Under normal physiological conditions, the endothelium. Relationship Between Carbamoyl-Phosphate Synthetase Genotype and Systemic Vascular Function

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1 Relationship Between Carbamoyl-Phosphate Synthetase Genotype and Systemic Vascular Function Marshall L. Summar, James V. Gainer, Mias Pretorius, Hector Malave, Stephanie Harris, Lynn D. Hall, Alec Weisberg, Douglas E. Vaughan, Brian W. Christman, Nancy J. Brown Abstract Endothelial cells can convert L-citrulline to L-arginine, the precursor of nitric oxide. The present study tests the hypothesis that a C-to-A nucleotide transversion (T1405N) in the gene-encoding carbamoyl-phosphate synthetase 1, the enzyme catalyzing the rate-limiting step in L-citrulline formation, influences nitric oxide metabolite concentrations or nitric oxide-mediated vasodilation in humans. Bradykinin (100, 200, and 400 ng/min) was infused via brachial artery in 106 (CC:AC:AA 40:54:12) healthy subjects. Sodium nitroprusside (1.6, 3.2, and 6.4 g/min) was also infused in 87 (CC:AC:AA 31:46:10) subjects. Forearm blood flow was measured by plethysmography and blood samples were collected for tissue-type plasminogen activator antigen, nitric oxide metabolites, and cyclic GMP. There was a significant relationship between carbamoyl-phosphate synthetase 1 genotype and nitric oxide metabolites, such that nitric oxide metabolite concentrations were highest in individuals homozygous for the C allele (mean SD, mol/l), lowest in individuals homozygous for the A allele ( mol/l), and intermediate ( mol/l) in heterozygotes (P 0.036). There was a significant effect of carbamoyl-phosphate synthetase 1 genotype on forearm blood flow during bradykinin (P 0.028), such that the vasodilator response was greatest in C allele homozygotes ( ml/min/100 ml at 400 ng/min), least in A allele homozygotes ( ml/min/100 ml), and intermediate ( ml/min/100 ml) in heterozygotes. Similarly, carbamoyl-phosphate synthetase 1 genotype influenced forearm blood flow during nitroprusside (maximal flow , , and ml/min/100 ml in the CC:AC:AA groups, respectively; P 0.022). In contrast, there was no effect of carbamoyl-phosphate synthetase 1 genotype on the nitric oxide independent tissue-type plasminogen activator response to bradykinin (P 0.943). These data indicate that a polymorphism in the gene encoding carbamoyl-phosphate synthetase 1 influences nitric oxide production as well as vascular smooth muscle reactivity. (Hypertension. 2004;43: ) Key Words: nitric oxide bradykinin genetics endothelium vasodilation Under normal physiological conditions, the endothelium plays a critical role in maintaining vascular homeostasis. The vascular endothelium converts L-arginine via nitric oxide synthase (NOS) to L-citrulline and nitric oxide 1 which, in turn, causes vasodilation, inhibits platelet aggregation and leukocyte adhesion, inhibits vascular smooth muscle cell proliferation, and modulates oxidative stress. 2 In recent years, studies measuring the vasodilation response to intraarterial infusion of NOSdependent agonists, such as acetylcholine and bradykinin, have demonstrated that endothelial function is impaired in individuals at risk for coronary artery disease. 3,4 Importantly, administration of exogenous L-arginine intraarterially, intravenously, orally improves endothelial dysfunction in these patients in a majority of studies. 5 7 Vascular endothelial cells can synthesize endogenous L-arginine by recycling L-citrulline, the byproduct of nitric oxide synthesis, using components of the urea cycle (argininosuccinic acid synthase and lyase, respectively). 8,9 In addition, endothelial cells may utilize circulating L-citrulline, formed by the mitochondrial enzymes of the urea cycle in the liver and proximal intestines. 10 Thus, a potential link exists between nitric oxide production and the urea cycle. The rate-limiting step in the urea cycle and L-citrulline production is catalyzed by the enzyme carbamoyl-phosphate synthetase 1 (CPS1). Recently, members of our group identified 14 polymorphisms in the CPS1 gene. 11 One of these, a C-to-A nucleotide transversion in exon 36, results in the substitution of asparagine (Asn) for threonine (Thr) at position 1405 (T1405N), in the critical N-acetylglutamate-binding domain. We have previously reported a significant relationship between this CPS1 genotype and serum concentration of L-arginine in a group of neonates. 12 Given that administration of exogenous L-arginine enhances NOS-dependent vasodilation and that endothelial cells Received October 14, 2003; first decision November 3, 2003; revision accepted December 1, From the Department of Pediatrics, Division of Medical Genetics (M.L.S., S.H., L.D.H.); the Department of Medicine, Divisions of Clinical Pharmacology (J.V.G., A.W., N.J.B.), Pulmonary Medicine (B.W.C.), and Cardiovascular Medicine (H.M., D.E.V.); and the Department of Anesthesia (M.P.), Vanderbilt University Medical Center, Nashville, Tenn. Correspondence to Nancy J. Brown, MD, 560 Robinson Research Building, Vanderbilt University Medical Center, Nashville, TN nancy.j.brown@vanderbilt.edu 2004 American Heart Association, Inc. Hypertension is available at DOI: /01.HYP

2 Summar et al CPS1 Genotype and Vasodilation 187 can convert circulating L-citrulline to endogenous L-arginine, the purpose of the present study was to determine the relationship between CPS1 T1405N genotype and plasma L-citrulline, L-arginine, and nitric oxide metabolite concentrations in adults, and to test the hypothesis that CPS1 genotype influences nitric oxide-dependent vasodilation. Methods Subjects One hundred six subjects participated in ongoing studies of bradykinin-stimulated vasodilation and t-pa release and provided DNA. All subjects gave written informed consent and underwent a history, physical examination, laboratory screening, and ECG. Subjects weighed within 25% of ideal body weight and were not taking medication. Subjects were defined as nonsmokers if they did not smoke any cigarettes and smokers if they smoked at least 5 cigarettes per day. No subject smoked more than 25 cigarettes per day. The protocol was approved by the Vanderbilt University Institutional Review Board and conducted according to the Declaration of Helsinki. Experimental Protocol Studies were performed in the morning, in a temperature-controlled room in the General Clinical Research Center. Subjects were studied in the supine position and in the fasting state. A 20-gauge polyurethane catheter (Cook, Inc, Bloomington, Ind) was inserted into the brachial artery of the nondominant arm, and an intravenous catheter was placed in the antecubital vein. Arterial catheter patency was maintained by infusion of 5% dextrose in water at a rate of 1 ml/min, and subjects were allowed to rest 30 minutes before baseline measurements were made and between drug infusions. Blood pressure was monitored in the contralateral arm using an automated blood pressure cuff. After measurement of basal forearm blood flow (FBF) and blood sampling, graded doses of sodium nitroprusside (SNP, Gensia Siccor Pharmaceuticals, Irvine, Calif) and bradykinin (Clinalfa AG, Läufelfingen, Switzerland) were infused in random order. Thirty-one subjects also received acetylcholine as a muscarinic control; an additional 62 received methacholine after it was determined that acetylcholine did not stimulate t-pa release. Because the muscarinic agonist and doses were not the same in all of the subjects, these data are not presented here. SNP, a nitric oxide donor, was infused at 1.6, 3.2, and 6.4 g/min. Bradykinin was infused at 100, 200, and 400 ng/min. At these doses, bradykinin causes vasodilation in part through a NOS-dependent pathway. 13 Each agonist dose was infused for 5 minutes and FBF was measured during the last 2 minutes of infusion. FBF was measured by silastic-in-mercury strain-gauge plethysmography, as previously described. 13 Forearm vascular resistance (FVR) was calculated as mean arterial pressure (MAP)/FBF. Blood Sampling and Biochemical Assays After measurement of FBF, arterial and venous samples were obtained from the infused arm before and after each dose of study drug. Blood for measurement of t-pa was collected on ice in tubes containing mol/L acidified sodium citrate, centrifuged immediately, and plasma was stored at -70 C until antigen levels were determined using a 2-site ELISA (Biopool AB, Umea, Sweden). Analysis of amino acids was performed on protein-free extracts of baseline venous plasma samples using cation-exchange chromatography (7300 amino acid analyzer, Beckmann, Palo Alto, Calif). Venous plasma concentrations of nitric oxide metabolites were measured using a modified Griess reaction as previously described. 12 3,5 guanosine monophosphate (cgmp) was measured by ELISA, using a commercially-available kit (Amersham Biosciences, Piscataway, NJ). Analysis of Polymorphisms in Carbamoyl-Phosphate Synthetase CPS1 genotypes were determined by single-strand conformation polymorphism analysis and electrophoresed with controls of known genotype, as previously described. 12 Genomic DNA was isolated from preparations of whole blood (Qiagen, Valencia, Calif) Oligonucleotide primers and the polymerase chain reaction were used to amplify a 253-bp fragment encompassing the C-to-A nucleotide transversion at position 4332 of exon 36 of the gene-encoding CPS1. After treatment with formamide, samples were subjected to electrophoresis for 5 hours at 4 C in a nondenaturing gel (FMC, Rockland, Md) and then stained with silver nitrate to detect DNA fragments. Genotypes were read by two blinded individuals. Statistical Analysis Data are presented as mean SD in tables and mean SEM in figures. To determine whether polymorphisms of the CPS1 gene were in Hardy-Weinberg equilibrium, the frequencies of alleles and of genotypes were analyzed, and actual and predicted genotype frequencies were compared by 2 analysis with one degree of freedom. Comparisons of continuous variables among genotype groups were made using ANOVA followed by post hoc comparisons using Dunnet T3 test to correct for multiple comparisons. Probability values for the ANOVA are provided in the text. Probability values for post hoc comparisons are provided in tables. For nitric oxide metabolites, analyses were completed with and without imputed values (series means) for missing values (n 7). The effect of ethnicity, sex, and smoking status (current smoker or not) on continuous variables such as the plasma concentration of nitric oxide metabolites was determined using an unpaired t test or Mann- Whitney U test, as appropriate. Relationships among continuous variables were assessed using Pearson correlation coefficient. The effect of CPS1 genotype on the vasodilator response to each agonist was determined using a general linear model-repeated measure ANOVA in which the within subject variable was dose and the between subject variables were genotype and/or ethnicity, gender, and smoking status, as appropriate on the basis of subgroup analyses. A 2-tailed probability value 0.05 was considered significant. All analyses were performed using SPSS for Windows (Version 11.0, SPSS, Chicago, Ill). Results Baseline Characteristics The distribution of the CPS1 genotypes was in Hardy- Weinberg equilibrium and similar to that reported previously 12 (CC:AC:AA 40:54:12 38%:51%:11%, Table 1). Although the frequency of the CC genotype tended to be decreased among the black Americans studied (CC:A- C:AA 9:18:3 30%:60%:10%) compared with the whites Americans studied (CC:AC:AA 31:36:9 41%:47%:12%), the difference was not statistically significant (P 0.086). CPS1 genotype significantly affected resting FVR (Table 1, P 0.023). There were no significant differences in other baseline characteristics among the 3 genotype groups. Urea Cycle Intermediates and Nitric Oxide Metabolites There was no effect of CPS1 genotype on plasma L-arginine or L-citrulline concentration, or the ornithine to citrulline (O/C) ratio, which varies inversely with CPS1 activity (Table 2). There was a significant relationship between CPS1 genotype and nitric oxide metabolites, measured before and during the 400-ng/min dose of bradykinin, such that nitric oxide metabolite concentrations were highest in subjects who were homozygous for the C allele, lowest in those subjects ho-

3 188 Hypertension February 2004 TABLE 1. Subject Characteristics Parameter CC (n 40) AC (n 54) AA (n 12) Age, y Gender, M:F 22:18 29:25 9:3 Race, W:B 31:9 36:18 9:3 BMI, kg/m SBP, mm Hg DBP, mm Hg Heart rate, bpm Cholesterol, mg/dl Smoker, N:Y 30:10 41:13 10:2 FBF, mlmin/100 ml FVR, mm Hg/ml/min/100 ml * Values are means SD. CC indicates homozygosity for the C-encoded Thr1405 variant; AA, homozygosity for the A-encoded Asn1405 variant; AC, heterozygosity for this polymorphism at position 1405; FBF, forearm blood flow; FVR, forearm vascular resistance; BMI, body mass index; SBP, systolic blood pressure; and DBP, diastolic blood pressure. *P 0.05 vs CC homozygotes. TABLE 2. Plasma Amino Acid, Nitric Oxide Metabolites, and Cyclic GMP Concentrations Parameter CC (n 39) AC (n 50) AA (n 10) L-arginine, M L-citrulline, M Ornithine to citrulline ratio Nitric oxide metabolite basal, M Nitric oxide metabolite * during bradykinin, M cgmp, pmol/ml Values are means SD. CC indicates homozygosity for the C-encoded Thr1405 variant; AA, homozygosity for the A-encoded Asn1405 variant; and AC, heterozygosity for this polymorphism. *P 0.05 vs CC homozygotes. Figure 1. A, Effect of the NOS-dependent vasodilator bradykinin and on forearm blood flow (FBF) according to carbamoylphosphate synthetase 1 (CPS1) genotype. B, Effect of the nitric oxide donor sodium nitroprusside on forearm blood flow (FBF) according to carbamoyl-phosphate synthetase 1 (CPS1) genotype. CC indicates homozygosity for the C-encoded Thr1405 variant; AA, homozygosity for the A-encoded Asn variant; and AC, heterozygosity for this polymorphism at position There was a significant effect of CPS1 genotype on FBF during both bradykinin (P 0.028) and sodium nitroprusside (P 0.022). mozygous for the A allele, and intermediate in heterozygotes (P 0.036). Venous nitric oxide metabolite concentrations did not increase during bradykinin (P 0.224), as expected, because of the dilutional effect of forearm vasodilation. There was no effect of CPS1 genotype on venous cgmp concentration (P 0.368). Plasma L-arginine and L-citrulline concentrations correlated with each other (correlation coefficient 0.389, P 0.001), but neither L-arginine nor L-citrulline concentration correlated with nitric oxide metabolites. There was no correlation between L-arginine or L-citrulline concentration and venous cgmp concentration; however, the O/C ratio correlated inversely with cgmp (correlation coefficient 0.394, P 0.001). Table 3 details the effects of ethnicity, gender, and smoking status on L-arginine and L-citrulline concentrations, O/C ratio, nitric oxide metabolites, and venous cgmp concentration. Because both CPS1 genotype and ethnicity affected nitric oxide metabolite concentrations, we analyzed the effect of CPS1 genotype separately in the white and black Americans studied. The relationship between genotype and nitric oxide metabolites was similar in the whites (eg, nitric oxide metabolites during bradykinin in CC , AC , and AA mol/l; P 0.076) and blacks (CC , AC , and AA mol/l; P 0.284) studied. Agonist-Stimulated Vasodilation Figure 1 shows the relationship between agonist-stimulated vasodilation and CPS1 genotype. Both bradykinin (P 0.001) and SNP (P 0.001) induced dose-dependent increases in FBF. There was a significant effect of CPS1 genotype on FBF during bradykinin (P 0.028, Figure 1), such that the vasodilator response was greatest in those individuals who were homozygous for the C allele, least in those homozygous for the A allele and intermediate in heterozygotes. Similarly, during SNP, FBF was diminished in those homozygous for the A allele compared with AC heterozygotes and AA homozygotes (P 0.022). The FBF response to bradykinin (P 0.004) and SNP (P 0.033) was significantly greater in white Americans compared with black Americans; however, inclusion of CPS1 genotype in the analysis abolished the effect of ethnicity on the FBF response to both vasodilators (P for an effect of ethnicity on the FBF response to either agonist). Table 4 provides the effect of CPS1 genotype on the vasodilator responses to bradykinin and SNP separately within ethnic group, gender group, and smokers versus nonsmokers (who may have endothelial dysfunction). 14 The trend toward the lowest FBF response to bradykinin and SNP in AA homozy-

4 Summar et al CPS1 Genotype and Vasodilation 189 TABLE 3. Effect of Sex, Ethnicity, and Smoking Status on Amino Acid Concentrations, Nitric Oxide Metabolites, and Cyclic GMP Parameter L-arginine, M L-citrulline, M Ornithine to Citrulline Ratio Nitric Oxide Metabolites Basal, M cgmp Basal, pmol/ml Gender Men Women * Ethnicity White Black Smoking status Nonsmoker Smoker Values are means SD. *P 0.05, P 0.01 vs men; P 0.05 vs whites; P 0.05 vs nonsmokers. gotes was similar across all groups. There was a significant effect of CPS1 on the percent change in FVR during bradykinin (P 0.003) but not SNP (P 0.072, Table 5). Tissue-type plasminogen activator (t-pa) release increased in a dose-dependent manner in response to bradykinin (P 0.001). However, there was no effect of the CPS1 genotype on bradykinin-stimulated t-pa release (Figure 2). TABLE 4. Effect of CPS1 Genotype on Forearm Blood Flow Within Subgroups Mean FBF During Bradykinin Parameter CC AC AA Gender Men * Women Ethnicity White Black Smoking status Nonsmoker Smoker Mean FBF During Sodium Nitroprusside CC AC AA Gender Men Women Ethnicity White Black Smoking status Nonsmoker * Smoker *P 0.01 vs CC homozygotes; P 0.05 vs CC homozygotes; P 0.05 vs heterozygotes. Discussion The present study examines the relationship between a C-to-A nucleotide transversion in exon 36 of the gene that encodes CPS1, the rate-limiting enzyme in the urea cycle production of L-citrulline, 12 and agonist-stimulated vasodilation. The data indicate that the CPS1 polymorphism significantly influences venous nitric oxide concentrations and both NOS-dependent and -independent vasodilation. On the one hand, the significant relationship between CPS1 genotype and venous nitric oxide metabolite concentrations supports the hypothesis that CPS1 genotype affects L citrulline production and cycling to L-arginine with subsequent nitric oxide production. Compatible with this hypothesis, CPS1 genotype did not affect the t-pa response to bradykinin, an endothelium-dependent but NOS-independent effect of the agonist. 13 On the other hand, there were no significant differences in L-arginine concentrations among genotype groups. Moreover, the major finding of the study that CPS1 genotype influenced the vasodilator response to both the endothelium-dependent vasodilator bradykinin and the endo- TABLE 5. Effect of CPS1 Genotype on the Percent Change in Forearm Vascular Resistance in Response to Agonists Percent Change in Forearm Vascular Resistance Dose CC AC AA Bradykinin 100 ng/min * 200 ng/min ng/min Sodium nitroprusside 1.6 g/min g/min g/min Values are means SD. CC indicates homozygosity for the C-encoded Thr1405 variant; AA, homozygosity for the A-encoded Asn1405 variant; and AC, heterozygosity for this polymorphism. *P vs CC homozygotes, P 0.05 vs heterozygotes, P 0.05 vs CC homozygotes.

5 190 Hypertension February 2004 Figure 2. Effect of bradykinin on t-pa release according to carbamoyl-phosphate synthetase 1 (CPS1) genotype. CC indicates homozygosity for the C-encoded Thr1405 variant; AA, homozygosity for the A-encoded Asn variant; and AC, heterozygosity for this polymorphism at position thelium-independent vasodilator SNP suggests that CPS1 genotype influences vascular smooth muscle reactivity directly. Given that shear force induces nitric oxide synthesis, 15 an alternative interpretation of the data regarding the relationship between CPS1 genotype and venous nitric oxide metabolite concentrations is that, in individuals homozygous for the C allele, increased nitric oxide concentrations were the consequence of, not the cause of, increased flow. The present study does not address the specific mechanism through which CPS1 T1405N genotype influences vascular smooth muscle reactivity. In addition to serving as a source of L-arginine for nitric oxide production, L-citrulline relaxes vascular smooth muscle through particulate guanylate cyclase. 16 However, although cgmp concentrations varied inversely with the O/C ratio, suggesting a relationship between cgmp and urea cycle efficiency, CPS1 genotype did not affect venous L-citrulline concentration, O/C ratio, or cgmp concentration. The disparity between the significant effect of CPS1 genotype on agonist-stimulated vasodilation and nitric oxide metabolite concentrations and the lack of effect of genotype on L-citrulline and cgmp concentrations may relate to methodological limitations in measuring urea cycle intermediates and second messengers. For example, circulating L-citrulline concentrations reflect a complex balance between de novo production, primarily via the urea cycle in the liver, 10 and metabolism to L-arginine, primarily in the kidney and in the vasculature. 17 Similarly, cgmp may be produced by stimulation of either soluble or particulate guanylate cylcase, and circulating concentrations of cgmp may not accurately reflect intracellular concentrations. 18 Likewise, nitric oxide metabolite concentrations may be influenced by dietary nitrate intake. 19 The lack of effect of CPS1 T1405N genotype on circulating L-citrulline concentrations in the present study confirms our prior observation in neonates. 12 In contrast, the lack of effect of CPS1 genotype on L-arginine concentrations in the present study contradicts our earlier observation that plasma L-arginine concentrations were significantly higher in infants homozygous for the A allele. 12 The difference in the ages of the two populations may underlie these divergent observations regarding the effect of CPS1 genotype on L-arginine concentrations. Whereas plasma L-arginine and L-citrulline concentrations measured in the present study were similar to those reported previously in young adults, 20 the L-arginine concentration was significantly lower (mean 30.5 mol/l) in the neonates studied. A limitation of the present study is the heterogeneity of the population with respect to ethnicity and gender, as well as smoking status. Conversely, analysis of the relationship between CPS1 genotype and agonist-stimulated vasodilation within subgroups provides the opportunity to generate new hypotheses. For example, African American race has been associated with impaired NOS-mediated as well as NOSindependent vasodilation. 21,22 Some investigators have proposed that ethnic differences in NOS-dependent vasodilation may result from interethnic differences in the distribution of enos variants. For example, a 4a variant in intron 4 of enos, which has been associated with decreased plasma NO metabolite concentrations, 23 is more prevalent in blacks than in whites. 24 The present study provides preliminary evidence that differences in distribution of CPS1 genotypes could also contribute to ethnic differences in NOS-dependent vasodilation. Thus, inclusion of CPS1 genotype in the analysis of agonist-stimulated vasodilation abolished the previously described ethnic differences in the FBF response to bradykinin and SNP. Additional large population studies are needed to determine the effect of ethnicity on the distribution of CPS1 genotype. Perspectives This present study is the first to examine the relationship between a C-to-A nucleotide transversion (T1405N) in the gene-encoding CPS1, the protein catalyzing the ratelimiting step in L-citrulline production by the urea cycle, and nitric oxide metabolite concentrations and vascular function. Although there was no effect of CPS1 genotype on plasma L-citrulline or L-arginine concentrations there was a significant effect of genotype on venous nitric oxide metabolites and on the vasodilation response to both the NOS-dependent vasodilator bradykinin and the NO-donor SNP. Because vascular function predicts the risk of subsequent cardiovascular events, 25 studies are needed to confirm this association in additional populations and to determine if CPS1 genotype predicts cardiovascular morbidity and mortality. Acknowledgments This research was funded by National Institutes of Health grants R01HL65193, R01HL60906, R01HL67308, T32GM007569, K23HL04445, and M01RR References 1. Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;333: Gewaltig MT, Kojda G. Vasoprotection by nitric oxide: mechanisms and therapeutic potential. 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