arteries.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by National Heart, Lung, and Blood Institute Grants HL-53524, HL-58080, and HL-066958 and the Mayo Foundation.


   ACKNOWLEDGMENTS
 
The authors thank Janet Beckman for editorial assistance and Timothy Peterson for technical assistance. The authors also thank Anthony Croatt for technical assistance with measurements of serum amyloid P component.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. S. Katusic, 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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beckman JS and Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol Cell Physiol 271: C1424–C1437, 1996.[Abstract/Free Full Text]
  2. Belcher JD, Bryant CJ, Nguyen J, Bowlin PR, Kielbik MC, Bischof JC, Hebbel RP, and Vercellotti GM. Transgenic sickle mice have vascular inflammation. Blood 101: 3953–3959, 2003.[Abstract/Free Full Text]
  3. Cai H and Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840–844, 2000.[Abstract/Free Full Text]
  4. Cosentino F and Luscher TF. Tetrahydrobiopterin and endothelial nitric oxide synthase activity. Cardiovasc Res 43: 274–278, 1999.[Free Full Text]
  5. Cosentino F, Patton W, d'Uscio LV, Werner ER, Werner-Felmayer G, Moreau P, Malinski T, and Luscher TF. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest 101: 1530–1537, 1998.[ISI][Medline]
  6. Didion SP, Ryan MJ, Didion LA, Fegan PE, Sigmund CD, and Faraci FM. Increased superoxide and vascular dysfunction in CuZnSOD-deficient mice. Circ Res 91: 938–944, 2002.[Abstract/Free Full Text]
  7. D'Uscio LV, Milstien S, Richardson D, Smith L, and Katusic ZS. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res 92: 88–95, 2003.[Abstract/Free Full Text]
  8. D'Uscio LV, Smith LA, and Katusic ZS. Hypercholesterolemia impairs endothelium-dependent relaxations in common carotid arteries of apolipoprotein E-deficient mice. Stroke 32: 2658–2664, 2001.[Abstract/Free Full Text]
  9. Fukushima T and Nixon JC. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 102: 176–188, 1980.[CrossRef][ISI][Medline]
  10. Hatake K, Kakishita E, Wakabayashi I, Sakiyama N, and Hishida S. Effect of aging on endothelium-dependent vascular relaxation of isolated human basilar artery to thrombin and bradykinin. Stroke 21: 1039–1043, 1990.[Abstract/Free Full Text]
  11. Higman D, Strachan A, Buttery L, Hicks R, Springall D, and Greenhalgh R. Smoking impairs the activity of nitric oxide synthase in saphenous vein. Arterioscler Thromb Vasc Biol 16: 546–552, 1996.[Abstract/Free Full Text]
  12. Hongo K, Nakagomi T, Kassell NF, Sasaki T, Lehman M, Vollmer DG, Tsukahara T, Ogawa H, and Torner J. Effects of aging and hypertension on endothelium-dependent vascular relaxation in rat carotid artery. Stroke 19: 892–897, 1988.[Abstract/Free Full Text]
  13. Imaoka Y, Osanai T, Kamada T, Mio Y, Satoh K, and Okumura K. Nitric oxide-dependent vasodilator mechanism is not impaired by hypertension but is diminished with aging in the rat aorta. J Cardiovasc Pharmacol 33: 756–761, 1999.[CrossRef][ISI][Medline]
  14. Kirsch M, Korth HG, Stenert V, Sustmann R, and De Groot H. The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen. J Biol Chem 278: 24481–2490, 2003.[Abstract/Free Full Text]
  15. Kitagawa S, Yamaguchi Y, Sameshima E, and Kunitomo M. Differences in endothelium-dependent relaxation in various arteries from Watanabe heritable hyperlipidaemic rabbits with increasing age. Clin Exp Pharmacol Physiol 21: 963–970, 1994.[ISI][Medline]
  16. Lakatta EG. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises. Part III: cellular and molecular clues to heart and arterial aging. Circulation 107: 490–497, 2003.[Free Full Text]
  17. Lakatta EG and Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises. Part I: aging arteries: a "set up" for vascular disease. Circulation 107: 139–146, 2003.[Free Full Text]
  18. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, and Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 103: 1282–1288, 2001.[Abstract/Free Full Text]
  19. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  20. Matz RL, de Sotomayor MA, Schott C, Stoclet JC, and Andriantsitohaina R. Vascular bed heterogeneity in age-related endothelial dysfunction with respect to NO and eicosanoids. Br J Pharmacol 131: 303–311, 2000.[CrossRef][ISI][Medline]
  21. Milstien S and Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun 263: 681–684, 1999.[CrossRef][ISI][Medline]
  22. Patel KB, Stratford MR, Wardman P, and Everett SA. Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate. Free Radic Biol Med 32: 203–211, 2002.[CrossRef][ISI][Medline]
  23. Paterno R, Faraci FM, and Heistad DD. Age-related changes in release of endothelium-derived relaxing factor from the carotid artery. Stroke 25: 2457–2460, 1994.[Abstract]
  24. Pieper GM. Acute amelioration of diabetic endothelial dysfunction with a derivative of the nitric oxide synthase cofactor, tetrahydrobiopterin. J Cardiovasc Pharmacol 29: 8–15, 1997.[CrossRef][ISI][Medline]
  25. Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, and Kikkawa R. Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2– imbalance in insulin-resistant rat aorta. Diabetes 48: 2437–2445, 1999.[Abstract]
  26. Shinozaki K, Nishio Y, Okamura T, Yoshida Y, Maegawa H, Kojima H, Masada M, Toda N, Kikkawa R, and Kashiwagi A. Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ Res 87: 566–573, 2000.[Abstract/Free Full Text]
  27. Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Luscher T, and Rabelink T. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest 99: 41–46, 1997.[ISI][Medline]
  28. Szabo C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett 140–141: 105–112, 2003.
  29. Ueda S, Matsuoka H, Miyazaki H, Usui M, Okuda S, and Imaizumi T. Tetrahydrobiopterin restores endothelial function in long-term smokers. J Am Coll Cardiol 35: 71–75, 2000.[Abstract/Free Full Text]
  30. Van der Loo B, Labugger R, Skepper JN, Bachschmid M, Kilo J, Powell JM, Palacios-Callender M, Erusalimsky JD, Quaschning T, Malinski T, Gygi D, Ullrich V, and Luscher TF. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med 192: 1731–1744, 2000.[Abstract/Free Full Text]
  31. Vann LR, Twitty S, Spiegel S, and Milstien S. 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. J Biol Chem 275: 13275–13281, 2000.[Abstract/Free Full Text]
  32. Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, and Pritchard KA Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci USA 95: 9220–9225, 1998.[Abstract/Free Full Text]
  33. Vasquez-Vivar J, Martasek P, Whitsett J, Joseph J, and Kalyanaraman B. The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogs controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study. Biochem J 362: 733–739, 2002.[CrossRef][ISI][Medline]
  34. Wei EP, Kontos HA, Christman CW, DeWitt DS, and Povlishock JT. Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilation after acute hypertension. Circ Res 57: 781–787, 1985.[Abstract/Free Full Text]
  35. Wever RM, van Dam T, van Rijn HJ, de Groot F, and Rabelink TJ. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun 237: 340–344, 1997.[CrossRef][ISI][Medline]
  36. Xia Y, Tsai AL, Berka V, and Zweier JL. Superoxide generation from endothelial nitric oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem 273: 25804–25808, 1998.[Abstract/Free Full Text]
  37. Yu PK, Yu DY, Cringle SJ, and Su EN. Tetrahydrobiopterin reverses the impairment of acetylcholine-induced vasodilatation in diabetic ocular microvasculature. J Ocul Pharmacol Ther 17: 123–129, 2001.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
G. L. Pierce and T. J. LaRocca
Reduced vascular tetrahydrobiopterin (BH4) and endothelial function with ageing: is it time for a chronic BH4 supplementation trial in middle-aged and older adults?
J. Physiol., June 1, 2008; 586(11): 2673 - 2674.
[Full Text] [PDF]

Home page
 HeartHome page
F Cosentino, D Hurlimann, C Delli Gatti, R Chenevard, N Blau, N J Alp, K M Channon, M Eto, P Lerch, F Enseleit, et al.
Chronic treatment with tetrahydrobiopterin reverses endothelial dysfunction and oxidative stress in hypercholesterolaemia
Heart, April 1, 2008; 94(4): 487 - 492.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. D. Delp, B. J. Behnke, S. A. Spier, G. Wu, and J. M. Muller-Delp
Ageing diminishes endothelium-dependent vasodilatation and tetrahydrobiopterin content in rat skeletal muscle arterioles
J. Physiol., February 15, 2008; 586(4): 1161 - 1168.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
Z. S. Katusic
Mechanisms of Endothelial Dysfunction Induced by Aging: Role of Arginase I
Circ. Res., September 28, 2007; 101(7): 640 - 641.
[Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
K. A. Brown, S. P. Didion, J. J. Andresen, and F. M. Faraci
Effect of Aging, MnSOD Deficiency, and Genetic Background on Endothelial Function: Evidence for MnSOD Haploinsufficiency
Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1941 - 1946.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
T. Matsumoto, D. J. Baker, L. V. d'Uscio, G. Mozammel, Z. S. Katusic, and J. M. van Deursen
Aging-Associated Vascular Phenotype in Mutant Mice With Low Levels of BubR1
Stroke, March 1, 2007; 38(3): 1050 - 1056.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
F. de Nigris, S. Williams-Ignarro, V. Sica, L. O. Lerman, F. P. D'Armiento, R. E. Byrns, A. Casamassimi, D. Carpentiero, C. Schiano, D. Sumi, et al.
Effects of a Pomegranate Fruit Extract rich in punicalagin on oxidation-sensitive genes and eNOS activity at sites of perturbed shear stress and atherogenesis
Cardiovasc Res, January 15, 2007; 73(2): 414 - 423.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
S. P. Didion, D. A. Kinzenbaw, L. I. Schrader, and F. M. Faraci
Heterozygous CuZn Superoxide Dismutase Deficiency Produces a Vascular Phenotype With Aging
Hypertension, December 1, 2006; 48(6): 1072 - 1079.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
G. Perez-Rivero, M. P. Ruiz-Torres, J. V. Rivas-Elena, M. Jerkic, M. L. Diez-Marques, J. M. Lopez-Novoa, M. A. Blasco, and D. Rodriguez-Puyol
Mice Deficient in Telomerase Activity Develop Hypertension Because of an Excess of Endothelin Production
Circulation, July 25, 2006; 114(4): 309 - 317.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
I. Eskurza, L. A Myerburgh, Z. D Kahn, and D. R Seals
Tetrahydrobiopterin augments endothelium-dependent dilatation in sedentary but not in habitually exercising older adults
J. Physiol., November 1, 2005; 568(3): 1057 - 1065.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/6/H2448    most recent
00248.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blackwell, K. A.
Right arrow Articles by Katusic, Z. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blackwell, K. A.
Right arrow Articles by Katusic, Z. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.