This Article Abstract Full Text (PDF) All Versions of this Article: 108/12/1425    most recent 01.CIR.0000091255.63645.98v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakamura, S.-i. Articles by Kugiyama, K. Search for Related Content PubMed PubMed Citation Articles by Nakamura, S.-i. Articles by Kugiyama, K. Pubmed/NCBI databases Gene GEO Profiles HomoloGene OMIM UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE Related Collections Endothelium/vascular type/nitric oxide

(Circulation. 2003;108:1425.)
© 2003 American Heart Association, Inc.

Brief Rapid Communications

Polymorphism in Glutamate-Cysteine Ligase Modifier Subunit Gene Is Associated With Impairment of Nitric Oxide–Mediated Coronary Vasomotor Function

Shin-ichi Nakamura, MD, PhD; Seigo Sugiyama, MD, PhD; Daisuke Fujioka, MD; Ken-ichi Kawabata, MD, PhD; Hisao Ogawa, MD, PhD; Kiyotaka Kugiyama, MD, PhD

From the 2nd Department of Internal Medicine (S.-i.N., D.F., K.-i.K., K.K.), Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan; and the Department of Cardiovascular Medicine (S.-i.N., S.S., H.O.), Graduate School of Medical Sciences, Kumamoto University, Japan.

Correspondence to Kiyotaka Kugiyama, MD, PhD, 2nd Department of Internal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Nakakoma-gun, Yamanashi, 409-3898 Japan. E-mail kugiyama{at}yamanashi.ac.jp

Received June 4, 2003; revision received July 29, 2003; accepted July 30, 2003.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— The minor -588T allele of polymorphism -588C/T of a modifier subunit gene in glutamate-cysteine ligase (GCLM), a rate-limiting enzyme for glutathione (GSH) synthesis, was associated with lower plasma GSH levels and was a risk factor for myocardial infarction.

Methods and Results— We examined effects of the -588C/T polymorphism on coronary arterial diameter and blood flow responses to intracoronary infusion of acetylcholine in 157 consecutive subjects who had normal coronary angiograms. In multivariate linear regression analysis with covariates including traditional risk factors, the minor -588T allele had an independent association with impaired dilation or enhanced constriction of epicardial coronary arteries in response to acetylcholine, and it was independently associated with blunted increase in coronary flow response to acetylcholine. In a subgroup of 59 consecutive subjects, constrictor responses of epicardial coronary diameter to intracoronary infusion of N^G-monomethyl-L-arginine, reflecting the presence of coronary nitric oxide (NO) bioactivity, had an inverse and independent association with the -588T allele in multivariate analysis.

Conclusions— The -588T polymorphism of the GCLM gene causes a decrease in endothelial NO bioactivity, leading to impairment of endothelium-dependent vasomotor function in large and resistance coronary arteries. The GCL–GSH–NO axis may play a role in the defense system against coronary artery disease.

Key Words: antioxidants • genetics • nitric oxide • acetylcholine

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Exposure to oxidants may initiate an adaptive intracellular antioxidant response, such as induction of antioxidant genes. Glutathione (GSH), tripeptide thiols, is a major and naturally occurring antioxidant, and it has a predominant role in the regulation of intracellular redox state and protects cells from oxidative injury.¹ Previous in vitro studies demonstrated that GSH depletion inhibits nitric oxide (NO) production in endothelial cells.² Furthermore, we and others have shown that supplementation of GSH or its precursor improved endothelial vasomotor dysfunction in patients with coronary risk factors in which oxidative stress has a pathogenic role.^3,4

Glutamate-cysteine ligase (GCL) is a rate-limiting enzyme for GSH synthesis.^1,5–7 GCL is a heterodimer composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). GCLM has a physiologically important regulatory function. Recently, we found polymorphism -588C/T in the 5'-flanking region of the GCLM gene.⁸ The minor -588T allele of the polymorphism suppresses oxidant-induced upregulation of GCLM gene expression and is associated with lower plasma GSH levels, and this polymorphism is a genetic risk factor for myocardial infarction (MI).⁸ However, mechanisms by which this polymorphism is linked to the genesis of MI remain undefined. The present study thus examined whether this polymorphism may be implicated in coronary endothelial vasomotor dysfunction, which plays an important role in the pathogenesis of coronary artery disease.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Study Subjects
This study enrolled 157 consecutive subjects who had quantitative coronary angiography with intracoronary injection of acetylcholine (ACh) at Kumamoto University Hospital. All of the subjects were ethnic Japanese and underwent diagnostic cardiac catheterization for evaluation of atypical chest pain. All of the 157 subjects had angiographically documented normal coronary arteries (<10% stenosis), normal ventriculography, and no coronary spasm during intracoronary infusion of ACh. No subject had previous MI, congestive heart failure, cardiomyopathy, valvular heart disease, left ventricular hypertrophy, or other serious diseases. These study subjects included most of those examined in our previous study.⁹ All of the subjects gave written, informed consent for this study and genetic analysis. The study protocol followed the national guidelines for genetic analysis in Japan and was approved by the ethics committee at Kumamoto University Hospital.

Study Protocol
After baseline angiography, incremental doses of ACh (10, 50, and 100 µg/min) were infused directly into the left coronary artery through the Judkins catheter for 2 minutes, as described previously.^3,9 Fifteen minutes after the ACh infusion, incremental doses of N^G-monomethyl-L-arginine (L-NMMA; 25 and 50 µmol/min, each for 4 minutes), an inhibitor of NO synthase, were infused into the left coronary artery through the Judkins catheter in a subgroup of 59 consecutive subjects. Subsequently, L-NMMA (50 µmol/min) was infused for an additional 5 minutes into the left coronary artery, and at the last minute of the L-NMMA infusion, 50 µg/min of ACh was simultaneously injected into the left coronary artery in the same manner as before the L-NMMA infusion.⁹ Finally, intracoronary injection of isosorbide dinitrate (1 mg) was given. Hemodynamic measurements and coronary angiography were repeated before and at each of the infusions.

Quantitative Coronary Angiography and Measurement of Coronary Blood Flow
A quantitative coronary angiographic study was performed in all of the 157 subjects in the same manner, as described.^3,9 The lumen diameter at the center of the left anterior descending coronary artery (LAD) was measured quantitatively by two observers who were blinded to the clinical data of the study subjects.

Blood flow velocity in the proximal segment of the LAD was measured in a subgroup of 99 consecutive subjects using a 0.014-inch wire equipped with a Doppler crystal at its tip, as described previously.^3,9 Responses of coronary artery diameter and blood flow to the infusion of the drugs were expressed as percentage changes from baseline coronary diameter and blood flow, respectively, which were measured just before each infusion.

Genotyping
Genomic DNA was extracted from peripheral blood lymphocytes. Genotyping was performed by a polymerase chain reaction–based method of restriction fragment length polymorphism with forward (5'-CTCAAGGGCAAAGACTCA-3') and reverse (5'-CCGCCTGGTGAGGTAGACAC-3') primers, as described previously.⁸

Statistical Analysis
Data are expressed as mean (±SEM) unless otherwise indicated. For comparison of coronary responses between subjects with and without the -588T allele, 2-way analysis of variance for repeated measures was used with Bonferroni’s multiple-comparison tests. Analyses for the associations were performed using the multivariate linear regression technique, with coronary arterial responses as the dependent variable. Of the independent variables, age, body mass index (kg/m²), and total cholesterol levels (mg/dL) were treated as continuous variables. Other independent variables, including male sex, smoking (>10 cigarettes per day for 1 year), hypertension (defined as blood pressure 140/90 mm Hg or as taking an antihypertensive medication), diabetes mellitus (defined according to the American Diabetes Association report or as taking an antidiabetic medication), and the -588T polymorphism, were treated as categorical variables. Statistical significance was defined as P<0.05. Statistical analysis was performed with StatView 5.0 (SAS Institute).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Genotypes of Study Subjects
The -588TT, CT, and CC genotypes were present in 1 (0.6%), 34 (22%), and 122 (78%) of 157 subjects studied, respectively. The frequencies of traditional coronary risk factors were comparable between subjects with and without the -588T allele (data not shown).

Responses of Epicardial Coronary Diameter and Coronary Blood Flow to ACh
Subjects with CT and TT genotypes had an impaired dilation or enhanced constriction of epicardial coronary arteries and a blunted increase in coronary blood flow in response to ACh infusion, as compared with those with the CC genotype, as shown in Figure 1. The dilator response of epicardial coronary arteries to nitrate was comparable between subjects with and without the -588T allele (26±4% in subjects with CC genotype versus 25±4% in those with CT and TT genotypes; P=NS). In multivariate linear regression analysis, the -588T allele was independently associated with constrictor response of epicardial coronary diameter and with impairment of coronary flow increase in response to ACh at all of doses among the covariates including traditional risk factors (diameter response to 50 µg/min of ACh: standardized regression coefficient=-0.34; P<0.001; flow response to 10 µg/min of ACh: standardized regression coefficient=-0.39; P<0.01). Among the covariates with a significant association, the association rank-ordered as follows: (1) for diameter response to 50 µg/min of ACh: age > -588T polymorphism > smoking; and (2) for flow response to 10 µg/min of ACh: -588T polymorphism > diabetes > total cholesterol levels.

View larger version (23K): [in this window] [in a new window]   Figure 1. Left, Percent changes (mean±SEM) from baseline in epicardial diameter of the LAD in response to intracoronary infusion of ACh. Right, Percent changes (mean±SEM) from baseline in coronary blood flow response to ACh infusion.

Responses of Epicardial Arterial Diameter to L-NMMA
Subjects with CT and TT genotypes had less constrictor response to intracoronary infusion of L-NMMA than did those with CC genotype, as shown in Figure 2. In multivariate linear regression analysis, the -588T allele was independently associated with decrease in diameter response to L-NMMA at both doses among the covariates (response to 50 µmol/min of L-NMMA: standardized regression coefficient=0.42; P<0.01). The simultaneous infusion of L-NMMA with ACh augmented the constrictor response of epicardial coronary arteries to ACh (50 µg/min) in subjects with CC genotype, whereas the simultaneous L-NMMA infusion had minimum effect on the constrictor response to ACh in those with CT and TT genotypes, as shown in Fig-ure 2.

View larger version (24K): [in this window] [in a new window]   Figure 2. Left, Percent changes (mean±SEM) from baseline in epicardial diameter of the LAD in response to intracoronary infusion of L-NMMA alone. Right, Percent changes (mean±SEM) from baseline in the epicardial coronary diameter in response to ACh alone (50 µg/min) and combined infusion of ACh (50 µg/min) and L-NMMA (50 µmol/min).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study indicated that the -588T polymorphism of the GCLM gene had a significant and independent association with abnormal vasomotor reactivity to ACh in large and resistance coronary arteries. The epicardial coronary diameter response to nitrates was not significantly different between subjects with and without the T allele. Thus, the -588T polymorphism of GCLM gene has an important and causative role in impairment of endothelium-dependent vasomotor function in large and resistance coronary arteries. The present study further showed that the constrictor response of epicardial coronary arteries to L-NMMA at basal conditions, reflecting basal coronary NO bioactivity, was independently decreased with the presence of the -588T polymorphism. Furthermore, the combined infusion of L-NMMA did not significantly augment the constrictor response of epicardial diameter to ACh in subjects with the T allele. Thus, these results suggest that the -588T polymorphism of the GCLM gene may cause a decrease in the basal and stimulated release of endothelial NO, leading to impairment of endothelium-dependent dilation and enhancement of constriction in response to ACh in coronary arteries.

It has been shown that GSH has a central role in protection of endothelial cells from oxygen free radicals and preservation of endothelium-derived NO in arteries exposed to oxidative stress.^1–7 When cells are exposed to oxidative stress, GSH synthesis is increased through upregulation of GCL gene expression, providing a protective/adaptive mechanism against oxidative stress.^1,5–7 However, we have previously shown that the -588T polymorphism suppresses the increase in GCLM gene expression in response to oxidative stress and that this polymorphism is associated with low plasma GSH concentration.⁸ Therefore, the -588T polymorphism may weaken the intracellular production of GSH in response to oxidative stress, leading to an increase in the susceptibility to impairment of NO-mediated endothelial vasomotor function. In addition, endothelium-derived NO has various antiatherothrombogenetic properties. Thus, a decrease in NO bioactivity due to the blunted GSH induction may partly play a role in the genesis of coronary artery disease in patients with the -588T polymorphism of the GCLM gene. The associations in the present study were, however, marginal and based on a small population size. Although it is necessary to replicate our findings in a larger number of patients, the -588T polymorphism of GCLM gene could have a causative role in oxidant-induced vascular dysfunction.

The present data are consistent with our previous report showing that polymorphism in the GCLC gene is also associated with coronary vasomotor dysfunction and MI.¹⁰ Thus, the previous and present data support our hypothesis that the GCL–GSH–NO axis may act as a defense system against oxidant-induced cardiovascular complications.

	Acknowledgments

 
This study was supported by grants-in-aid for (B) (2)-15390244, Priority Areas (C) "Medical Genome Science 15012222" from the Ministry of Education, Culture, Sports, Science, and Technology; H15-Chozy-012 Health and Labor Sciences Research Grants for Comprehensive Research on Aging and Health; the Smoking Research Foundation; and Arteriosclerosis Prevention Fund, Tokyo, Japan.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983; 52: 711–760.[CrossRef][Medline] [Order article via Infotrieve]
   
2. Murphy ME, Piper HM, Watanabe H, et al. Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment. J Biol Chem. 1991; 266: 19378–19383.[Abstract/Free Full Text]
   
3. Kugiyama K, Ohgushi M, Motoyama T, et al. Intracoronary infusion of reduced glutathione improves endothelial vasomotor response to acetylcholine in human coronary circulation. Circulation. 1998; 97: 2299–2301.[Abstract/Free Full Text]
   
4. Vita JA, Frei B, Holbrook M, et al. L-2-Oxothiazolidine-4-carboxylic acid reverses endothelial dysfunction in patients with coronary artery disease. J Clin Invest. 1998; 101: 1408–1414.[Medline] [Order article via Infotrieve]
   
5. Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke–induced airway disease. Am J Pathol. 1999; 277: L1067–L1088.
   
6. Cho S, Hazama M, Urata Y, et al. Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells. Free Radic Biol Med. 1999; 26: 589–602.[CrossRef][Medline] [Order article via Infotrieve]
   
7. Moinova HR, Mulcahy RT. An electrophile responsive element (EpRE) regulates -naphthoflavone induction of the human -glutamylcysteine synthetase regulatory subunit gene. J Biol Chem. 1998; 273: 14683–14689.[Abstract/Free Full Text]
   
8. Nakamura S, Kugiyama K, Sugiyama S, et al. Polymorphism in the 5'-flanking region of human glutamate-cysteine ligase modifier subunit gene is associated with myocardial infarction. Circulation. 2002; 105: 2968–2973.[Abstract/Free Full Text]
   
9. Kugiyama K, Doi H, Motoyama T, et al. Association of remnant lipoprotein levels with impairment of endothelium-dependent vasomotor function in human coronary arteries. Circulation. 1998; 97: 2519–2526.[Abstract/Free Full Text]
   
10. Koide S, Kugiyama K, Sugiyama S, et al. Association of polymorphism in glutamate-cysteine ligase catalytic subunit gene with coronary vasomotor dysfunction and myocardial infarction. J Am Coll Cardiol. 2003; 41: 539–545.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				A.-L. Levonen, E. Vahakangas, J. K. Koponen, and S. Yla-Herttuala Antioxidant Gene Therapy for Cardiovascular Disease: Current Status and Future Perspectives Circulation, April 22, 2008; 117(16): 2142 - 2150. [Abstract] [Full Text] [PDF]

				L. A. McConnachie, I. Mohar, F. N. Hudson, C. B. Ware, W. C. Ladiges, C. Fernandez, S. Chatterton-Kirchmeier, C. C. White, R. H. Pierce, and T. J. Kavanagh Glutamate Cysteine Ligase Modifier Subunit Deficiency and Gender as Determinants of Acetaminophen-Induced Hepatotoxicity in Mice Toxicol. Sci., October 1, 2007; 99(2): 628 - 636. [Abstract] [Full Text] [PDF]

				G. Dai, S. Vaughn, Y. Zhang, E. T. Wang, G. Garcia-Cardena, and M. A. Gimbrone Jr Biomechanical Forces in Atherosclerosis-Resistant Vascular Regions Regulate Endothelial Redox Balance via Phosphoinositol 3-Kinase/Akt-Dependent Activation of Nrf2 Circ. Res., September 28, 2007; 101(7): 723 - 733. [Abstract] [Full Text] [PDF]

				G. Giordano, C. C. White, L. A. McConnachie, C. Fernandez, T. J. Kavanagh, and L. G. Costa Neurotoxicity of Domoic Acid in Cerebellar Granule Neurons in a Genetic Model of Glutathione Deficiency Mol. Pharmacol., December 1, 2006; 70(6): 2116 - 2126. [Abstract] [Full Text] [PDF]

				D. Botta, S. Shi, C. C. White, M. J. Dabrowski, C. L. Keener, S. L. Srinouanprachanh, F. M. Farin, C. B. Ware, W. C. Ladiges, R. H. Pierce, et al. Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice J. Biol. Chem., September 29, 2006; 281(39): 28865 - 28875. [Abstract] [Full Text] [PDF]

				Y. Chen, H. G. Shertzer, S. N. Schneider, D. W. Nebert, and T. P. Dalton Glutamate Cysteine Ligase Catalysis: DEPENDENCE ON ATP AND MODIFIER SUBUNIT FOR REGULATION OF TISSUE GLUTATHIONE LEVELS J. Biol. Chem., October 7, 2005; 280(40): 33766 - 33774. [Abstract] [Full Text] [PDF]

				S. Kann, C. Estes, J. F. Reichard, M.-y. Huang, M. A. Sartor, S. Schwemberger, Y. Chen, T. P. Dalton, H. G. Shertzer, Y. Xia, et al. Butylhydroquinone Protects Cells Genetically Deficient in Glutathione Biosynthesis from Arsenite-Induced Apoptosis Without Significantly Changing Their Prooxidant Status Toxicol. Sci., October 1, 2005; 87(2): 365 - 384. [Abstract] [Full Text] [PDF]

				Y. Ichigi, H. Takano, K. Umetani, K. Kawabata, J.-e. Obata, Y. Kitta, Y. Kodama, A. Mende, T. Nakamura, D. Fujioka, et al. Increased Ambulatory Pulse Pressure Is a Strong Risk Factor for Coronary Endothelial Vasomotor Dysfunction J. Am. Coll. Cardiol., May 3, 2005; 45(9): 1461 - 1466. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 108/12/1425    most recent 01.CIR.0000091255.63645.98v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakamura, S.-i. Articles by Kugiyama, K. Search for Related Content PubMed PubMed Citation Articles by Nakamura, S.-i. Articles by Kugiyama, K. Pubmed/NCBI databases Gene GEO Profiles HomoloGene OMIM UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE Related Collections Endothelium/vascular type/nitric oxide