Am J Physiol Heart Circ Physiol 285: H2165-H2170, 2003.
First published July 10, 2003; doi:10.1152/ajpheart.00253.2003
0363-6135/03 $5.00
GTP cyclohydrolase 1 inhibition attenuates vasodilation and increases blood pressure in rats
Brett M. Mitchell,
Anne M. Dorrance, and
R. Clinton Webb
Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-3000
Submitted 20 March 2003
; accepted in final form 9 July 2003
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ABSTRACT
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GTP cyclohydrolase 1 is the rate-limiting enzyme in production of tetrahydrobiopterin, a necessary cofactor for endothelial nitric oxide synthase. We tested the hypothesis that inhibition of tetrahydrobiopterin synthesis impairs endothelium-dependent relaxation and increase blood pressure in rats. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a GTP cyclohydrolase 1 inhibitor, was given in drinking water (
120 mg · kg–1 · day–1) to male Sprague-Dawley rats for 3 days. Systolic blood pressures were measured (tail-cuff procedure) for 3 days before and each day during DAHP treatment. Blood pressure was significantly increased after DAHP treatment (122 ± 2 vs. 154 ± 3 mmHg before and after DAHP, respectively; P < 0.05). Endothelium-intact aortic segments from pentobarbital sodium-anesthetized rats were isolated and hung in organ chambers for measurement of isometric force generation. Aortas from DAHP-treated rats exhibited a decreased maximal relaxation to ACh compared with controls [% relaxation from phenylephrine (10–7 M)-induced contraction: DAHP 57 ± 6% vs. control 79 ± 4%; P < 0.05]. Relaxation responses to A-23187 were also decreased in aortas from DAHP-treated rats compared with controls. Incubation with sepiapterin (10–4 M, 1 h), which produces tetrahydrobiopterin via a salvage pathway, restored relaxation to ACh in aortas from DAHP-treated rats. Superoxide dismutase significantly increased ACh-induced relaxation in aortas from DAHP-treated rats, whereas catalase had no effect. Endothelium-independent relaxation to sodium nitroprusside in aortas from DAHP-treated rats was not different from control rats; however, nitric oxide synthase inhibition increased sensitivity to sodium nitroprusside in aortas from DAHP-treated rats. These results support the hypothesis that GTP cyclohydrolase 1 inhibition decreases relaxation and increases blood pressure in rats.
tetrahydrobiopterin; nitric oxide synthase; endothelium; experimental hypertension
NITRIC OXIDE (NO) plays a key role in vascular tone, and alterations in NO production modulate endothelium-dependent vasodilation and blood pressure. NO is produced from the conversion of arginine and oxygen to citrulline via NO synthase (NOS), of which three isoforms have been discovered: neuronal (NOS I), inducible (NOS II), and endothelial (NOS III). All NOS isoforms require certain cofactors for optimal catalytic activity, and one of these cofactors is tetrahydrobiopterin (BH4). BH4 is necessary for all NOS isoforms, aids in the stabilization of the NOS dimer, and increases the affinity of NOS for arginine (4). BH4 is produced via a de novo pathway from GTP by GTP cyclohydrolase 1 (GTPCH1), the first and rate-limiting of a three-enzyme pathway. GTPCH1 activity is regulated by end-product feedback via the GTP cyclohydrolase feedback regulatory protein (22). BH4 can also be produced via a salvage pathway in which sepiapterin is converted to the intermediate dihydrobiopterin by sepiapterin reductase and then to BH4 (9).
Previous research showed that in the presence of low levels of BH4, NOS III becomes uncoupled and generates superoxide anion (2, 17, 19, 20). In addition to increased superoxide production by NOS, low BH4 levels lead to decreased NO production, both of which contribute to endothelial dysfunction. Exogenous BH4 has been shown to restore endothelial function in humans with coronary artery disease, hypercholesterolemia, atherosclerosis, and a history of cigarette smoking (8, 12, 13, 16). Exogenous BH4 given to the spontaneously hypertensive rat, which has a genetic form of hypertension, has been shown to suppress the development of elevated blood pressure (5).
GTPCH1 has been mostly studied in the context of neurotransmitter and catecholamine production, of which BH4 is also a cofactor. Genetic defects in GTPCH1 can lead to dopa dystonia. However, the role of GTPCH1 in vascular biology is still not fully understood. Previous studies using cell cultures showed that GTPCH1 inhibition with 2,4-diamino-6-hydroxypyrimidine (DAHP) does indeed lower BH4 levels and decrease NO production (10, 17). In addition, studies using isolated aorta, coronary, and cerebral arteries showed that inhibition of BH4 production causes endothelial dysfunction (2, 3, 7). Kinoshita et al. (7) showed that a 6-h incubation of isolated canine basilar arteries with DAHP depleted 95% of intracellular BH4. However, in vivo studies exploring the alteration of BH4 biosynthesis to support these findings have not been performed. Therefore, the purpose of this study was to examine the role of GTPCH1 in vascular reactivity and blood pressure in rats. We hypothesized that DAHP, a known inhibitor of BH4 synthesis, would decrease endothelium-dependent relaxation and increase blood pressure in rats.
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METHODS
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Animals and blood pressure measurements. Male Sprague-Dawley rats (obtained from Harlan; 300–324 g) were used. All procedures were approved by the Medical College of Georgia's Animal Use for Research and Education Committee. All rats were maintained on a 12:12-h light-dark cycle and had access to standard rat chow ad libitum throughout the study. Systolic blood pressures were measured by tail-cuff plethysmography (pneumatic transducer). All rats underwent 3 days of habituation to the procedure followed by a 3-day baseline period consisting of daily blood pressure measurements while being given tap water ad libitum. On the next 3 days, rats were given DAHP (10–2 M; n = 6) in their drinking water or continued receiving tap water (controls; n = 6). The effective concentration (120 mg · kg–1 · day–1) was determined by calculating the normal intake of water in 300-g rats (41 ± 1 ml/day) and using the molecular weight of DAHP and the body weight of the rats. Blood pressure measurements were taken at the same time each day during the DAHP or tap water treatment.
A previous report examined the mechanism of DAHP inhibition of GTPCH1 and found that, in low concentrations, DAHP mimics BH4 in an end-product feedback inhibition mechanism (21). The binding of DAHP to the 9.5-kDa protein GTP cyclohydrolase feedback regulatory protein forms an inhibitory complex with GTPCH1 and decreases its activity. At high concentrations, such as that used in the present study, DAHP was shown to compete directly with GTP for GTPCH1 binding.
Organ chamber experiments. On the day of experiments, rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The thoracic aorta was excised and immediately placed in cold physiological salt solution (PSS; composition in mM: 130.0 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4 · 7H2O, 14.9 NaHCO3, 5.5 dextrose, 0.026 EDTA, 1.6 CaCl2). The isolated endothelium-intact aortic segment was cleaned free of connective tissue and cut into 3- to 4-mm rings. The aortic rings were then connected to an isometric force transducer in a 50-ml organ chamber filled with 37°C PSS and bubbled with 95% O2-5% CO2. Aortic rings from one DAHP-treated and one control rat were studied in parallel. All experiments were performed in the presence of indomethacin (10–5 M) to inhibit cyclooxygenase. Vessels were set at a passive force of 3.5–4.0 g, and isometric force generation was recorded continuously. After a 60-min equilibration period, all vessels were contracted with phenylephrine (PE; 10–7 M) to test viability. ACh (10–6 M) was administered to test the functional integrity of endothelium as measured by relaxation. Concentration-response curves were obtained in a half-log, cumulative fashion. Responses to ACh, A-23187, and sodium nitroprusside (SNP) were generated after contraction to PE (10–7 M). Incubation time was 10 min for superoxide dismutase (SOD) and catalase, 20 min for N
-nitro-L-arginine (L-NNA), and 60 min for indomethacin and sepiapterin. Relaxation responses to ACh, A-23187, and SNP were expressed as percent relaxation from submaximal PE (10–7 M)-induced contraction. To determine EC50 values for relaxation responses to SNP after NOS inhibition, data were expressed as a percentage of maximal relaxation. Regression analysis with three data points along the linear section of the concentration-response curve was used to generate an equation from which the EC50 value was determined. These values were then averaged, and the geometric mean was compared between groups.
RT-PCR. Thoracic aorta was removed and cleaned free of adherent fat, connective tissue, and blood before being snap frozen. RNA was extracted with TRIzol reagent following the manufacturer's protocol (GIBCO). The RNA was quantified by spectrophotometry, and 1 µg of RNA was used to produce cDNA. Contaminating cDNA was removed with DNase enzyme before reverse transcription with avian myeloblastosis virus reverse transcriptase and oligo(dT) as a primer. Occasional RNA samples were subjected to the PCR procedure without prior reverse transcription to control for the presence of contaminating genomic DNA in the sample. PCR amplifications were carried out on a portion of the cDNA produced. Each PCR reaction contained each oligonucleotide primer at 5 pM, 200 µM dNTP, and 0.2 U Taq in the manufacturer's buffer. Optimum annealing temperature was assessed with a gradient block thermal cycler. Cycle number and template dilution factor were determined for each amplicon before experimentation to ensure linearity. The cDNA produced was resolved on a 2% agarose gel, and the amount of DNA present was identified with ethidium bromide staining. The results were quantified with KODAK 1D software and an EDAS 290 imaging system (Eastman Kodak, Rochester, NY). The specific oligonucleotide primers were designed with an Internet-based primer design program, GeneFisher. Rat GTPCH1 (forward: 5'-ATTTGTGGGAAGGGTCCA-3', reverse: 5'-CAGATAACGCTGGCCTCA-3') primers were obtained from Biosource (Camarillo, CA).
Reagents. The following compounds were purchased from Sigma (St. Louis, MO): ACh, calcium ionophore A-23187, catalase, indomethacin, L-NNA, PE, sepiapterin, SNP, and SOD (from bovine erythrocytes, 3,700 U/mg protein). L-NNA and sepiapterin were dissolved in PSS, and a stock solution of indomethacin was dissolved in ethanol (<0.1% final concentration in organ chamber). All other drugs were dissolved in distilled water, and all reagents were prepared fresh on the day of experiments.
Statistical analyses. Results are presented as means ± SE. Blood pressures were averaged over the 3 days of baseline and DAHP or tap water treatment. An analysis of variance was used for multiple comparisons followed by the Newman-Keuls post hoc test when necessary. Comparison between two values was analyzed with Student's t-test when appropriate. mRNA levels are expressed as the ratio of GTPCH1 to GAPDH in arbitrary units. The significance level was set at 0.05.
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RESULTS
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Blood pressure measurements. Before administration of DAHP, baseline systolic blood pressure did not differ between the two groups of rats (P > 0.05; Fig. 1). After DAHP (10–2 M) administration in the drinking water, systolic blood pressure increased significantly over 3 days (average 3-day baseline = 122 ± 2 mmHg vs. average 3-day DAHP treatment = 154 ± 3 mmHg; P < 0.001) and was significantly higher than systolic blood pressure in control rats each day (P < 0.001).
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Fig. 1. 2,4-Diamino-6-hydroxypyrimidine (DAHP; 10–2 M), given in the drinking water, increased systolic blood pressure in male Sprague-Dawley rats. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. control (ANOVA with Newman-Keuls multiple-comparison test).
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Effect of in vivo GTPCH1 inhibition on endothelium-dependent relaxation. The magnitude of force generation to PE (10–7 M) in aortas from DAHP-treated rats was not different compared with controls (DAHP 975 ± 165 vs. control 974 ± 120 mg of isometric force; P > 0.05). Maximal aortic relaxation to ACh was significantly decreased in DAHP-treated compared with control rats (57 ± 6% vs. 79 ± 4% relaxation from 10–7 M PE; P < 0.05; Fig. 2). EC50 values for ACh in the vessels from DAHP-treated and control rats were similar (10–7 M). Similarly, relaxation to the calcium ionophore A-23187 was significantly decreased in aortas from DAHP-treated rats (Fig. 3). NOS inhibition via L-NNA (10–5 M) decreased maximal relaxation to ACh in both groups (Fig. 2). Sepiapterin (10–4 M), a BH4 donor, restored maximal relaxation responses to ACh in the segments from DAHP-treated rats to that of controls (57 ± 6% to 81 ± 4% relaxation from 10–7 M PE; Fig. 4). The dose-response curve and EC50 value for aortas from DAHP-treated rats after sepiapterin were similar to those of the vessels from the control rats. SOD (150 U/ml) significantly increased ACh-induced relaxation in aortas from DAHP-treated rats; however, catalase (1,200 U/ml) had no effect (Fig. 5). In the control rats, aside from one data point, SOD had no significant effect on relaxation responses to ACh (data not shown).
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Fig. 2. Acetylcholine-induced relaxation was decreased in aortas from 3-day DAHP-treated rats and abolished in both groups after nitric oxide synthase (NOS) inhibition with N-nitro-L-arginine (L-NNA, 10–5 M). Results are expressed as means ± SE (n = 5–6). *P < 0.05 vs. control (ANOVA with Newman-Keuls multiple-comparison test). [Acetylcholine], acetylcholine concentration.
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Fig. 3. Relaxation to the calcium ionophore A-23187 was decreased in aortas from 3-day DAHP-treated rats. Results are expressed as means ± SE (n = 5–6). *P < 0.05 vs. control (ANOVA with Newman-Keuls multiple-comparison test).
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Fig. 4. Restoration of tetrahydrobiopterin (BH4, sepiapterin; 10–4 M) increased acetylcholine-induced relaxation in aortas from 3-day DAHP-treated rats. Results are expressed as means ± SE (n = 5–6). *P < 0.05 vs. control (ANOVA with Newman-Keuls multiple-comparison test).
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Fig. 5. Acetylcholine-induced relaxation after incubation with superoxide dismutase (SOD; 150 U/ml) was increased but unchanged with catalase (1,200 U/ml) in aortic rings from 3-day DAHP-treated rats. Results are expressed as means ± SE (n = 6–11). *P < 0.05 vs. DAHP (ANOVA with Newman-Keuls multiple-comparison test).
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Effect of GTPCH1 inhibition on endothelium-independent relaxation. SNP was used to analyze endothelium-independent vascular reactivity. Relaxation in aortic segments from DAHP-treated rats to SNP was not statistically different compared with controls (data not shown). However, NOS inhibition by L-NNA (10–5 M) significantly increased the sensitivity of the DAHP-treated vessels to SNP compared with aortas from control rats (Fig. 6). The EC50 value for aortas from the DAHP-treated rats (–8.89 ± 0.09, antilog = 1.3 x 10–9) was markedly decreased compared with the control vessels (–8.41 ± 0.07, antilog = 3.9 x 10–9; P < 0.05).
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Fig. 6. Sensitivity to sodium nitroprusside after NOS inhibition (L-NNA; 10–5 M) was increased in aortic tissues from 3-day DAHP-treated rats. Values are expressed as % of maximal relaxation. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. control + L-NNA (ANOVA with Newman-Keuls multiple-comparison test).
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Effect of 3-day DAHP-water treatment on GTPCH1 mRNA expression. Figure 7 shows that aortic GTPCH1 mRNA expression levels were not significantly different between DAHP-treated rats and controls (intensity in arbitrary units corrected for GAPDH: DAHP-treated 1.81 ± 0.09 vs. controls 1.55 ± 0.17; P > 0.05).
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Fig. 7. Aortic GTP cyclohydrolase 1 (GTPCH1) mRNA was not different in 3-day (3d) DAHP-treated rats compared with controls. Results are expressed as means ± SE (n = 6) and normalized to GAPDH mRNA.
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DISCUSSION
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This study tested the role of GTPCH1 in vascular reactivity and blood pressure regulation. First, we showed that in vivo GTPCH1 inhibition increases systolic blood pressure. We also demonstrated that endothelium-dependent relaxation is decreased as a result of in vivo BH4 depletion and can be restored with sepiapterin, a BH4 donor, and SOD.
NO plays an important role in blood pressure regulation, and inhibition of NO production leads to elevated blood pressures as demonstrated in rats given exogenous L-NNA and NOS III knockout mice (6, 11). It has been suggested that BH4 may dictate the rate of production of NO (21). Indeed, DAHP treatment increased systolic blood pressures 35 mmHg within 1 day, and pressures remained elevated for up to 3 days (Fig. 1). This extends the findings of an elevated systolic blood pressure in the hph-1 mouse, a hyperphenylalaninemic mouse mutant that displays a 90% deficiency of GTPCH1 (2).
It is known that a decrease in NO can elicit alterations in vascular reactivity. Previous studies reported that cultured endothelial cells and isolated arteries incubated with DAHP have decreased NO production (3, 10, 17, 18). The present study further demonstrated that ACh- and A23187
[GenBank]
-induced relaxations were significantly decreased in aortas from rats treated with a GTPCH1 inhibitor (Figs. 2 and 3) and were abolished after NOS inhibition (Fig. 2). These results support the in vitro studies of Kinoshita et al. (7), who found that endothelium-dependent relaxation to the calcium ionophore A-23187 and bradykinin were significantly reduced in canine basilar arteries incubated with DAHP. Tiefenbacher et al. (13) also showed decreased relaxation to serotonin and substance P in pig coronary arteries incubated with DAHP. In contrast, several studies reported no differences in vasodilation after DAHP incubation or in the hph-1 mouse mutant (2, 3). Increased hydrogen peroxide, a known vasodilator, was able to maintain vasodilation despite decreased BH4 biosynthesis either by DAHP incubation or genetic manipulation of GTPCH1. This finding suggests that the observed increase in blood pressure in the hph-1 mouse mutant may be due to additional mechanisms other than decreased endothelium-dependent relaxation.
Previous studies showed that exogenous BH4 can improve endothelial function in humans and animals (8, 12, 13, 16). Sepiapterin, which restores BH4 through a salvage pathway, has been shown to be effective as a pharmacological tool to increase BH4 levels (7, 14). In the present study, sepiapterin restored ACh-induced relaxation in aortas from DAHP-treated rats to that of control vessels (Fig. 4). This supports a previous study in which pig coronary arteries incubated with DAHP had restored relaxation when incubated with sepiapterin (14). In contrast, incubation with BH4 in hph-1 mouse aortas had no effect on ACh-induced relaxation (2). Together, these results support the concept proposed by Tsutsui et al. (15) that increased availability of BH4 may activate NOS III and lead to increased relaxation.
In the presence of low levels of BH4, superoxide can be produced by "uncoupled" NOS III (2, 17, 19, 20). In addition, 16-wk-old spontaneously hypertensive rats also exhibit decreased BH4 and increased superoxide levels (5). In the current study, we examined the superoxide levels in DAHP-treated rats with a pharmacological approach. If superoxide levels were elevated in the DAHP-treated rats, then SOD, which converts superoxide into the vasodilator hydrogen peroxide, should improve endothelium-dependent relaxations to ACh. Indeed, after incubation with SOD we observed a significantly increased aortic relaxation in aortas from DAHP-treated rats (maximal relaxation increased from 57 ± 6% to 90 ± 2%; Fig. 5), but SOD incubation had no effect in aortas from control rats. This extends the findings in the hph-1 mouse, which also displayed a significant increase in ACh-induced relaxation after SOD (2). Catalase (1,200 U/ml) had no effect on relaxation in the DAHP-treated rats (Fig. 5), which contrasts with findings in the hph-1 mouse mutant. Although Cosentino et al. (2) found no significant differences in relaxations to ACh between hph-1 and wildtype mice, catalase was shown to reduce ACh-induced relaxation only in the hph-1 mice. The authors concluded that reactive oxygen species mediated endothelium-dependent relaxation in the genetic model of BH4 deficiency. This finding differs from the current study because we observed no change in relaxation after incubation with catalase. These data suggest that genetic mutations in the hph-1 mouse cause an alteration in vasodilator mechanisms to maintain vasodilation, whereas our DAHP-treated rats had decreased vasodilation as a result of in vivo GTPCH1 inhibition.
The decreased relaxation in DAHP-treated rats is an endothelium-dependent mechanism demonstrated by the lack of difference in the SNP doseresponse curves between DAHP-treated and control animals (data not shown). This supports previous studies that found no differences in endothelium-independent relaxation in canine basilar arteries incubated with DAHP or in the hph-1 mouse (2, 7). However, we did find a significantly increased sensitivity to the NO donor after NOS inhibition with L-NNA (10–5 M) in aortas from DAHP-treated rats (Fig. 6). Together, these data support previous findings that, in the presence of low NO bioavailability, downstream mediators of NO-induced vasodilation may become upregulated (i.e., guanylate cyclase, cGMP, and/or cGMP-dependent protein kinase) (1).
A previous study showed that incubation of rat aortic smooth muscle cells with DAHP (10–2 M) completely abolished GTPCH1 protein levels (21). However, no one to date has studied the effects of in vivo DAHP administration on GTPCH1 mRNA expression in isolated vascular segments. Because DAHP has been shown to compete with GTP for GTPCH1 binding (21), we hypothesized that DAHP administered in the drinking water would have no effect on mRNA levels. To confirm this, we performed mRNA expression studies using RT-PCR analysis. Indeed, GTPCH1 mRNA levels were not significantly different in the DAHP-treated rats compared with controls (Fig. 7). On the basis of this result, we speculate that GTPCH1 activity and protein levels are also unchanged and that the decreased use of the substrate, GTP, results in decreased BH4 biosynthesis.
These data support the hypothesis that in vivo GTPCH1 inhibition decreases BH4 and NO bioavailability, leading to increased superoxide production, decreased vasodilation, and elevated blood pressure. GTPCH1 activity may, in part, contribute to vascular tone and blood pressure regulation. In addition to insulin and cytokines, discovery of other regulators of GTPCH1 activity will be important in understanding the enzyme's contribution to cardiovascular disease states.
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DISCLOSURES
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This work was supported by National Heart, Lung, and Blood Institutegrant HL-18575 (R. C. Webb), by an American Heart Association (Southeast Affiliate) Predoctoral Fellowship (B. M. Mitchell), and by American Heart Association Scientist Development Grant 0130364N (A. M. Dorrance).
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ACKNOWLEDGMENTS
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Portions of this study were presented at the Experimental Biology 2002 Meeting, New Orleans, LA, April 2002, and published in abstract form (FASEB J 16: A96, 2002).
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FOOTNOTES
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Address for reprint requests and other correspondence: B. M. Mitchell, Dept. of Physiology, CL-3162, Medical College of Georgia, 1120 15th St., Augusta, GA 30912 (E-mail: brettmitchell{at}hotmail.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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