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


     

Am J Physiol Heart Circ Physiol 286: H570-H574, 2004. First published October 9, 2003; doi:10.1152/ajpheart.00669.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/2/H570    most recent
00669.2003v1
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 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 Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hynes, S. O.
Right arrow Articles by Katusic, Z. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hynes, S. O.
Right arrow Articles by Katusic, Z. S.

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

Sean O. Hynes,1 Leslie A. Smith,1 Darcy M. Richardson,1 Imre Kovesdi,2 Timothy O'Brien,3 and Zvonimir S. Katusic1

1Departments of Anesthesiology, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905; 2GenVec, Gaithersburg, Maryland 20878; and 3Department of Medicine, National University of Ireland, Galway, Ireland

Submitted 25 July 2003 ; accepted in final form 5 October 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 109 plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or {beta}-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10–5 to 10–9 M), endothelium-dependent relaxation to acetylcholine (10–5 to 10–9 M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.

nitric oxide synthase; tetrahydrobiopterin; adenovirus

NITRIC OXIDE (NO) is a potent vasodilator and an important regulator of vascular homeostasis (4, 8, 16). Endothelial cells produce NO by the constitutively expressed enzyme endothelial NO synthase (eNOS) (5, 6). Tetrahydrobiopterin (BH4) is an essential cofactor required for optimal enzymatic activity of NOS and in particular eNOS. Although the exact mechanisms underlying the effects of BH4 on NO synthesis are not completely understood, existing evidence suggests that BH4 can function as both an allosteric and redox cofactor (2, 7, 15, 19, 23). Numerous studies have suggested that reduced availability of BH4 could contribute to pathogenesis of endothelial dysfunction, and these are reviewed elsewhere (9).

BH4 is produced from GTP in a series of enzymatic steps (7). The rate-limiting enzyme in this process is GTP cyclohydrolase I (GTPCH I). It appears that in cultured endothelial cells, intracellular concentrations of BH4 are subsaturating for eNOS enzymatic activity (20, 21). On the basis of this premise, it would appear that eNOS activity could be augmented through increased expression of GTPCH I. The direct delivery of sepiaterin, a precursor of biopterin, has proven beneficial in cases of endothelial dysfunction potentially acting through this mechanism (22). A recent study by Cai and colleagues (2) demonstrated that gene transfer of GTPCH I could be used to enhance BH4 production and increase eNOS activity in cultured endothelium. In the present study, we hypothesized that in vivo overexpression of GTPCH I in endothelial cells could increase bioavailability of BH4, stimulate biosynthesis of NO, and thereby enhance endothelium-dependent relaxations mediated by NO.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. The following pharmacological agents were used: phenylephrine, acetylcholine, indomethacin, and papaverine hydrochloride (Sigma; St. Louis, MO). Reagents were dissolved in distilled water, and volumes of <0.15 ml were added to the organ chambers. Concentrations of all drugs are expressed as the final moles per liter concentration in distilled water. A modified Krebs solution was used in the present study with the following composition (in mM): 1.2 KH2PO4, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 118.3 NaCl, and 11.1 dextrose. Protein levels throughout the study were determined with the use of a DC protein assay kit (Bio-Rad; Hercules, CA).

Viral vectors for GTPCH I and reporter gene. A recombinant adenovirus (Ad)-encoding human GTPCH I gene (AdGTPCH) driven by a cytomegalovirus promoter was generated as previously described (10). A recombinant adenoviral vector encoding the Escherichia coli-{beta} galactosidase gene (AdLacZ) driven by the cytomegalovirus promoter was used as a control. The virus was purified with double cesium chloride gradient ultracentrifugation and was dialyzed against 10 mM Tris, 1.0 mM MgCl2, 1.0 mM HEPES, and 10% glycerol for 4 h at 4°C. Viral titer was determined by plaque assay. Viral stocks were stored at –80°C.

Gene transfer into experimental animals. Two- to three-month-old male New Zealand White rabbits (2–3 kg) were obtained from Harlan (Indianapolis, IN). Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and are described elsewhere (11, 24).

Rabbits were anesthetized with ketamine (35 mg/kg), xylazine (5 mg/kg), and acepromazine (2.3 mg/kg) before surgery. A midline neck incision was made and the common carotid arteries were exposed. The arterial branches were cauterized, and heparin (100 U/kg) was administered intravenously. The artery was clamped, and after an arteriotomy was performed, an intraarterial catheter (24 GA) was introduced. An adenoviral load of 109 plaque-forming units (pfu) in 100 µl of sterile PBS was then injected, the arteriotomy was sutured, and the virus was allowed to incubate in situ for 20 min. The clamps were then removed and the wound sutured. The rabbits were euthanized 72 h later, and tissues were harvested.

Analysis of vasomotor function. Isolated carotid arterial rings (3–5 mm) were connected to a force transducer for recording of isometric force and placed in organ baths filled with 25 ml of Krebs solution at 37°C (94% O2-6% CO2; pH 7.4). Isometric force was recorded continuously, and the rings were allowed to stabilize at 0.2 to 0.4 g for 1 h. Each ring was then gradually stretched to 3.0 g. All experiments were carried out in the presence of 10–5 M indomethacin to eliminate the influence of endogenous cyclooxygenase. The rings were incubated with indomethacin for 30 min before exposure to other reagents. Contractions to phenylephrine (10–5 to 10–9 M) were examined before exposure to acetylcholine. The relaxation responses to acetylcholine (10–5 to 10–9 M) were studied during contractions to a submaximal concentration of phenylephrine. Papaverine (3 x 10–4 M) was used to induce complete relaxation of the vessels.

Histochemical analysis. Expression of the {beta}-galactosidase reporter gene was determined with the use of BluoGal and histochemical staining. After BluoGal staining, the arterial rings were frozen in optimum cutting temperature compound (Sakura; Torrance, CA). Five micrometer cross sections were cut onto slides. The sections were then counterstained with neutral red (11).

HPLC analyses. BH4 levels in carotid arteries were determined after differential oxidation in acid and base conditions by reversephase HPLC (3). GTPCH I activity was assessed as a function of neopterin production under standard conditions with GTP as a substrate. Results were normalized against tissue protein levels. cGMP. Radioimmunoassay kits (Amersham; Buckinghamshire, UK) were used to perform the measurements. Rings were initially incubated in minimum essential medium supplemented with 0.1% bovine serum, 24 U penicillin, and 24 µg streptomycin in a 5% CO2 incubator for 30 min and were then incubated a further 30 min in minumum essential medium with 3-isobutyl-1-methylxanthine (10–4 M) to inhibit degradation of cyclic nucleotides by phosphodiesterases. The rings were then removed from the medium and immediately snap frozen with liquid N2. After homogenization, cGMP levels were assessed with the use of a cGMP radioimmunoassay kit and normalized against tissue protein levels.

Statistical analyses. Results are represented as means ± SE. Statistical analysis was performed by two-way repeated-measures ANOVA with a Bonferroni t-test to detect differences in multiple comparisons and by an unpaired t-test for comparisons between two groups or Mann-Whitney test where appropriate to the data. The tests applied are as stated in the figure legends. A P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Intraluminal delivery of recombinant proteins. Expression of the {beta}-galactosidase reporter gene in AdLacZ-transduced arteries was confirmed with the use of histochemical analysis. Vessels from rabbits treated with 109 pfu of AdLacZ and harvested 3 days later showed that {beta}-galactosidase activity was localized in the endothelial cells (Fig. 1A). In contrast, no staining was noted in the AdGTPCH-treated arteries (Fig. 1B).



View larger version (61K):
[in this window]
[in a new window]
  		Fig. 1. Histological localization of {beta}-galactosidase reporter gene expression in rabbit carotid arteries after exposure to adenovirus (Ad) vector encoding the Escherichia coli-{beta}-galactosidase gene (AdLacZ) (A) and in control (B) arteries exposed to AdGTP cyclohydrolase (AdGTPCH). Blue staining demonstrates endothelium-specific gene transfer of {beta}-galactosidase. Magnification, x40.

 

Effect of recombinant proteins on GTPCH I activity and BH4. In AdGTPCH-transduced carotid arteries, basal levels of GTPCH I activity were significantly increased by more than twofold compared with both AdLacZ and untreated controls (P < 0.05; Fig. 2). In addition, levels of total biopterins (32.22 ± 7.6 vs. 11.79 ± 1.9 pmol/mg protein; P < 0.05) and BH4 (P < 0.05; Fig. 3A) were significantly increased by almost threefold in AdGTPCH-transduced arteries compared with AdLacZ-transduced arteries. Despite no significant difference noted in oxidized biopterin levels between the two treatment groups and untreated controls, a trend toward a significant increase was noted for AdGTPCH-transduced arteries (P < 0.1) (Fig. 3B). BH2, an oxidized form of BH4 can be an important factor in determining eNOS activity, because it may compete for the same binding site as BH4 in the eNOS molecule but will result in an inactive enzyme. Moreover, BH4, determined as a percentage of total biopterin (Fig. 3C), did not change significantly after AdGTPCH delivery. Collectively, these results demonstrate the successful expression and function of recombinant GTPCH I to increase BH4 production in vivo.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. GTPCH I activity levels in rabbit carotid arteries determined as a function of neopterin production are shown after transduction with 109 plaqueforming units (pfu) AdGTPCH. Levels of GTPCH I increased over twofold in AdGTPCH-treated arteries compared with controls. Results are shown as means ± SE (n = 7 for untreated controls, n = 8 for both AdLacZ or AdGTPCH-treated arteries). *P < 0.05.

 


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 3. The effect of GTPCH I gene delivery on biopterin levels in the rabbit carotid artery. Bar graphs show tetrahydrobiopterin (BH4) levels (A), oxidized biopterin levels (B), and BH4 (C) as a percentage of total biopterin in rabbit carotid arteries after transduction with 109 pfu AdGTPCH. Levels of BH4 increased over twofold compared with controls. Results are shown as means ± SE (n = 9 for untreated controls, n = 16 for AdLacZ and AdGTPCH). *P < 0.05.

 

Effect of recombinant proteins on vasomotor function. Maximal contractions and sensitivity to phenylephrine were similar in arteries after delivery of AdGTPCH (n = 9 rings, 4 animals) or AdLacZ (n = 7 rings, 4 animals) (5.2 ± 0.50 vs. 6.6 ± 0.63 g; P > 0.05) (Fig. 4A). Furthermore, during submaximal contraction to phenylephrine, endothelium-dependent relaxations to acetylcholine (10–9 to 10–5 M) were not significantly affected in AdGTPCH-transduced carotid arteries compared with either AdLacZ-transduced arteries or untreated controls (Fig. 4B). The results from these functional assays suggest that eNOS activity was not affected by transduction of AdGTPCH because the relaxations induced using acetylcholine preceded by indomethacin are mediated by NO.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4. A: concentration response curves to phenylephrine treatment. Data are shown as means ± SE and are expressed as percentage of maximal contraction induced by phenylephrine (10–5 M; 100% = 5.69 ± 0.33, 6.64 ± 0.63, and 5.2 ± 0.50 g for untreated, AdLacZ, and AdGTPCH, respectively). B: concentration response curves for acetylcholine. Values are shown as means ± SE and are expressed as the percentage of the maximal relaxation for papaverine (3 x 10–4 M; 100% = 3.75 ± 0.20, 3.60 ± 0.34, and 3.33 ± 0.29 g for untreated, AdLacZ, and AdGTPCH, respectively). P > 0.05 for repeatedmeasures two-way ANOVA for both sets of curves.

 

Effects of recombinant proteins on intracellular cGMP levels. In AdGTPCH-transduced arteries, mean basal levels of cGMP did not significantly differ from arteries transduced with AdLacZ (n = 11; Fig. 5), further indicating that eNOS activity did not increase in AdGTPCH-transduced arteries.



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. Bar graph showing cGMP levels in rabbit carotid arteries after exposure to 109 pfu. No significant differences were noted between either AdGTPCH- or AdLacZ-treated arteries with the use of a t-test. Results are shown as means ± SE (n = 11; P > 0.05).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
This is the first study to characterize the in vivo expression and function of recombinant GTPCH I in vascular endothelial cells. We report several novel findings. Intraluminal delivery of adenovirus-encoding GTPCH I into carotid artery resulted in increased enzymatic activity of GTPCH I, and a significantly higher intracellular level of BH4. In contrast, expression of the recombinant reporter gene {beta}-galactosidase did not affect enzymatic activity of GTPCH I or BH4 level, ruling out the possibility that a replication-incompetent adenovirus may affect vascular expression and function of GTPCH I. Interestingly, the ratio between BH4 and oxidized biopterin remained the same in control, AdLacZ, and AdGTPCH-transduced arteries. This finding indicates that adenoviral-mediated gene delivery into endothelial cells does not affect BH4 metabolism or oxidation. Our study also provides evidence that in the carotid artery, endothelium-dependent relaxations mediated by NO are not affected by increased availability of BH4. Although indirect, this evidence refutes our hypothesis that subsaturating levels of BH4 for eNOS enzymatic activity are present in endothelium under physiological conditions.

In several previous studies (11, 24, 26), we demonstrated that intraluminal delivery of an adenovirus in the rabbit carotid artery resulted in expression of recombinant proteins in endothelial cells. Consistent with these findings, we detected expression of {beta}-galactosidase in the endothelium (Fig. 1). We used a viral titer of 109 pfu per artery for both the adenovirus encoding the reporter gene and GTPCH I. This titer does not have a cytotoxic effect on endothelial cells, as illustrated by the fact that endothelium-dependent relaxations to acetylcholine were not affected in arteries transduced with {beta}-galactosidase. Furthermore, contractions to phenylephrine were also not affected in arteries treated with 109 pfu of adenovirus, reinforcing our conclusion that in rabbit carotid artery, this viral titer is not cytotoxic. BH4 is one of the most potent naturally occurring reducing substances and may be oxidized with peroxynitrite (12, 14, 17). In vivo oxidation of BH4 has been proposed as a possible mechanism underlying the uncoupling of eNOS and endothelial dysfunction in experimental models of hypertension and hypercholesterolemia (13, 14). It is interesting and important to note that we did not detect any increase in BH4 oxidation in arteries exposed to AdLacZ. The lack of BH4 oxidation is further evidence to suggest that adenovirus (109 pfu) did not have a cytotoxic effect on endothelial cells. However, we provide evidence that 109 pfu of adenovirus-encoding GTPCH I is sufficient to increase local production of BH4 in the vascular wall. We observed an approximately two- to threefold increase in both GTPCH I enzymatic and biosynthesis of BH4 in arteries transduced with AdGTPCH.

In contrast to our findings, previous studies using cultured endothelial cells demonstrated that overexpression of GTPCH I resulted in an increased intracellular BH4 level, along with a subsequent increase in NO production (2). The stabilization of functional eNOS dimers by BH4 was demonstrated and suggested as a potential mechanism of increased NO biosynthesis. The discrepancy between in vitro and in vivo results is most likely because expression and enzymatic activity of GTPCH I are both very low (in some studies undetectable) in cultured endothelial cells. Indeed, a study by Rosenkranz-Weiss (21) demonstrated that BH4 levels are significantly higher in freshly isolated endothelial cells compared with cultured endothelium. The reason for reduction of GTPCH I expression and activity observed during culturing of endothelial cells is unclear. Nevertheless, this observation may explain the beneficial effect of BH4 supplementation in cultured endothelium (1, 2, 21).

In the present study, despite a successful expression of recombinant GTPCH I and subsequent increase in BH4 bioavailability, we did not observe any augmentation of endothelium-dependent relaxations mediated by the release of NO. Consistent with this observation, levels of cGMP, a secondary messenger for NO, did not change in arteries expressing recombinant GTPCH I. We did not directly measure NO and enzymatic activity of NOS; however, endothelium-dependent relaxations to acetylcholine and cGMP levels have traditionally served as very good indexes of NO production (25, 27). Our findings suggest that in the rabbit carotid artery, eNOS is saturated with BH4 under physiological conditions.

In conclusion, the results of this study demonstrate that expression of recombinant GTPCH I in rabbit carotid artery results in increased enzymatic activity of GTPCH I and biosynthesis of BH4. Our findings suggest that in the vascular endothelium eNOS is saturated with BH4 under physiological conditions. In a similar manner, it has been demonstrated previously that L-arginine, the substrate for eNOS activity, may also prove saturating for recombinant eNOS activity (18). The relatively high basal levels of BH4, lack of viral toxicity, and an in vivo setting suggest that manipulation of vascular endothelium with adenovirus-mediated gene transfer may provide a useful model in studies designed to characterize mechanisms responsible for metabolism of BH4. Our results provide the basis for future studies designed to characterize the effects of GTPCH I gene transfer on endothelial dysfunction.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
I. Kovesdi owns equity in GenVec, Inc., Gaithersburg, MD.


   ACKNOWLEDGMENTS
 
The authors are grateful for the editorial assistance of Janet Beckman.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-53524, HL-58080, and HL-66958, the American Heart Association, Bugher Foundation award for the investigation of stroke, and the Mayo Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. S. Katusic, Anesthesia Research, 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
 DISCLOSURES
 REFERENCES
 

  1. Baker TA, Milstien S, and Katusic ZS. Effect of vitamin C on the availability of tetrahydrobiopterin in human endothelial cells. J Cardiovasc Pharmacol 37: 333–338, 2001.[CrossRef][Web of Science][Medline]
  2. Cai S, Alp NJ, McDonald D, Smith I, Kay J, Canevari L, Heales S, and Channon KM. GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerization. Cardiovasc Res 55: 838–849, 2002.[Abstract/Free Full Text]
  3. 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]
  4. Furchgott RF and Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376, 1980.[CrossRef][Medline]
  5. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, and Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 392: 821–824, 1998.[CrossRef][Medline]
  6. Gesierich A, Niroomand F, and Tiefenbacher CP. Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism. Basic Res Cardiol 98: 69–75, 2003.[CrossRef][Web of Science][Medline]
  7. Gross SS, Jones CL, Hattori Y, and Raman CS. Tetrahydrobiopterin: an essential cofactor of nitric oxide synthase with an elusive role. In: Nitric Oxide, edited by Ignarro LJ. San Diego, CA: Academic, 2000.
  8. Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 65: 1–21, 1989.[Free Full Text]
  9. Katusic ZS. Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am J Physiol Heart Circ Physiol 281: H981–H986, 2001.[Abstract/Free Full Text]
  10. Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Crotty TB, Barber DA, Katusic ZS, and O'Brien T. Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity. Circulation 96: 2254–2261, 1997.[Abstract/Free Full Text]
  11. Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Tsutsui M, Katusic ZS, and O'Brien T. Enhanced endothelium-dependent relaxations after gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries. Hypertension 30: 314–320, 1997.[Abstract/Free Full Text]
  12. Kuzkaya N, Weissmann N, Harrison DG, and Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid and thiols: implications for uncoupling endothelial nitric oxide synthase. J Biol Chem 278: 22546–22554, 2003.[Abstract/Free Full Text]
  13. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, and Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111: 1201–1209, 2003.[CrossRef][Web of Science][Medline]
  14. 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]
  15. Mayer B and Hemmens B. Biosynthesis and action of nitric oxide in mammalian cells. Trends Biochem Sci 22: 477–481, 1997.[CrossRef][Web of Science][Medline]
  16. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, and Kadowitz PJ. Evidence for the inhibitory role of guanosine 3',5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 57: 946–955, 1981.[Free Full Text]
  17. Milstien S and Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun 263: 681–684, 1999.[CrossRef][Web of Science][Medline]
  18. Mohacsi T, Mozes G, Sato J, Gloviczki P, Katusic Z, and O'Brien T. L-Arginine availability is not limiting for nitric oxide generation from recombinant endothelial nitric oxide synthase. J Vasc Res 36: 437–444, 532–534, 1999.[CrossRef]
  19. Panda K, Rosenfeld RJ, Ghosh S, Meade AL, Getzoff ED, and Stuehr DJ. Distinct dimer interaction and regulation in nitric-oxide synthase types I, II, and III. J Biol Chem 277: 31020–31030, 2002.[Abstract/Free Full Text]
  20. Pollock JS, Forstermann U, Mitchell JA, Warner TD, Schmidt HH, Nakane M, and Murad F. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci USA 88: 10480–10484, 1991.[Abstract/Free Full Text]
  21. Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, and Pober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. J Clin Invest 93: 2236–2243, 1994.[Web of Science][Medline]
  22. Tarpey MM. Sepiapterin treatment in atherosclerosis. Arterioscler Thromb Vasc Biol 22: 1519–1521, 2002.[Free Full Text]
  23. Wei CC, Wang ZQ, Meade AL, McDonald JF, and Stuehr DJ. Why do nitric oxide synthases use tetrahydrobiopterin? J Inorg Biochem 91: 618–624, 2002.[CrossRef][Web of Science][Medline]
  24. Zanetti M, D'Uscio LV, Kovesdi I, Katusic ZS, and O'Brien T. In vivo gene transfer of inducible nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits. Stroke 34: 1293–1298, 2003.[Abstract/Free Full Text]
  25. Zanetti M, Katusic ZS, and O'Brien T. Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells. J Vasc Res 37: 449–456, 2000.[CrossRef][Web of Science][Medline]
  26. Zanetti M, Sato J, Jost CJ, Gloviczki P, Katusic ZS, and O'Brien T. Gene transfer of manganese superoxide dismutase reverses vascular dysfunction in the absence but not in the presence of atherosclerotic plaque. Hum Gene Ther 12: 1407–1416, 2001.[CrossRef][Web of Science][Medline]
  27. Zanetti M, Sato J, Katusic ZS, and O'Brien T. Gene transfer of endothelial nitric oxide synthase alters endothelium-dependent relaxations in aortas from diabetic rabbits. Diabetologia 43: 340–347, 2000.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 HypertensionHome page
T. E. Peterson, L. V. d'Uscio, S. Cao, X.-L. Wang, and Z. S. Katusic
Guanosine Triphosphate Cyclohydrolase I Expression and Enzymatic Activity Are Present in Caveolae of Endothelial Cells
Hypertension, February 1, 2009; 53(2): 189 - 195.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C.-F. Lam, T. E. Peterson, D. M. Richardson, A. J. Croatt, L. V. d'Uscio, K. A. Nath, and Z. S. Katusic
Increased blood flow causes coordinated upregulation of arterial eNOS and biosynthesis of tetrahydrobiopterin
Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H786 - H793.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
O. Suda, L. A. Smith, L. V. d'Uscio, T. E. Peterson, and Z. S. Katusic
In Vivo Expression of Recombinant Vascular Endothelial Growth Factor in Rabbit Carotid Artery Increases Production of Superoxide Anion
Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 506 - 511.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/2/H570    most recent
00669.2003v1
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 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 Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hynes, S. O.
Right arrow Articles by Katusic, Z. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hynes, S. O.
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.