NOS3 gene encodes the enzyme endothelial nitric oxide synthase (eNOS), which catalyzes the formation of endogenous nitric oxide (NO) from the amino acid L-arginine (Fig. 1).
Uncharged and ubiquitously present, the NO gas is a bioactive signaling molecule in human body. It is best known as a vasodilator thanks to the mechanistic studies of many blood pressure control medicines that function through the NO signaling pathways. Examples of these medicines include the widely used nitroglycerin against heart attack and the well known Viagra against erectile dysfunction. In addition, NO is also involved in the inhibition of platelets aggregation and adhesion, neuronal signal transmission, cytotoxicity against pathogens and tumors, coordination of heart rhythm and the regulation of cellular respiration activity.
Imbalance of the NO level leads to endothelial dysfunctions and many other diseases. On one hand, reduction in basal NO release may predispose to hypertension, pre-eclampsia, thrombosis, vasospasm, atherosclerosis, and metabolic syndrome. On the other hand, high NO levels in circulating blood are associated with endotoxic shock, inflammation, acute hepatic dysfunction, glomerulonephritis, asthma, cardiomyopathy and a number of other disorders.
NOS3 encoded eNOS enzyme is critical for the NO level regulation in the cardiovascular system. Knockout mice deficient in eNOS developed hypertension as adults while transgenic mice over-expressing eNOS reduced blood pressure. Meanwhile, the eNOS+/- mice that have only one copy of the NOS3 gene are healthy under normal conditions but develop hypertension and insulin resistance under dietary stress such as high fat diet.
There are three common polymorphisms in the NOS3 gene that are associated with hypertension and other cardiovascular diseases: T-786C; VNTR 4b/a; and Glu298Asp. The -786C and the Asp298 minor alleles are more common in Caucasians while the 4a minor allele is more common in African-Americans (Table 1). In African-Americans and Caucasians, 54% of the population carries at least one of the three minor alleles. In Asians, about 23% of the population carries at least one of the minor alleles.
The eNOS catalyzed NO biosynthesis is one of the most complicated reactions, requiring three co-substrates (L-arginine, NADPH, and O2) and five cofactors (flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin (CaM), heme and tetrahydrobiopterin(BH4)). Many dietary factors regulate eNOS promoted NO production by modulating the bioavailability of these co-substrates and cofactors. For example, the bioavailability of BH4, NADPH, FAD and FMN is regulated by redox balance, which is modulated by antioxidants found in fruits and vegetables. The biosynthesis of FAD and FMN is also depending on their precursor vitamin B2. Three dietary minerals are directly involved in eNOS function: Fe2+ regulates the function of heme; Ca2+ regulates the function of CaM; and Zn2+ is directly involved in the interaction of eNOS and BH4.
Among the known dietary factors that regulate eNOS activity and NO production, significant interactions between sodium intake, antioxidants and fatty acids with the three common NOS3 polymorphisms have been reported. Dietary sodium regulates blood pressure and kidney function through the renin–angiotensin system that is described in the ACE and Blood Pressure review. A down-stream target of the renin–angiotensin system, bradykinin, regulates blood vessel dilatation through the NO signaling pathway. High sodium level normally induced high blood pressure. But for the -786C and the 4a minor allele carriers, the degree of blood pressure increase in responding to high sodium is much greater. Antioxidants compensate the lower eNOS level in the -786C minor allele and the Asp298 minor allele carriers. It has been proposed that ω-3 polyunsaturated fatty acids improves the membrane localization of the defective Asp298 enzyme, supported by the observation that blood triglycerides reduction by ω-3 polyunsaturated fatty acids supplementation was dramatic in the Asp298 minor allele carriers was not significant in the homozygous Glu/Glue genotype individuals. Therefore, low sodium, high antioxidants (from fruits and vegetables) and increased ω-3 fatty acids are especially important for the minor allele carriers of these three polymorphisms to maintain an optimal eNOS function. L-arginine, the nitrogen donor in the NO production reaction, is generally abundant in protein rich diet and can be readily converted from other amino acids in human body. It is normally sufficient for the eNOS function. L-arginine supplement is NOT necessary unless recommended by medical doctors for clinically diagnosed symptoms.
Cigarette smoking induced stresses reduce the bioavailability of NO and impair the vascular dilatation function of eNOS. It also interact with NOS3 polymorphisms by exacerbating the harmful effects in the minor allele carriers. A range of 2- to 4-fold increased risks for smoking induced coronary heart disease have been reported for all of the three minor allele carriers, for whom it is strongly recommended to stay away from smoking.
In responding to physical exercise, minor allele carriers generally do not get as much health benefits as major allele genotypes carriers do. One exception is the male -786C allele carriers who response more favorably to aerobic exercise than the homozygous major allele 786TT genotype. Therefore, for most of the minor allele carriers (except -786C male), keep physical exercise at moderate level is recommended.
Taking all the information together, a guideline for NOS3 polymorphism specific diet regimen recommends (Table 2):
NOS, eNOS and NO signaling
The eNOS is the principle enzyme responsible for NO production in cardiovascular system. There are three NOSs in human body. The same reaction illustrated in Fig. 1 can also be catalyzed by the other two: the neuronal (nNOS) and induced (iNOS) ones. These three NOSs are encoded by three different genes and are distinguished by their tissue distribution, physiological function and the mechanisms of regulation (Table 3). In brief, eNOS and nNOS are expressed constitutively and their activities are regulated by calcium through the calmodulin (CaM) interaction while the iNOS is inducible in response to pathogen infection and its activity is calcium-independent.
Table 3. A comparison of the three nitric oxide synthase genes in human.
| Gene |
NOS3 |
NOS1 |
NOS2 |
| Protein |
eNOS (Endothelial NOS) |
nNOS (Neuronal NOS) |
iNOS (Inducible NOS) |
| Tissue Distribution |
Endothelium, cardiac myocytes, blood platelets, mast cells, erythrocytes, and leukocytes. |
Neurons, skeletal muscle cells, Endothelial cells |
Immune system, cardiovascular system, heptocytes, chondrocytes, keratinocytes, respiratory epithelia,
and macrophages |
| Physiological Function |
Vasodilatation |
Neurotransmitter |
Inflammatory response |
| Chromosomal location |
7q35-36 |
12q24.2 |
17cen-q12 |
| Expression |
Constitutive |
Constitutive |
Inducible |
| Enzyme Activity |
Ca2+ dependent |
Ca2+ dependent |
Ca2+ independent |
In the cardiovascular system, eNOS is critical for the blood NO level control, thus the vascular tone maintenance. Knockout mice deficient in eNOS developed hypertension as adults (Huang et al, 1995). In contrast, transgenic mice over-expressing bovine eNOS reduced blood pressure (Ohashi et al, 1995). In a mouse model, it was found that when fed a low fat (12%) diet, eNOS+/- mice had normal insulin sensitivity and were normotensive. When fed a high fat (72%) diet for 8 weeks, however, eNOS+/- mice developed exaggerated high blood pressure and insulin resistance. Therefore, one eNOS gene provides sufficient eNOS protein expression and activity to maintain normal insulin sensitivity and arterial blood pressure under normal conditions. However, during a metabolic stress such as high fat diet, eNOS deficiency amplified a pathological mechanism observed under normal conditions and led to exaggerated insulin resistance and arterial hypertension (Cook et al, 2004).
In human body, eNOS is constitutively expressed in the epithelium. Post-translational myristoylation and palmitoylation enable the eNOS to localize in caveolae of endothelial cells. Caveolae are distinct plasma membrane structures featured by the abundance of a scaffolding protein called caveolin and diverse receptors and signaling molecules from a variety of signal transduction pathways, including G protein coupled receptors, G proteins, growth factor receptors, and calcium regulatory proteins. Localization of eNOS in caveolae serves to communicate signals between different pathways. Two sequential enzymes argininosuccinate lyase and argininosuccinate synthase, responsible for recycling citrulline to arginine, are also co-localized with eNOS in the caveolae. The membrane anchored eNOS interacts with caveolin and stays in an inactive state. At elevated Ca2+ concentration due to a variety of stimuli such as shear force induced by blood flow, bioactive peptides bradykinin, protease thrombin, hormones such as estrogen and insulin, eNOS dissociates from caveolin and binds to CaM through the same binding site.
Like other NOSs, eNOS functions as a homodimer with each monomer comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds a heme cofactor and a BH4 cofactor while the reductase domain binds an FMN cofactor, an FAD cofactor and a CaM molecule. The dimerization occurs upon the binding of L-arginine, heme and BH4. The BH4 cofactor is critical for the dimer formation and its binding to NOS is stabilized by an auto-inhibitory loop structure of the enzyme aided by ZnS4. During the synthesis of NO, NADPH-derived electrons pass into the flavins FAD and FMN in the reductase domain and then are transferred to the heme in the oxygenase domain so that the heme iron can bind O2 and catalyses stepwise NO synthesis from L-arginine. CaM activates NOS by bridging the reductase domain and oxygenase domain so the electrons transfer could occur (Fig.2).
Figure 2. A diagram of the eNOS dimer illustrating the electron transfer pathway and the interactions among cofactors. CaM, calmodulin; H4B, tetrahydrobiopterin; AL, an auto-inhibitory loop that binds ZnS4 and stabilizes the H4B cofactor (Fleming & Busse, 1999).
After its formation in the endothelium, NO spreads to the target tissue such as smooth muscle and activates the heme group of soluble guanylate cyclase (sGC). The activated sGC synthesizes the cyclic guanosine monophosphate (cGMP) from the guanosine triphosphate (GTP), leading to an accumulation of cGMP, which is a secondary messenger that activates a series of intracellular signal transduction cascades. Among effects of signal transduction are decreased vascular smooth muscle contraction and consequently vascular vessel relaxation.
Figure 3. The NO - cGMP signally transduction (Adopted from Denninger & Marletta, 1999).
Since the biosynthesis of NO requires three co-substrates (L-arginine, NADPH, and O2) and five cofactors or prosthetic groups (FAD, FMN, calmodulin, tetrahydrobiopterin, and heme), any environmental or genetic factors that regulate the activity and bioavailability of these substrates or cofactors will impact the activity of NOS and consequently level of NO in human body. For example, in the absence L-arginine or BH4, the electron transfer is “uncoupled” in the eNOS dimer, resulting in the production of superoxide (O2-) rather than NO, thus increased intracellular oxidative stress. Therefore, the activity of eNOS is also associated with the redox status of the human body. Certain physiological stresses such as cigarette smoking, diabetes, hypertension and atherosclerosis also lead to the “uncoupling effect” of eNOS (Alp & Channon, 2004). From a nutrition point of view, dietary L-arginine, BH4 and their biosynthesis precursors, anti-oxidants, the minerals Fe2+, Ca2+ and Zn2+ are all critical modulators of eNOS catalyzed NO production.
NOS3 polymorphisms
In human genome, the NOS3 gene spans about 24 Kb on chromosome 7. It contains 26 exons and 25 introns. A number of polymorphic sites, including variable number of tandem repeats (VNTRs), dinucleotides repeats (CA)n and SNPs in the NOS3 gene have been identified. Three common and clinically relevant polymorphisms in the eNOS gene have been widely studied because they may affect NO formation in healthy subjects and in patients: the T-786C (rs2070744) in the promoter region; the 4b/a VNTR in intron 4; and the Glu298Asp (rs1799983) in exon 7 (Fig.4).
Figure 4. The organization of NOS3 gene and localization of the three common polymorphisms. The top line and the numbers represents the scale of the DNA in kilobases. The bottom line and the solid bars on it represent the exons and their relative locations (adopted from Tanus-Santos et al, 2001).
The T-786C polymorphism, due to a thymine nucleotide replacement by a cytosine in 786 nucleotides upstream of the coding region, reduces the expression of eNOS gene by about 50% (Nakayama et al, 1999; Miyamoto et al, 2000). The reduced expression might be the result of the altered transcription factor binding caused by the minor allele. Although eNOS is normally constitutively expressed at basal level, it is up-regulated by the cytokine IL-10 through the transcription factor STAT-3 upon shear stress stimulation. A cell based study showed that in the -786 CC genotype cells, the IL-10 response is abolished. It was suggested that this insensitivity to IL-10 stimulation played a role in the increased risk for rheumatoid arthritis in the CC genotype carriers (Melchers & Blaschke, 2006).
In the 4b/a VNTR polymorphism, the wild-type allele (4b) has five 27-bp repeats while the minor allele (4a) has four. It has been proposed that the 27-bp repeats acted as small RNA in controlling the eNOS expression through a negative feedback since endothelial cells with the wild type allele (five repeats) had lower levels of eNOS expression than the cells with the minor allele (four repeats) (Zhang et al, 2008). However, the NO level in the 4a carriers was found to be lower (Tsukada et al, 1998). Therefore the molecular mechanism of this polymorphism is inconclusive so far. In addition to 4b/a polymorphism, two other VNTR polymorphisms in intron 4, 4c that correspond to six 27-bp repeats and 4y that has only two 27-bp repeats were also reported in rare cases, confined to the group of African-Americans with severe cardiovascular diseases (Tanus-Santos et al, 2001; Sigusch et al., 2000).
The Glu298Asp polymorphism, also known as G894T, is characterized by a guanine conversion to thymine at position 894 of the gene. Consequently the amino acid residue at 298 of the protein eNOS is converted from glutamine to aspartate. This amino acid change reduces the binding of eNOS to caveolin-1, the major protein located in the caveolae of epithelial cells, resulting in less eNOS protein storage there and subsequently a reduced NO production upon activation (Joshi et al, 2007).
Haplotype analysis shows that these three polymorphisms are not randomly distributed. The Asp298 allele is negatively associated with the 4a allele, meaning that an Asp298 minor allele carrier is very unlikely to carry the VNTR 4a minor allele. The Asp298 allele is also positively associated with the -786C allele in Caucasians, with the Asp298-786C-4b haplotype for 24% frequency in the population (Table 4).
Table 4. Haplotype distribution of the three polymorphisms in major ethnic groups. The minor alleles are color coded and the three most frequent minor allele carrying haplotypes are highlighted. All the numbers represent in percentage. The original data are from Tanus-Santos et al, 2001.
| Glu298Asp |
T-786C |
VNTR 4b/a |
African-Americans |
Caucasians |
Asians |
Comment |
| Glu |
T |
4b |
46 |
46 |
77 |
All wild type |
| Glu |
T |
4a |
27 |
2 |
3 |
4a minor allele |
| Glu |
C |
4b |
7 |
4 |
2 |
T-786C minor allele |
| Glu |
C |
4a |
5 |
14 |
9 |
Two minor alleles 786C & 4a |
| Asp |
T |
4b |
9 |
10 |
6 |
298Asp minor allele |
| Asp |
T |
4a |
0 |
0 |
0 |
Two minor alleles Asp298&4a |
| Asp |
C |
4b |
6 |
24 |
2 |
Two minor alleles Asp298&786C |
| Asp |
C |
4a |
0 |
0 |
1 |
Three minor alleles |
Disease Association
Both the T-786C and Glu298Asp polymorphisms were first discovered in patients with coronary spasm and the VNTR 4b/a polymorphism was first associated with acute myocardial infarction. All three polymorphisms have been associated with a variety of cardiovascular diseases since their discovery (Cooke et al, 2007; Silva et al, 2011).
To date, hypertension represents the only cardiovascular disease that is consistently associated with all three polymorphisms. There have been more than 223 individually association studies published on endothelial nitric oxide synthase (eNOS) polymorphisms in association with hypertension. A recent meta-analysis of these studies, including a total 19,284 cases and 26,003 controls for Glu298Asp, and 6890 cases and 6858 controls for 4b/a, and 5346 cases and 6392 controls for T-786C polymorphism, showed an overall 16% increased risk for hypertension by Asp298 allele in comparison to the Glu298 allele. This increased risk was significantly higher in Asian (a 32% increased risk) and especially so in Chinese (a 40% increased risk). There were an overall increased risk (29%) for hypertension by 4a allele versus 4b allele in all study populations, and this risk was even higher in Asians (42% increased risk). For T-786C, ethnicity-stratified analyses suggested a significantly increased risk for -786C allele carriers (25% increase in single C allele genotype and 69% increase in homozygous CC genotype) in Caucasians (Niu & Qi, 2011).
Other diseases have been associated with two or one of the three polymorphisms. Both the Glu298Asp and VNTR 4b/a, but not the T-786C, are associated with myocardial infarction and ischemic heart disease. Both the T-786C and VNTR 4b/a, but not the Glue298Asp, are associated with rheumatoid arthritis and prostate cancer. Both the Glue298Asp and T-786C, but not the VNTR 4b/a, are associated with coronary spasm and coronary artery disease. For some diseases such as diabetic nephropathy and pre-eclampsia, the association studies result in contradicting conclusions for all three polymorphisms (Table 5).
Table 5. Disease association of the three polymorphisms of NOS3 gene. All the effects represent that of the minor alleles.
| Diseases |
Glu298Asp |
T-786C |
VNTR 4b/a |
| Coronary Artery Disease |
Increased |
Increased |
No effect |
| Coronary spasm |
Increased |
Increased |
Unknown |
| Diabetic nephropathy |
Inconclusive |
Inconclusive |
Inconclusive |
| Erectile dysfunction |
Increased |
Unknown |
No effect |
| Ischemic heart disease |
Increased |
No effect |
Increased |
| Myocardial infarction |
Increased |
Unknown |
Increased |
| Pre-eclampsia |
Inconclusive |
Inconclusive |
Inconclusive |
| Prostate cancer |
No effect |
Increased |
Increased |
| Recurrent pregnancy loss |
Increased |
Unknown |
Unknown |
| Rheumatoid arthritis |
Unknown |
Increased |
Increased |
| Systemic lupus erythematosus |
No effect |
No effect |
Decreased |
Dietary response
As mentioned earlier, the eNOS catalyzed biosynthesis of NO requires three co-substrates (L-arginine, NADPH, and O2) and five cofactors (FAD, FMN, CaM, BH4, and heme), all but O2 are modulated by diet. L-arginine is a component of proteins hence is rich in high protein diet. It is also available as a muscle training supplement. The bioavailability of BH4, NADPH, FAD and FMN is regulated by redox balance, which is modulated by anti-oxidants. FAD and FMN are also depending on their biosynthesis precursor vitamin B2. Fe2+ regulates the function of heme, Ca2+ regulates the function of calmodulin and Zn2+ is directly involved in the interaction of eNOS and BH4. Other dietary components (such as fatty acids) and life style factors (such as smoking and exercise) also regulates the activity of eNOS. Most of the dietary and life style factors impact the function of eNOS independent of the NOS3 polymorphisms. Only the factors that related to the three NOS3 polymorphisms are discussed in the following sections.
Dietary sodium regulates blood pressure and renal hemodynamics through the renin–angiotensin system described in the ACE and Blood Pressure review. One of the regulators of eNOS is bradykinin, a down-stream target of the renin–angiotensin system. It is therefore not surprising to see the interaction of dietary sodium interaction with eNOS polymorphism. In a controlled interventional study of the interaction between the T-786C polymorphism and dietary sodium, the renal hemodynamic parameters glomerular filtration rate (GFR) and renal plasma flow (RPF), blood pressure (BP), and plasma nitric oxide (NOx) levels of 28 hypertensive individuals were determined after eight days of low (20 mEq) and high (200 mEq) sodium diets. The minor C allele carrier group had a significantly greater increase in diastolic and mean arterial blood pressure and a significant decline in both GFR and RPF compared to the homozygote wild type TT genotype group in response to Na+ loading. Furthermore, Na+ loading resulted in a significant increase in plasma NOx in the TT, but not in the C allele carrier group, corresponding to an increase of urine NOx in C allele carrier, but not in the TT genotype group (Dengel et al, 2007). The elevated plasma NOX level indicates renal NOx production and the consequently increased GFR and RPF. Vice versa, the increased NOx production in urea indicates impaired eNOS function and the consequently increased GFR and RPF. These data suggest low-sodium diet should be recommended for carriers of the minor allele of the T-786C polymorphism. The same recommendation is applicable to the 4a carriers of the VNTR 4b/a polymorphism since it was associated with sodium sensitivity in a report, in which no association was detected between sodium sensitivity and the Glu298Asp polymorphism (Hoffmann et al, 2005).
Dietary antioxidant counters oxidative stress that contributes to the endothelial dysfunction by disrupting the normal function of eNOS. The reactive oxygen species (ROS) react with NO, resulting in the formation of peroxynitrile and a reduction of the bioavailability of NO. More importantly, increase of the ROS and peroxynitrile can also lead to the oxidation of the BH4 cofactor of eNOS, leading to a decoupling of this enzyme. The decoupling of eNOS produces superoxide instead of NO, leading to more oxidative stress and less NO availability. The malicious cycle goes on and ultimately results in various types of cardiovascular disorders. Therefore, sufficient amount of antioxidant in diet is critical in fending off oxidative stress, modulating the physiological function of eNOS and NO bioavailability, and consequently maintaining a healthy cardiovascular system.
Sufficient amount of antioxidant intake is especially important for minor allele carriers of the Glu298Asp and T-786C polymorphisms. In a controlled study, the interaction of phenolic compounds content in virgin olive oil and the Glu298Asp was investigated. Phenolic compounds function as antioxidants and present as soluble minor components in wine and virgin olive oil. In this study, 57 subjects with metabolic syndrome received three breakfasts based on virgin olive oil with different phenolic content (398, 149, and 70 ppm respectively). The results showed that homozygous 298Asp/Asp subjects showed lower values of eNOS, NO, and postocclusive skin reactive hyperemia (blood flow recovery after blockage) comparing to the wild type allele (298Glu) carriers at low-phenol diet. However, most of these differences were attenuated when high-phenol virgin olive oil was consumed, suggesting that in a population with a compromised endothelial function, sufficient intake of antioxidants such as phenolic compounds in dietary compensates the reduced enzyme availability associated with the minor allele of NOS3 Glu298Asp polymorphism (Jiménez-Morales et al, 2011). In another controlled study of interactions between antioxidant vitamins and NOS3 polymorphism, five vitamins (vitamin A retinol, vitamin A β-carotene, vitamin E, vitamin C and folic acid) were investigated in relation to risk for breast cancer in women. All but β-carotene reduced the risk for breast cancer more in the minor allele carriers of the Glu298Asp polymorphism. Vitamin E and vitamin C reduced the risk for breast cancer more in the minor allele carriers of the T-786C polymorphism (Lee et al, 2012). The report on antioxidant and the VNTR 4b/a polymorphism interaction is lacking.
Omega-3 polyunsaturated fatty acids (ω-3 PUFA) in general have many beneficial effects on cardiovascular function. Several publications have reported the beneficial effect of polyunsaturated ω-3 fatty acid intake on the minor allele carriers of the Glu298Asp polymorphism. In a dietary intervention study, 450 individuals with metabolism syndrome subject to 12 weeks of dietary fat modification. Following ω-3 PUFA supplementation, plasma triglycerides concentrations in carriers of the Asp298 minor allele showed significant reduction (70%) comparing to hardly any change in the homozygous major allele carriers (Ferguson et al, 2010). The results echoed a previous population study involving 248 healthy young adults that concluded a positive association between plasma ω-3 fatty acids and flow-mediated dilatation in Asp298 carriers while not association between plasma ω-3 fatty acids and flow-mediated dilatation in Glu/Glu genotype carriers (Fig. 5). The exact mechanism of the interaction between ω-3 PUFA and eNOS is not clear. One theory hypothesizes that ω-3 PUFA impact the eNOS function through the caveolae of endothelial cells. The defective caveolae localization of the Asp298 allele may be compensated by higher ω-3 PUFA content. The restored Asp298 enzyme in return may improve the vasodilatation function and consequently the increased lipoprotein metabolism. In the situation of wild type Glu298 allele, since the enzyme is already at its full function, ω-3 PUFA content may not exert much effect.
Figure 5. A positive relationship between ω-3 fatty acid levels and flow-mediated dilation in carriers of the Asp298 allele (right diagram) and no overall relationship between n-3 fatty acids and flow-mediated dilation in Glu298 homozygote (left diagram). Plasma EPA+DHA refer to ω-3 fatty acids (Adapted from Lesson et al, 2002).
L-Arginine supplementation is overwhelming in the muscle training exercise supplements market. Since it is the primary substrate for NO production, it is logical to assume L-arginine supplementation would enhance the production of NO, especially in the NOS3 minor allele carriers. The matter of fact is that L-arginine supplementation has been associated with paradoxes and controversies. A conditionally essential amino acid, L-arginine can be derived from proline or glutamate, with the ultimate synthetic step catalyzed by the enzyme argininosuccinate lyase. L-arginine is catabolized by arginases, nitric oxide synthase, and arginine:glycine amidinotransferase, and possibly also by arginine decarboxylase, resulting ultimately in the production of urea, proline, glutamate, polyamines, nitric oxide, creatine, or agmatine. Consequently, the interplay among these enzymes in the regulation of specific aspects of arginine metabolism can be quite complex. The 'arginine paradox' is the fact that, despite intracellular physiological concentration of arginine being hundreds fold more than it is needed to achieve the maximum reaction rate for eNOS, the acute supply of exogenous arginine still increases NO production. This paradox is the foundation for the claims for L-arginine supplements benefits. Another paradox is that the largest controlled study on chronic oral arginine supplementation in patients after myocardial infarction had to be interrupted for excess mortality in treated patients. An even more puzzling paradox is that long-term arginine supplementation is ineffective in improving eNOS activity. Even worse, exogenous arginine increased mortality when arginine was given to promote vasodilatation in patients after a myocardial infarction Expression and activity of arginases, which produce urea and divert arginine from NOS, are positively related to exogenous arginine supplementation. Therefore, the more arginine is introduced, the more it is destroyed, eventually leading to an impaired NO production. In addition, oral arginine supplementation not only increases arginine disposal through urea synthesis, but also promotes insulin resistance, a detrimental condition that reduces the availability of components necessary for both de novo and recycling synthesis (Dioguardi, 2011). Despite of the paradoxes, no publication has shown the effect of L-arginine on eNOS polymorphism interactions. In conclusion, L-arginine is NOT recommended to improve eNOS activity, regardless the polymorphisms.
Smoking response
Cigarette smoking influences endothelial function by increasing vascular oxidative stress, reducing bioavailability nitric oxide and inhibiting eNOS expression (Guo et al, 2006; Su et al, 1998). The smoking induced stresses also interact with NOS3 polymorphisms, exacerbating the deleterious effect in the minor allele carriers.
In a case-cohort design study to determine whether cigarette smoking modified the association between T-786C and Glu298Asp polymorphisms and risk of coronary heart disease or stroke, 1085 incident coronary heart disease cases, 300 incident ischemic stroke cases and 1065 reference individuals were analyzed. It was found that in Caucasians, association between Glu298Asp genotype and risk of coronary heart disease was significantly modified by current smoking status, with the highest risk observed in smokers carrying the variant Asp298 allele relative to nonsmokers carrying two E298 alleles (Odd ratio = 2.07). In African-Americans, association between T-786C genotype and risk of ischemic stroke was significantly modified by smoking history, with the highest risk observed in ≥20 pack-year smokers carrying the variant C-786 allele relative to <20 pack-year smokers carrying the homozygous wild type genotype (odds ratio 4.03) (Lee et al, 2006). Smoking also reduced the endothelial function in the Asp298 allele carriers when measured by FMD (flow-mediated dilatation). In contrast, there was no FMD difference between smokers and nonsmokers in Glu298 homozygote carriers (Lesson et al, 2002).
The eNOS4a/b polymorphism and smoking interaction has previously been reported for association with coronary artery disease. In current and ex-cigarette smokers, but not nonsmokers, there was a significant excess of minor allele homozygote in patients with severely stenosed arteries, compared with those with no or mild stenosis (Wang et al, 1996).
Therefore, avoiding smoking is especially important for NOS minor allele carriers.
Exercise response
Exercise training increases the bioavailability of NO through the shear stress induced up-regulation of eNOS and SOD (superoxide dismutase) expression. Up-regulated eNOS increases the production of NO and up-regulated SOD activity decreases NO inactivation. So the net result is the increased NO production upon exercise. The three polymorphisms of eNOS impact the response to exercise through altered eNOS activity and hence the NO level.
In a controlled study of the effect of eNOS polymorphism on aerobic exercise involving 49 postmenopausal women trained in sessions of 30-40 min, 3 days a week for 8 weeks, it was found that blood pressure values were significantly reduced after exercise, but the reduction was genotype independent. However, total and LDL cholesterol levels in women carrying the minor alleles of T-786C or VNTR intron 4b/a responded less favorably than the homozygous major allele genotypes (-786TT or intron 4bb). The Glu298Asp polymorphism did not show any effect in this aspect (Esposti et al, 2011). In another controlled double blind study involving 59 postmenopausal women trained in aerobic exercise sessions of 60 min, , 3 days a week for 6 months, it was found that the -786T>C polymorphism had no effect on NO production at basal conditions. But when physical exercise is applied, an evident -786T>C polymorphism effect is detected, showing the minor C allele carriers response less favorably than the wide type genotype (Sponton et al, 2010).
Interestingly, the T-786C polymorphism responses to aerobic exercise more favorably in men carrying the C minor allele. In a study of aerobic exercise in 49 pre- to stage-1 hypertension (145.6±1.5/85.9±1.1 mmHg) men performing a non-exercise control session and two cycle exercise bouts at 40% (LIGHT) and 60% (MODERATE) of peak oxygen consumption, the systolic BP (SBP) was significantly reduced in the minor C allele carriers than in the TT genotype after LIGHT (4.6±2.9 mmHg) as well as MODERATE training (5.3±2.4 mmHg) while no significant SBP was observed in the wile type TT genotype (Augeri et al, 2009). Therefore, the impact of T-786C polymorphism on aerobic exercise is gender-dependent, with the minor allele carriers showing unfavorable results in women and favorable results in men when compared to the wild type allele carriers.
In a study of Glu298Asp polymorphism effect on hand gripping (anaerobic) exercise-induced reflex muscle vasodilatation, it was found that the baseline forearm blood flow rates were similar among genotypes. However, in response to exercise, changes of the forearm flood flow rates were significantly lower in Asp/Asp comparing to Glu/Asp and Glu/Glu genotypes. This observation concluded that the Asp298 allele of the eNOS enzyme have attenuated non-exercising muscle vasodilatation in response to exercise due to reduced eNOS function and NO-mediated vasodilatation (Diaz et al, 2009).
In summary, minor allele carriers generally respond to exercise not as well as the major allele genotypes carriers do, except for the male -786C allele carriers who response more favorably to aerobic exercise.
Drug response
Many cardiovascular improving drugs act by regulate the endogenous level of NO by direct targeting the NO – cGMP pathway or as a side effect. These drugs include the ACE inhibitors, calcium channel blockers, statins, β-blockers, phosphodiesterase inhibitors and hormones. Different studies showed various degree of association of eNOS polymorphisms with variations in NO formation and response to these drugs. Results are far from conclusive and further investigations are certainly needed. There are several detailed reviews on this topic (Silva et al, 2011; Cook et al, 2007; Ignarro et al 2002). In a over simplified summary, it seems that the minor alleles in the T-786C and VNTR 4b/a response favorably to statin drugs while the Asp298 minor allele response unfavorably to the estradiol, hdrochlorothiazide and sildenafil.
Summary
In human cardiovascular system, NOS3 is the key gene in regulating the endogenous NO level that maintains the homeostasis of vascular tone. Three common polymorphisms of NOS3 (T-786C, VNTR 4b/a, and Glu298Asp) associated with a variety of diseases and response distinctly to dietary sodium, antioxidant and ω-3 PUFA. Knowing each of the specific interactions, scientists at GB Lifesciences are able to design personalized diet regimen tailored specifically to different NOS genotypes.
References cited
Alp NJ, Channon KM (2004). Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease. Arterioscler Thromb Vasc Biol. Mar;24(3):413-20. PMID:14656731
Augeri AL, Tsongalis GJ, Van Heest JL, Maresh CM, Thompson PD, Pescatello LS (2009). The endothelial nitric oxide synthase -786 T>C polymorphism and the exercise-induced blood pressure and nitric oxide responses among men with elevated blood pressure. Atherosclerosis. 204(2):e28-34. PMID: 19155013
Casas JP, Bautista LE, Humphries SE, Hingorani AD (2004). Endothelial nitric oxide synthase genotype and ischemic heart disease: meta-analysis of 26 studies involving 23028 subjects. Circulation. 109(11):1359-65. PMID:15007011
Cooke GE, Doshi A, Binkley PF (2007). Endothelial nitric oxide synthase gene: prospects for treatment of heart disease. Pharmacogenomics. 8:1723–34. PMID: 18086002
Cook S, Hugli O, Egli M, Ménard B, Thalmann S, Sartori C, Perrin C, Nicod P, Thorens B, Vollenweider P, Scherrer U, Burcelin R (2004). Partial gene deletion of endothelial nitric oxide synthase predisposes to exaggerated high-fat diet-induced insulin resistance and arterial hypertension. Diabetes. 53(8):2067-72.
Dengel DR, Brown MD, Ferrell RE, Reynolds TH, Supiano MA (2007). A preliminary study on T-786C endothelial nitric oxide synthase gene and renal hemodynamic and blood pressure responses to dietary sodium. Physiol Res. 56(4):393-401. PMID:16925467
Denninger JW, Marletta MA (1999). Guanylate cyclase and the .NO/cGMP signaling pathway. Biochim Biophys Acta. 1411(2-3):334-50. PMID:10320667
Dias RG, Alves MJ, Pereira AC, Rondon MU, Dos Santos MR, Krieger JE, Krieger MH, Negrão CE (2009). Glu298Asp eNOS gene polymorphism causes attenuation in nonexercising muscle vasodilatation. Physiol Genomics. 37(2):99-107. PMID: 19158254
Dioguardi FS (2011). To give or not to give? Lessons from the arginine paradox. J Nutrigenet Nutrigenomics. 4(2):90-8. PMID:21625171
Esposti RD, Sponton CH, Malagrino PA, Carvalho FC, Peres E, Puga GM, Novais IP, Albuquerque DM, Rodovalho C, Bacci M, Zanesco A (2011). Influence of eNOS gene polymorphism on cardiometabolic parameters in response to physical training in postmenopausal women. Braz J Med Biol Res. 44(9):855-63. PMID: 21956531
Fleming I, Busse R (1999). Signal transduction of eNOS activation. Cardiovasc Res. 43(3):532-41. PMID:10690325
Ferguson JF, Phillips CM, McMonagle J, Pérez-Martínez P, Shaw DI, Lovegrove JA, Helal O, Defoort C, Gjelstad IM, Drevon CA, Blaak EE, Saris WH, Leszczyńska-Gołabek I, Kiec-Wilk B, Risérus U, Karlström B, Lopez-Miranda J, Roche HM (2010). NOS3 gene polymorphisms are associated with risk markers of cardiovascular disease, and interact with omega-3 polyunsaturated fatty acids. Atherosclerosis. 211(2):539-44. PMID:20409549
Guo X, Oldham MJ, Kleinman MT, Phalen RF, Kassab GS (2006). Effect of cigarette smoking on nitric oxide, structural, and mechanical properties of mouse arteries. Am J Physiol Heart Circ Physiol. 291(5):H2354-61. PMID:16815989
Hoffmann IS, Tavares-Mordwinkin R, Castejon AM, Alfieri AB, Cubeddu LX (2005). Endothelial nitric oxide synthase polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics. J Hum Hypertens. 19(3):233-40. PMID:15565175
Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. (1995). Hypertension in mice lacking the gene for endothelial nitric oxide synthase.
Nature 377: 239–242. PMID:7545787
Ignarro LJ, Napoli C, Loscalzo J (2002). Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res. 90:21–8. PMID: 11786514
Jiménez-Morales AI, Ruano J, Delgado-Lista J, Fernandez JM, Camargo A, López-Segura F, Villarraso JC, Fuentes-Jiménez F, López-Miranda J, Pérez-Jiménez F (2011). NOS3 Glu298Asp polymorphism interacts with virgin olive oil phenols to determine the postprandial endothelial function in patients with the metabolic syndrome. J Clin Endocrinol Metab. 96(10):E1694-702. PMID:21816783
Joshi MS, Mineo C, Shaul PW, Bauer JA (2007). Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear. Faseb J. 21 (11): 2655-63. PMID:17449720
Lee CR, North KE, Bray MS, Avery CL, Mosher MJ, Couper DJ, Coresh J, Folsom AR, Boerwinkle E, Heiss G, Zeldin DC (2006). NOS3 polymorphisms, cigarette smoking, and cardiovascular disease risk: the Atherosclerosis Risk in Communities study. Pharmacogenet Genomics. 16(12):891-9. PMID:17108813
Lee SA, Lee KM, Yoo KY, Noh DY, Ahn SH, Kang D (2012). Combined effects of antioxidant vitamin and NOS3 genetic polymorphisms on breast cancer risk in women. Clin Nutr. 31(1):93-8. PMID:21872972
Leeson CP, Hingorani AD, Mullen MJ, Jeerooburkhan N, Kattenhorn M, Cole TJ, Muller DP, Lucas A, Humphries SE, Deanfield JE (2002). Glu298Asp endothelial nitric oxide synthase gene polymorphism interacts with environmental and dietary factors to influence endothelial function. Circ Res. 90(11):1153-8. PMID:12065317
Melchers I, Blaschke S, Hecker M, Cattaruzza M (2006). The -786C/T single-nucleotide polymorphism in the promoter of the gene for endothelial nitric oxide synthase: insensitivity to physiologic stimuli as a risk factor for rheumatoid arthritis. Arthritis Rheum. 2006 Oct;54(10):3144-51. PMID:17009241
Miyamoto Y, Saito Y, Nakayama M, Shimasaki Y, Yoshimura T, Yoshimura M, Harada M, Kajiyama N, Kishimoto I, Kuwahara K, Hino J, Ogawa E, Hamanaka I, Kamitani S, Takahashi N, Kawakami R, Kangawa K, Yasue H, Nakao K (2000). Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a -786T-->C mutation associated with coronary spastic angina. Hum Mol Genet. 9(18):2629-37. PMID:11063722
Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, Nakao K (1999). T-786-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 99(22):2864-70. PMID:10359729
Niu W, Qi Y(2011). An updated meta-analysis of endothelial nitric oxide synthase gene: three well-characterized polymorphisms with hypertension. PLoS One. 2011;6(9):e24266. PMID:21912683
Ohashi Y, Kawashima S, Hirata K, Yamashita T, Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y, Yokoyama M (1998). Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J Clin Invest 102:2061–2071. PMID:9854041
Silva PS, Lacchini R, Gomes Vde A, Tanus-Santos JE (2011). Pharmacogenetic implications of the eNOS polymorphisms for cardiovascular action drugs. Arq Bras Cardiol. 96(2):e27-34. PMID:21445464
Sponton CH, Rezende TM, Mallagrino PA, Franco-Penteado CF, Bezerra MA, Zanesco A (2010). Women with TT genotype for eNOS gene are more responsive in lowering blood
pressure in response to exercise. Eur J Cardiovasc Prev Rehabil. 17(6):676-81. PMID: 20436351
Su Y, Han W, Giraldo C, De Li Y, Block ER (1998). Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol. 19(5):819-25. PMID:9806747
Tanus-Santos JE, Desai M, Flockhart DA (2001). Effects of ethnicity on the distribution of clinically relevant endothelial nitric oxide variants. Pharmacogenetics. 11(8):719-25. PMID:11692081
Tesauro M, Thompson WC, Rogliani P, Qi L, Chaudhary PP, Moss J (2000). Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298. Proc Natl Acad Sci U S A. 97(6):2832-5.PMID:10717002
Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, Yamada A, Kawaguchi Y, Hosoya T, Igari J (1998).Evidence of association of the eNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun 1998; 245:190-193. PMID: 9535806
Wang XL, Sim AS, Badenhop RF, McCredie RM, Wilcken DE (1996). A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med. 2(1):41-5. PMID: 8564837
Zhang MX, Zhang C, Shen YH, Wang J, Li XN, Chen L, Zhang Y, Coselli JS, Wang XL (2008). Effect of 27nt small RNA on endothelial nitric-oxide synthase expression. Mol Biol Cell. 19 (9): 3997-4005. PMID:18614799
