© 1996 American Heart Association, Inc.
Articles |
Effect of Tetrahydrobiopterin on Endothelial Function in Canine Middle Cerebral Arteries
the Department of Anesthesiology and Pharmacology, Mayo Clinic, Rochester, Minn (M.T., Z.S.K.), and the Laboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Md.
Correspondence to Dr Zvonimir S. Katusic, Department of Anesthesiology and Pharmacology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail katusic.zvonimir@mayo.edu.
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Abstract |
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Tetrahydrobiopterin is an essential cofactor required for activation of NO synthase. However, in intact arteries, the exact role of tetrahydrobiopterin in the regulation of NO synthase activity is not fully understood. The present study was designed to determine the effect of increasing intracellular tetrahydrobiopterin levels on endothelial function in isolated canine middle cerebral arteries. The arterial segments were incubated in MEM for 24 hours at 37°C in the presence or absence of a tetrahydrobiopterin precursor, sepiapterin (10-4 mol/L), and/or superoxide dismutase (150 U/mL). The rings were suspended for isometric tension recording. Tetrahydrobiopterin levels were assayed by high-performance liquid chromatography. Production of cGMP was measured by radioimmunoassay. Incubation with sepiapterin markedly increased intracellular tetrahydrobiopterin levels. In sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly reduced. Superoxide dismutase alone did not affect either relaxation to A23187 or production of cGMP. However, when arteries were incubated with superoxide dismutase plus sepiapterin, endothelium-dependent relaxations to A23187, as well as cGMP production, were significantly augmented. The augmentation of cGMP was observed in rings with (but not without) endothelium. Incubation of arteries in calcium-free medium almost abolished the synergistic effect of tetrahydrobiopterin and superoxide dismutase on cGMP production. These results demonstrate that increased availability of tetrahydrobiopterin may activate endothelial NO synthase. This effect appears to be critically dependent on the presence of superoxide dismutase.
Key Words: NO • NO synthase • superoxide anion • superoxide dismutase
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Introduction |
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Tetrahydrobiopterin is a key cofactor required for activation of NO synthase and production of NO.1 2 3 4 Existing evidence suggests that tetrahydrobiopterin may function as an allosteric and/or reducing cofactor.3 5 More recent findings6 demonstrated that tetrahydrobiopterin is needed for formation and stabilization of active dimeric NO synthase. However, the precise role of this pterin in regulation of NO synthase catalytic activity is still not completely understood.
In isolated rat aorta, exogenously administered tetrahydrobiopterin causes relaxations.7 A NO synthase inhibitor, NG-nitro-L-arginine methyl ester, reduced these relaxations, suggesting that tetrahydrobiopterin may activate NO synthase and increase production of NO.7 In contrast, our previous study on canine cerebral arteries demonstrated that exogenous tetrahydrobiopterin causes endothelium-dependent contractions, mediated by auto-oxidation of tetrahydrobiopterin and subsequent generation of superoxide anions.8 Because of these conflicting findings, the present study was designed to determine the effect of increasing intracellular tetrahydrobiopterin on endothelial function in canine middle cerebral arteries. To this end, we treated arteries with sepiapterin, a well-characterized precursor of tetrahydrobiopterin.9 Intracellularly, sepiapterin is converted to tetrahydrobiopterin via the so-called salvage pathway9 (Fig 1
).
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Materials and Methods |
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Rings (4 mm long) of middle cerebral arteries were taken from dogs (15 to 20 kg) anesthetized with 30 mg/kg IV sodium pentobarbital. All procedures were in accordance with institutional guidelines. In certain rings, the endothelium was removed mechanically by gentle rubbing of the intimal surface. The rings were incubated with sepiapterin (10-4 mol/L) or superoxide dismutase (150 U/mL) for 24 hours at 37°C in 2.5 mL MEM (with Earle's salts, containing 0.1% BSA, 100 U/mL penicillin, and 100 µg/mL streptomycin) in a CO2 incubator (5% CO2/95% air; Wedco). They were then used for experiments. Several rings cut from the same artery were studied in parallel.
Measurement of Intracellular Tetrahydrobiopterin
To terminate incubation, rings were removed from MEM and frozen in liquid nitrogen. The rings were homogenized in buffer containing 10 mmol/L Tris (pH 7.4), 1 mmol/L dithiothreitol, and 1 mmol/L EDTA at 4°C. After centrifugation at 16 000g for 20 minutes at 4°C, tetrahydrobiopterin levels were determined by differential oxidation in acid and base with reverse-phase high-performance liquid chromatography, as described previously.10
Organ-Chamber Experiments
After incubation, rings were placed in modified Krebs-Ringer bicarbonate solution (control solution, mmol/L: NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, calcium EDTA 0.0026, and glucose 11.1). Each ring was connected to an isometric force transducer (Gould) and suspended in an organ chamber filled with 25 mL of control solution (37°C, pH 7.4) gassed with 94% O2/6% CO2. Isometric tension was recorded continuously. The arteries were allowed to stabilize at a resting tension of 200 to 400 mg for 1 hour. Each ring was then gradually stretched to the optimal point of its length-tension curve as determined by the contraction to UTP (10-5 mol/L). To inhibit cyclooxygenase activity, all experiments were carried out in the presence of indomethacin (10-5 mol/L). The functional integrity of endothelium was tested by the presence of relaxations to 10-6 mol/L bradykinin.
Concentration-response curves to calcium ionophore A23187 were cumulatively obtained during submaximal contractions to UTP (10-5 mol/L). Only one concentration-response curve was made per preparation. In arteries incubated 24 hours with superoxide dismutase or sepiapterin plus superoxide dismutase, organ-chamber experiments were performed in the presence of superoxide dismutase. The incubation time with indomethacin was 30 minutes. The relaxations were expressed as a percentage of maximal relaxations induced by papaverine (3x10-4 mol/L).
Measurement of Intracellular cGMP
A radioimmunoassay technique was used to determine the levels of cGMP. After incubation for 24 hours, indomethacin (10-5 mol/L) and 3-isobutyl-1-methylxanthine (IBMX, 10-4 mol/L) were added to MEM and the preparations were incubated for another 30 minutes to inhibit cyclooxygenase activity and the degradation of cyclic nucleotides by phosphodiesterases, respectively. The rings were then removed from the medium and frozen in liquid nitrogen. A cGMP radioimmunoassay kit (Amersham) was used to perform the measurements.
The role of calcium in production of cGMP was studied in rings incubated in MEM without calcium in the presence of an extracellular calcium chelator (EGTA, 10-3 mol/L) and an intracellular calcium chelator (BAPTA/AM, 2x10-5 mol/L)11 12 for 1 hour at 37°C. This step was followed by 24 hours' incubation in the absence or in the presence of superoxide dismutase and/or sepiapterin.13
Measurement of Superoxide Anions
Tetrahydrobiopterin (3x10-6 to 10-4 mol/L) was added to Krebs-Ringer bicarbonate solution bubbled with 94% O2/6% CO2 in the organ chamber. After 5 minutes, the level of superoxide anions in the solution was determined by reduction of ferricytochrome c (3x10-5 mol/L) at 550 nm for 15 minutes in a spectrophotometer (Beckman, DU 640) as reported previously.14 15 Superoxide anion production was calculated on the basis of the molar extinction coefficient of reduced ferricytochrome c [
=21 000 mol/L-1cm-1] and the portion that was inhibited by superoxide dismutase (400 U/mL).14 15
Drugs
The following pharmacological agents were used: sepiapterin and tetrahydrobiopterin from Dr B. Schircks Laboratories; superoxide dismutase from dog erythrocytes, indomethacin, IBMX, calcium ionophore A23187, UTP, papaverine hydrochloride, EDTA, extracellular calcium chelator EGTA, fraction V bovine albumin, DMSO, sodium nitroprusside, and cytochrome c (horse heart: type VI) all from Sigma Chemical Co; intracellular calcium chelator BAPTA/AM11 12 from Calbiochem Biochemicals; and MEM and penicillin-streptomycin from GIBCO-BRL. Stock solutions of the drugs were freshly prepared every day. A23187, IBMX, and BAPTA/AM were dissolved in DMSO. To minimize the influence of DMSO on the arteries, the final concentration of DMSO in all media and solutions was <0.5%. Stock solutions of indomethacin were prepared in equal molar concentrations of Na2CO3. All concentrations are expressed as final molar concentration in medium or solution.
Statistical Analysis
Results are expressed as mean±SEM; in each set of experiments, n indicates the number of animals studied. Statistical evaluation of the data was performed by ANOVA, followed by Fisher's post hoc test. A value of P<.05 was considered statistically significant.
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Stimulatory Effect of Sepiapterin on Tetrahydrobiopterin Biosynthesis
Under basal conditions, significantly higher endogenous levels of tetrahydrobiopterin were detected in arteries with endothelium (Fig 2
). Sepiapterin (10-4 mol/L; incubation time, 24 hours) increased levels of tetrahydrobiopterin in arteries with endothelium. The removal of endothelial cells significantly reduced the effect of sepiapterin (Fig 2).
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significant difference between arteries with and without endothelium (P<.05).
Basal levels of tetrahydrobiopterin obtained in the present study were comparable with the levels detected in freshly isolated canine cerebral arteries,16 indicating that availability of intracellular tetrahydrobiopterin is not affected by 24 hours' incubation in MEM (Table 1).
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Effect of Sepiapterin on Endothelium-Dependent Relaxations and Production of cGMP
Contractions to UTP (10-5 mol/L) were preserved in arteries incubated for 24 hours in the absence or presence of sepiapterin (10-4 mol/L) and/or superoxide dismutase (150 U/mL; Figs 3, 4, and 5). In sepiapterin-treated arteries, maximal relaxations to A23187 were significantly reduced (Fig 3) and intracellular cGMP levels decreased (Fig 6). Superoxide dismutase (150 U/mL) alone did not affect either relaxations to A23187 or production of cGMP (Figs 4 and 7). In addition, in arteries with endothelium, superoxide dismutase did not affect production of tetrahydrobiopterin from sepiapterin (n=5; data not shown). However, when arteries were incubated with superoxide dismutase plus sepiapterin, endothelium-dependent relaxations to A23187 were significantly augmented (Fig 5) and production of cGMP was concomitantly enhanced (Fig 8). In contrast, in arteries without endothelium, sepiapterin or superoxide dismutase did not affect either endothelium-independent relaxation to a guanylate cyclase activator, sodium nitroprusside (Table 2), or production of cGMP (Table 3).
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Lower concentrations of sepiapterin (10-5 to 10-7 mol/L) did not significantly affect endothelium-dependent relaxations to A23187 (n=6; data not shown).
Effect of Calcium-Free Medium on Production of cGMP
When arteries were incubated in calcium-free MEM plus an extracellular calcium chelator (EGTA, 10-3 mol/L) and an intracellular calcium chelator (BAPTA/AM, 2x10-5 mol/L),11 12 the synergistic effect of superoxide dismutase and sepiapterin on cGMP production was almost abolished (Fig 9).
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Production of Superoxide Anions by Tetrahydrobiopterin Auto-oxidation
Incubation of tetrahydrobiopterin (3x10-6 to 10-4 mol/L) in Krebs-Ringer bicarbonate solution bubbled with 94% O2/6% CO2 caused a concentration-dependent increase in production of superoxide anions (Fig 10).
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Discussion |
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This is the first study to examine the effect of increased intracellular levels of tetrahydrobiopterin on endothelial function in isolated arteries. As an experimental model, we used canine middle cerebral arteries incubated with sepiapterin. The major new findings are that (1) sepiapterin stimulates tetrahydrobiopterin production in endothelial and smooth muscle cells of intact arteries and (2) high tissue levels of tetrahydrobiopterin have an inhibitory effect on endothelium-dependent relaxations, whereas (3) in the presence of superoxide dismutase, tetrahydrobiopterin augments endothelium-dependent relaxations.
Under basal conditions, measurements of tetrahydrobiopterin levels revealed a detectable amount of NO synthase cofactor in cerebral arterial wall preparations incubated for 24 hours. Similar levels were also detected in freshly isolated basilar arteries,16 suggesting that tetrahydrobiopterin production had not been affected by incubation in MEM. The removal of endothelial cells caused a 60% reduction in the total amount of tetrahydrobiopterin, indicating that the endothelium has a relatively higher rate of tetrahydrobiopterin production than media and adventitia. More importantly, in arteries treated with sepiapterin, very high levels of tetrahydrobiopterin were detected, demonstrating that sepiapterin could be used as a pharmacological tool to activate tetrahydrobiopterin production. Our results also demonstrate that the endothelium contains almost 80% of the total amount of tetrahydrobiopterin in arteries incubated with sepiapterin. This finding is similar to results obtained under basal conditions and suggests that in cerebral arteries, endothelial cells have a high capacity to synthesize tetrahydrobiopterin.
A number of previous studies demonstrated that the relaxations to A23187 are due to translocation of calcium into endothelial cells, activation of NO synthase, and release of NO.17 18 19 20 21 22 It is also well established that in smooth muscle cells, cGMP is a major second messenger for NO and that changes in cGMP levels reflect NO production.17 18 19 20 21 22 23 In contrast to our expectations, high levels of NO synthase cofactor reduced rather than augmented endothelium-dependent relaxations to A23187. Similarly, levels of cGMP were significantly reduced in arteries exposed to sepiapterin. Auto-oxidation of tetrahydrobiopterin and subsequent formation of superoxide anions, which in turn chemically inactivate NO,24 may explain our results. Our previous study on isolated canine basilar arteries demonstrated that exogenous tetrahydrobiopterin causes endothelium-dependent contractions, primarily due to inactivation of NO with superoxide anions generated in the course of tetrahydrobiopterin auto-oxidation.8 Indeed, in the present study we confirmed that tetrahydrobiopterin is capable of producing superoxide anions under our experimental conditions. This finding is in agreement with observations that under in vitro conditions, tetrahydrobiopterin reacts with oxygen to yield dihydrobiopterin and superoxide anions.25 We therefore decided to determine whether superoxide dismutase may prevent sepiapterin-induced impairment of endothelial function. It was somewhat surprising that superoxide dismutase not only corrected impaired endothelial function but significantly augmented endothelium-dependent relaxations and production of cGMP. These effects were selective for the arteries that were incubated with sepiapterin and had high levels of tetrahydrobiopterin. The selectivity was confirmed by the fact that superoxide dismutase did not affect endothelial function or cGMP synthesis in control arteries. Conversion of sepiapterin to tetrahydrobiopterin was also not affected by the presence of superoxide dismutase. Furthermore, in arteries without endothelium, sepiapterin and superoxide dismutase did not affect either endothelium-independent relaxation to a nitrovasodilator, sodium nitroprusside, or production of cGMP, excluding the possibility that sepiapterin and superoxide dismutase alter activity of guanylate cyclase or general vascular responsiveness. Thus, it appears that in isolated arteries, increased availability of tetrahydrobiopterin may stimulate production of NO, provided that activity of superoxide dismutase in the surrounding environment is high enough to prevent auto-oxidation of tetrahydrobiopterin and inactivation of NO by generated superoxide anions.
The ability of tetrahydrobiopterin to stimulate endothelial NO synthase and to increase production of NO has been demonstrated in cultured human umbilical vein endothelial cells.26 In this experimental model, tetrahydrobiopterin levels were increased by 24 hours' incubation with tumor necrosis factor, interleukin-1, and interferon-
. It is, however, interesting that tumor necrosis factor is a very potent inducer of superoxide dismutase activity in vitro and in vivo.27 More importantly, in cultured human umbilical vein endothelial cells, tumor necrosis factor and interleukin-1 stimulate expression of manganese–superoxide dismutase.28 These findings demonstrate that in vascular endothelium, increased tetrahydrobiopterin biosynthesis is associated with simultaneous induction of superoxide dismutase. Whether high superoxide dismutase activity is needed to protect endothelial cells from excessive production of superoxide anions generated in the chemical reaction between tetrahydrobiopterin and oxygen remains to be determined.
Because superoxide dismutase is a large molecule29 and cannot cross the cell membrane, addition of exogenous enzyme to the organ chamber increases enzymatic activity in the extracellular space. On the other hand, a high tissue concentration of tetrahydrobiopterin may favor movement of pterin across the concentration gradient into the extracellular space, creating conditions for auto-oxidation in Krebs-Ringer bicarbonate solution bubbled with 94% oxygen. In addition, superoxide anions formed intracellularly may escape to the extracellular space via anion channels.30 Thus, despite the fact that it cannot enter the intracellular space, exogenous superoxide dismutase may significantly decrease the concentration of superoxide anions and prevent inactivation of NO in the extracellular space.31
Three different NO synthase isoforms have been identified by molecular cloning: neuronal,32 33 macrophage,34 35 36 and endothelial.37 38 The activity of endothelial and neuronal NO synthase is regulated by intracellular calcium levels.39 40 In contrast, the macrophage isoform binds calmodulin in the absence of calcium and does not require calcium for production of NO.41 42 In the present study, the augmentation of cGMP production induced by tetrahydrobiopterin plus superoxide dismutase was observed only in arteries with endothelium. More importantly, incubation of arteries in calcium-free medium almost abolished this effect. These results demonstrate that in arteries treated with sepiapterin and superoxide dismutase, increased activity of endothelial NO synthase is responsible for augmentation of endothelium-dependent relaxations.
The precise mechanisms underlying tetrahydrobiopterin-induced stimulation of endothelial NO synthase are still unknown. Experiments with purified neuronal isoform revealed that tetrahydrobiopterin may have dual roles as both an allosteric and a redox cofactor.5 Previous studies also demonstrated that tetrahydrobiopterin converts the L-arginine binding site of the enzyme to a high-affinity state43 44 and that only redox-active derivatives of tetrahydrobiopterin enhance NO synthase activity.3 A recent study reported a stabilizing effect of tetrahydrobiopterin on neuronal NO synthase homodimers.6 The precise mechanism responsible for the apparent stimulation of endothelial NO synthase reported in the present study remains to be determined.
The concentration of tetrahydrobiopterin for half-maximal stimulation (Km) of NO synthase is 2x10-8 mol/L.45 Our results do not allow any conclusion regarding the concentration of tetrahydrobiopterin in endothelial and smooth muscle cells of cerebral arteries. However, stimulation with sepiapterin induced a 300-fold increase in tissue levels of tetrahydrobiopterin (Fig 2
). Under physiological conditions, the concentrations of tetrahydrobiopterin in cerebral arteries are unknown. The high capacity of vascular tissue to synthesize tetrahydrobiopterin may serve to fully activate endothelial NO synthase and to support macrophage (inducible) NO synthase activity that is expressed under pathological conditions.21 Indeed, elevated tetrahydrobiopterin has been detected in rat aortic smooth muscle cells exposed to lipopolysaccharide and interferon-.2 46
Our previous study demonstrated that in isolated canine cerebral arteries, auto-oxidation of exogenous tetrahydrobiopterin causes endothelium-dependent contractions.8 The present study revealed dual action of high intracellular tetrahydrobiopterin on vascular tone: (1) superoxide anion–mediated inhibition of endothelium-dependent relaxations and (2) augmentation of endothelium-dependent relaxations by activation of endothelial NO synthase. Thus, increased availability of tetrahydrobiopterin may stimulate NO production; however, this action appears to be critically dependent on the levels of superoxide dismutase. In cerebral arteries, the balance between synthesis of tetrahydrobiopterin and activity of superoxide dismutase may be an important determinant of the NO amount available for control of vascular tone.
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Acknowledgments |
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This work was supported in part by the National Heart, Lung, and Blood Institute (grant HL-53542) and the Mayo Foundation. We would like to thank Dr J. Cristopher Sill for valuable comments regarding our study, Adele Stelter and Leslie Smith for technical assistance, and Janet Beckman for typing the manuscript.
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Footnotes |
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Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 12-16, 1995.
Received February 5, 1996; accepted April 29, 1996.
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References |
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1. Mayer B, John M, Bohme E. Purification of a Ca2+/calmodulin–dependent nitric oxide synthase from porcine cerebellum: cofactor role of tetrahydrobiopterin. FEBS Lett. 1990;277:215-219.[Medline] [Order article via Infotrieve]
2. Gross SS, Jaffe EA, Levi R, Kilbourn RG. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun. 1991;178:823-829.[Medline] [Order article via Infotrieve]
3. Hevel JM, Marletta MA. Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity. Biochemistry. 1992;31:7160-7165.[Medline] [Order article via Infotrieve]
4. Schmidt K, Werner ER, Mayer B, Wachter H, Kukovetz WR. Tetrahydrobiopterin-dependent formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells. Biochem J. 1992;281:297-300.
5. Mayer B, Werner ER. In search of a function for tetrahydrobiopterin in the biosynthesis of nitric oxide. Naunyn Schmiedebergs Arch Pharmacol. 1995;351:453-463.[Medline] [Order article via Infotrieve]
6. Klatt P, Schmidt K, Lehner D, Glatter O, Bachinger HP, Mayer B. Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer. EMBO J. 1995;14:3687-3695.[Medline] [Order article via Infotrieve]
7. Van Amsterdam JGC, Wemer J. Tetrahydrobiopterin induces vasodilatation via enhancement of cGMP level. Eur J Pharmacol. 1992;215:349-350.[Medline] [Order article via Infotrieve]
8. Kinoshita H, Katusic ZS. Exogenous tetrahydrobiopterin causes endothelium-dependent contractions in isolated canine basilar artery. Am J Physiol. 1996. In press.
9. Nichol CA, Smith GK, Duch DS. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. 1985;54:729-764.[Medline] [Order article via Infotrieve]
10. Fukushima T, Nixon JC. Analysis of reduced forms of biopterin in biological fluids and tissues. Anal Biochem. 1980;102:176-188.[Medline] [Order article via Infotrieve]
11. Lew VL, Tsien RY, Miner C, Bookchin RM. Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator. Nature. 1982;298:478-481.[Medline] [Order article via Infotrieve]
12.
Trilivas I, McDonough PM, Brown JH. Dissociation of protein kinase C redistribution from the phosphorylation of its substrates. J Biol Chem. 1991;266:8431-8438.
13.
Katusic ZS. Endothelial L-arginine pathway and regional cerebral arterial reactivity to vasopressin. Am J Physiol. 1992;262:H1557-H1562.
14.
Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995;77:510-518.
15.
Rosen GM, Freeman BA. Detection of superoxide generated by endothelial cells. Proc Natl Acad Sci U S A. 1984;81:7269-7273.
16. Katusic ZS, Milstien S, Stelter A. Endothelium and tetrahydrobiopterin levels in isolated arteries and veins. Circulation. 1995;92(suppl I):I-391. Abstract.
17. Ignarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J. 1989;3:31-36.[Abstract]
18.
Ignarro LJ. Nitric oxide: a novel signal-transduction mechanism for transcellular communication. Hypertension. 1990;16:477-483.
19. Ignarro LJ. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol. 1990;30:535-560.[Medline] [Order article via Infotrieve]
20. Knowles RG, Moncada S. Nitric oxide synthase in mammals. Biochem J. 1994;298:249-258.
21. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109-142.[Medline] [Order article via Infotrieve]
22. Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest. 1991;21:361-374.[Medline] [Order article via Infotrieve]
23. Ignarro LJ, Kadowitz PJ. The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Annu Rev Pharmacol Toxicol. 1985;25:171-191.[Medline] [Order article via Infotrieve]
24. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986;320:454-456.[Medline] [Order article via Infotrieve]
25.
Mayer B, Klatt P, Werner ER, Schmidt K. Kinetics and mechanism of tetrahydrobiopterin-induced oxidation of nitric oxide. J Biol Chem. 1995;270:655-659.
26. Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, Pober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. J Clin Invest. 1994;93:2236-2243.
27.
Wong GHW, Goeddel DV. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science. 1988;242:941-944.
28.
Suzuki K, Tatsumi H, Satoh S, Senda T, Nakata T, Fujii J, Taniguchi N. Manganese-superoxide dismutase in endothelial cells: localization and mechanism of induction. Am J Physiol. 1993;265:H1173-H1178.
29. McCord JM, Fridovich I. Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J Biol Chem. 1969;144:6049-6055.
30.
Kontos HA, Wei EP, Ellis EF, Jenkins LW, Povlishock JT, Rowe GT, Hess ML. Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats. Circ Res. 1985;57:142-151.
31.
Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. 1986;250:H822-H827.
32. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991;351:714-718.[Medline] [Order article via Infotrieve]
33. Nakane M, Schmidt HHHW, Pollock JS, Forstermann U, Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett. 1993;316:175-180.[Medline] [Order article via Infotrieve]
34.
Xie Q, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992;256:225-228.
35.
Greller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Wang SC, Nakayama DK, Simmons RL, Snyder SH, Billiar TR. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci U S A. 1993;90:3491-3495.
36. Nunokawa Y, Ishida YN, Tanaka S. Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem Biophys Res Commun. 1993;191:89-94.[Medline] [Order article via Infotrieve]
37.
Sessa WC, Harrison JK, Barber CM, Zeng D, Durieux ME, D'Angelo DD, Lynch KR, Peach MJ. Molecular cloning and expression of cDNA encoding endothelial cell nitric oxide synthase. J Biol Chem. 1992;267:15274-15276.
38. Marsden PA, Schappert KT, Chen HS, Flowers M, Sundell CL, Wilcox JN, Lamas S, Michel T. Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett. 1992;307:287-293.[Medline] [Order article via Infotrieve]
39. Busse R, Mulsch A. Calcium-dependent nitric oxide synthase in endothelial cytosol is mediated by calmodulin. FEBS Lett. 1990;265:133-134.[Medline] [Order article via Infotrieve]
40.
Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990;87:682-690.
41.
Stuehr DJ, Cho HJ, Kwon NS, Weise M, Nathan CF. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991;88:7773-7777.
42.
Cho HJ, Xie QW, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Nathan C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med. 1992;176:599-604.
43.
Klatt P, Schmid M, Leopold E, Schmidt K, Werner ER, Mayer B. The pteridine binding site of brain nitric oxide synthase: tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. J Biol Chem. 1994;269:13861-13866.
44.
Klatt P, Schmidt K, Brunner F, Mayer B. Inhibitors of brain nitric oxide synthase: binding kinetics, metabolism, and enzyme inactivation. J Biol Chem. 1994;269:1674-1680.
45. Kaufman S. New tetrahydrobiopterin-dependent systems. Annu Rev Nutr. 1993;13:261-286.[Medline] [Order article via Infotrieve]
46. Gross SS, Levi R. Tetrahydrobiopterin synthesis: an absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992;276:25722-25729.
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 S. Kawashima and M. Yokoyama Dysfunction of Endothelial Nitric Oxide Synthase and Atherosclerosis Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 998 - 1005. [Abstract] [Full Text] [PDF]
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Z. S. Katusic and L. V. d'Uscio Tetrahydrobiopterin: Mediator of Endothelial Protection Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 397 - 398. [Full Text]
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N. J. Alp and K. M. Channon Regulation of Endothelial Nitric Oxide Synthase by Tetrahydrobiopterin in Vascular Disease Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 413 - 420. [Abstract] [Full Text]
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 E. R. Werner, A. C.F. Gorren, R. Heller, G. Werner-Felmayer, and B. Mayer Tetrahydrobiopterin and Nitric Oxide: Mechanistic and Pharmacological Aspects Experimental Biology and Medicine, December 1, 2003; 228(11): 1291 - 1302. [Abstract] [Full Text] [PDF]
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B. M. Mitchell, A. M. Dorrance, and R. C. Webb GTP cyclohydrolase 1 inhibition attenuates vasodilation and increases blood pressure in rats Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H2165 - H2170. [Abstract] [Full Text] [PDF]
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Z. Bagi, A. Koller, and G. Kaley Superoxide-NO interaction decreases flow- and agonist-induced dilations of coronary arterioles in Type 2 diabetes mellitus Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1404 - H1410. [Abstract] [Full Text] [PDF]
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 J.-S. Zheng, X.-Q. Yang, K. J. Lookingland, G. D. Fink, C. Hesslinger, G. Kapatos, I. Kovesdi, and A. F. Chen Gene Transfer of Human Guanosine 5'-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension Circulation, September 9, 2003; 108(10): 1238 - 1245. [Abstract] [Full Text] [PDF]
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 L. V. d'Uscio, S. Milstien, D. Richardson, L. Smith, and Z. S. Katusic Long-Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity Circ. Res., January 10, 2003; 92(1): 88 - 95. [Abstract] [Full Text] [PDF]
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D. Yang, N. Levens, J. N. Zhang, P. M. Vanhoutte, and M. Feletou Specific Potentiation of Endothelium-Dependent Contractions in SHR by Tetrahydrobiopterin Hypertension, January 1, 2003; 41(1): 136 - 142. [Abstract] [Full Text] [PDF]
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M. M. Tarpey Sepiapterin Treatment in Atherosclerosis Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1519 - 1521. [Full Text] [PDF]
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J. Vasquez-Vivar, D. Duquaine, J. Whitsett, B. Kalyanaraman, and S. Rajagopalan Altered Tetrahydrobiopterin Metabolism in Atherosclerosis: Implications for Use of Oxidized Tetrahydrobiopterin Analogues and Thiol Antioxidants Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1655 - 1661. [Abstract] [Full Text] [PDF]
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O. Suda, M. Tsutsui, T. Morishita, A. Tanimoto, M. Horiuchi, H. Tasaki, P. L. Huang, Y. Sasaguri, N. Yanagihara, and Y. Nakashima Long-Term Treatment With N{omega}-Nitro-L-Arginine Methyl Ester Causes Arteriosclerotic Coronary Lesions in Endothelial Nitric Oxide Synthase-Deficient Mice Circulation, September 24, 2002; 106(13): 1729 - 1735. [Abstract] [Full Text] [PDF]
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S. Cai, N. J Alp, D. McDonald, I. Smith, J. Kay, L. Canevari, S. Heales, and K. M Channon GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation Cardiovasc Res, September 1, 2002; 55(4): 838 - 849. [Abstract] [Full Text] [PDF]
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P. J.M Best, J. C Burnett Jr., S. H Wilson, D. R Holmes Jr., and A. Lerman Dendroaspis natriuretic peptide relaxes isolated human arteries and veins Cardiovasc Res, August 1, 2002; 55(2): 375 - 384. [Abstract] [Full Text] [PDF]
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 M. Akiyama, D. Eguchi, D. Weiler, T. O'Brien, I. Kovesdi, R. S. Scotland, W. C. Sessa, and Z. S. Katusic Expression and Function of Recombinant S1179D Endothelial Nitric Oxide Synthase in Canine Cerebral Arteries Stroke, April 1, 2002; 33(4): 1071 - 1076. [Abstract] [Full Text] [PDF]
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L. V. d'Uscio, L. A. Smith, and Z. S. Katusic Hypercholesterolemia Impairs Endothelium-Dependent Relaxations in Common Carotid Arteries of Apolipoprotein E-Deficient Mice Stroke, November 1, 2001; 32(11): 2658 - 2664. [Abstract] [Full Text] [PDF]
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S. P. Didion, C. A. Hathaway, and F. M. Faraci Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1697 - H1703. [Abstract] [Full Text] [PDF]
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Z. S. Katusic Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H981 - H986. [Abstract] [Full Text] [PDF]
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L. V. d'Uscio, T. A. Baker, C. B. Mantilla, L. Smith, D. Weiler, G. C. Sieck, and Z. S. Katusic Mechanism of Endothelial Dysfunction in Apolipoprotein E-Deficient Mice Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 1017 - 1022. [Abstract] [Full Text] [PDF]
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D. Eguchi and Z. S. Katusic Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H892 - H898. [Abstract] [Full Text] [PDF]
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D. G. Johns, A. M. Dorrance, N. L. Tramontini, and R. C. Webb Glucocorticoids Inhibit Tetrahydrobiopterin-Dependent Endothelial Function Experimental Biology and Medicine, January 1, 2001; 226(1): 27 - 31. [Abstract] [Full Text]
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 S. Verma, F. Lovren, A. S. Dumont, K. J. Mather, A. Maitland, T. M. Kieser, C. R. Triggle, and T. J. Anderson Tetrahydrobiopterin improves endothelial function in human saphenous veins J. Thorac. Cardiovasc. Surg., October 1, 2000; 120(4): 668 - 671. [Abstract] [Full Text] [PDF]
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 M. KAKOKI, Y. HIRATA, H. HAYAKAWA, E. SUZUKI, D. NAGATA, A. TOJO, H. NISHIMATSU, N. NAKANISHI, Y. HATTORI, K. KIKUCHI, et al. Effects of Tetrahydrobiopterin on Endothelial Dysfunction in Rats with Ischemic Acute Renal Failure J. Am. Soc. Nephrol., February 1, 2000; 11(2): 301 - 309. [Abstract] [Full Text] [PDF]
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M. Tsutsui, H. Onoue, Y. Iida, L. Smith, T. O'Brien, and Z. S. Katusic B1 and B2 bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H367 - H372. [Abstract] [Full Text] [PDF]
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M. Tsutsui, H. Onoue, Y. Iida, L. Smith, T. O'Brien, and Z. S. Katusic Adventitia-dependent relaxations of canine basilar arteries transduced with recombinant eNOS gene Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H1846 - H1852. [Abstract] [Full Text] [PDF]
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K. M. Channon, H. Qian, V. Neplioueva, M. A. Blazing, E. Olmez, G. A. Shetty, S. A. Youngblood, J. Pawloski, T. McMahon, J. S. Stamler, et al. In Vivo Gene Transfer of Nitric Oxide Synthase Enhances Vasomotor Function in Carotid Arteries From Normal and Cholesterol-Fed Rabbits Circulation, November 3, 1998; 98(18): 1905 - 1911. [Abstract] [Full Text] [PDF]
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M. Tsutsui, A. F. Y. Chen, T. O'Brien, T. B. Crotty, and Z. S. Katusic Adventitial Expression of Recombinant eNOS Gene Restores NO Production in Arteries Without Endothelium Arterioscler Thromb Vasc Biol, August 1, 1998; 18(8): 1231 - 1241. [Abstract] [Full Text] [PDF]
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S. Ashwal, B. Tone, H. R. Tian, D. J. Cole, W. J. Pearce, and F. M. Faraci Core and Penumbral Nitric Oxide Synthase Activity During Cerebral Ischemia and Reperfusion • Editorial Comment Stroke, May 1, 1998; 29(5): 1037 - 1047. [Abstract] [Full Text] [PDF]
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R. M. F. Wever, T. F. Luscher, F. Cosentino, and T. J. Rabelink Atherosclerosis and the Two Faces of Endothelial Nitric Oxide Synthase Circulation, January 13, 1998; 97(1): 108 - 112. [Full Text] [PDF]
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Z. S. Katusic, A. Stelter, and S. Milstien Cytokines Stimulate GTP Cyclohydrolase I Gene Expression in Cultured Human Umbilical Vein Endothelial Cells Arterioscler Thromb Vasc Biol, January 1, 1998; 18(1): 27 - 32. [Abstract] [Full Text] [PDF]
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W. I. Rosenblum and Z. S. Katusic Tetrahydrobiopterin, a Cofactor for Nitric Oxide Synthase, Produces Endothelium-Dependent Dilation of Mouse Pial Arterioles Stroke, January 1, 1997; 28(1): 186 - 189. [Abstract] [Full Text]
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 R. Heller, A. Unbehaun, B. Schellenberg, B. Mayer, G. Werner-Felmayer, and E. R. Werner L-Ascorbic Acid Potentiates Endothelial Nitric Oxide Synthesis via a Chemical Stabilization of Tetrahydrobiopterin J. Biol. Chem., January 5, 2001; 276(1): 40 - 47. [Abstract] [Full Text] [PDF]
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