eNOS T-786C Genotype, Physical Activity,
and Peak Forearm Blood Flow in Females
SHERI A. DATA1, MARK H. ROLTSCH1,3, BRIAN HAND1, ROBERT E. FERRELL2,
JUNG-JUN PARK1, and MICHAEL D. BROWN1
Department of Kinesiology, University of Maryland, College Park, MD; 2Department of Human Genetics, University of
Pittsburgh Graduate School of Public Health, Pittsburgh, PA; and 3Howard University Cancer Center, Howard
University, Washington, DC
1
ABSTRACT
DATA, S. A., M. H. ROLTSCH, B. HAND, R. E. FERRELL, J-J. PARK, and M. D. BROWN1. eNOS T-786C Genotype, Physical
Activity, and Peak Forearm Blood Flow in Females. Med. Sci. Sports Exerc., Vol. 35, No. 12, pp. 1991–1997, 2003. Background: The
purpose of the present study was to determine interactive and main effects of the eNOS T-786C gene polymorphism and habitual
physical activity level on forearm vascular resistance (FVR) and forearm blood flow (FBF) at rest and during 3 min of reactive
hyperemia. Methods: We studied healthy, Caucasian (age 25 ⫾ 1 yr), sedentary (maximal oxygen consumption, V̇O2max: 33.8 ⫾ 1
mL·kg⫺1·min⫺1), and endurance-trained (V̇O2max: 45.3 ⫾ 1 mL·kg⫺1·min⫺1) women. FBF was measured using venous occlusion
plethysmography before (resting) and after 5 min of arm arterial occlusion at 1 (peak vasodilation), 2, and 3 min of reactive hyperemia.
V̇O2max was measured using a standard treadmill protocol, and skinfolds were measured to estimate body composition. Results: There
was a significant interaction between eNOS genotype and physical activity level on resting FVR (P ⫽ 0.0003). Sedentary subjects with
the TT genotype had the lowest resting FVR, but among the endurance-trained group, the TC⫹CC genotype group had the lowest
resting FVR. This interaction was reflected in the resting FBF values (P ⫽ 0.03). After accounting for important covariates, there was
a main effect of eNOS genotype on peak FBF (TT, 7.0 ⫾ 0.3 vs TC⫹CC, 5.9 ⫾ 0.4 mL·100 mL⫺1 FAV·min⫺1, P ⫽ 0.03) and the
percent decrease in FVR (TT, ⫺62 ⫾ 2 vs TC⫹CC, ⫺51 ⫾ 4%, P ⫽ 0.04) at minute 1. Conclusions: These results of the interactive
effects suggest that young females possessing a C allele may reduce their resting FVR by improving their cardiovascular fitness level,
but TT homozygotes, who may have normal eNOS gene function, may not improve their resting FVR with improvements in
cardiovascular fitness. Furthermore, regardless of physical activity level, the TT genotype showed a favorable hemodynamic response
during reactive hyperemia compared with the C allele carriers. Key Words: PHYSICAL ACTIVITY, GENETICS, NITRIC OXIDE,
BLOOD FLOW, FEMALES
T
he endothelium plays an important role in regulating
blood flow during various metabolic perturbations.
Studies have shown that nitric oxide (NO) contributes, in part, to reactive hyperemia in the forearm because
NO synthase inhibitors partially attenuate the forearm blood
flow (FBF) response (9,14). In particular, investigators have
focused on peak forearm vascular resistance (FVR) and FBF
during reactive hyperemia as a measure of peak vasodilatory
capacity. The primary factors contributing to peak vasodilatory capacity are local regulator such as adenosine and
prostaglandin (3,8,13). Although NO may have only a small
direct role in determining blood flow during reactive hyperemia, NO also interacts with adrenergic mechanisms
(29,35), adenosine (3,32), and prostaglandins (13,24) to
affect the blood flow response.
The eNOS gene consists of 26 exons and 25 introns
located on the long arm of chromosome 7 at 7q35336 (19).
In the promoter 5' flanking region, a single nucleotide polymorphism (SNP) with a T to C substitution occurring at
nucleotide position ⫺786 (T-786C) has been identified
(12,31). Recently, in vitro studies have shown that individuals with the C allele had significantly decreased promoter
activity compared with individuals without a C allele (31).
This mutation suppresses eNOS transcription, which is consistent with reduced NO production. In addition, a shear
stress responsive element has been identified in a region
near the eNOS promoter (12). These functional characteristics of the eNOS T-786C polymorphism suggest individuals with a C allele may demonstrate differences in FVR and
FBF during reactive hyperemia.
Aerobic exercise training has the capacity to improve
endothelial function (9,28). Two cross-sectional studies
found a greater FBF response during acetylcholine infusions
in healthy exercise trained individuals than their age- and
sex-matched controls (6,15). Testa et al. (28) reported an
increase in peak calf reactive hyperemia after 12 wk of
aerobic exercise training in chronic heart failure patients.
Interestingly, in this study, approximately half of the sub-
Address for correspondence: Sheri Data, M.A., University of Maryland,
Dept. of Kinesiology, HHP Bldg., Rm. 2128, College Park, MD 207422611; E-mail: [email protected].
Submitted for publication December 2002.
Accepted for publication July 2003.
0195-9131/03/3512-1991
MEDICINE & SCIENCE IN SPORTS & EXERCISE®
Copyright © 2003 by the American College of Sports Medicine
DOI: 10.1249/01.MSS.0000099105.99682.8B
1991
jects demonstrated an increase in eNOS gene expression
whereas there was no change in the remaining subjects. This
finding of heterogeneous responses in eNOS gene expression to a standardized exercise stimulus suggests that heterogeneity at the DNA level may be responsible for the
range of responses. Together, these studies suggest that the
eNOS gene interacts with aerobic exercise training to affect
FBF. There are no studies that have determined whether the
eNOS T-786C gene polymorphism interacts with physical
activity levels to affect FVR and FBF during reactive
hyperemia.
The purpose of the present study was to determine
whether the eNOS T-786C gene polymorphism interacts
with habitual physical activity level to affect FVR and FBF
at rest and at 1 (peak vasodilation), 2, and 3 min during
reactive hyperemia in healthy Caucasian women. We also
sought to determine whether there was a main effect of
eNOS genotype on FVR and FBF because the C allele has
been associated with reduced eNOS gene transcriptional and
promoter activity. We hypothesized that C allele carriers
(TC⫹CC genotypes) would have a greater FVR and a lower
FBF at rest and at each minute during reactive hyperemia
compared with the TT genotype group. In addition, because
high levels of cardiovascular fitness are associated with
better peripheral vascular function, we hypothesized that
that endurance-trained subjects with the TT genotype would
have the lowest FVR and the greatest FBF at min 1, 2, and
3 during reactive hyperemia compared with the sedentary
subjects with the CC genotype.
METHODS
Subjects. The study was approved by the University of
Maryland Human Subjects Review Board. All subjects signed
an informed written consent form. The subjects were part of a
larger project designed to assess the effects of genotype on
exercise cardiovascular hemodynamics in young females conducted in the Department of Kinesiology, University of Maryland College Park. Healthy sedentary and endurance-trained
18- to 30-yr-old Caucasian female volunteers were recruited
from the University of Maryland and the surrounding metropolitan area through print and media advertisements. A total of
63 subjects (35 endurance-trained, 28 sedentary) were recruited
for the study. Initially, potential subjects who responded to the
advertisements were interviewed by telephone using a screening questionnaire. Women with a history of the coronary artery
disease, other cardiovascular diseases, smoking, diabetes, medications affecting cardiovascular responses, or orthopedic conditions were excluded from participating in this study. In addition to their health history, the subject’s previous physical
activity level was recorded to preliminarily group them as
sedentary or endurance-trained. Sedentary requirements were
regularly participating in any aerobic exercise ⬍20 min·d⫺1
less than 3 d·wk⫺1 and endurance-trained requirements were
participating in aerobic exercise 45– 60 min·d⫺1 at least 5
d·wk⫺1 at a moderate to high intensity for a minimum of 2 yr.
During the first screening visit, subjects completed a
detailed medical history questionnaire to verify the tele1992
Official Journal of the American College of Sports Medicine
phone interview information. A 15-mL blood sample was
then obtained by venipuncture from the antecubital vein for
DNA analysis. Finally, the subject’s height, weight, and
skinfold measurements were recorded. The Jackson and
Pollock (1) seven-site skinfold technique was used to determine body composition.
Maximal oxygen consumption. The subject’s maximal oxygen consumption (V̇O2max), a surrogate measure of
habitual physical activity levels, was assessed utilizing a
maximal treadmill exercise test as previously described (5).
Throughout and at the termination of the test, blood pressure
and heart rate were monitored and recorded. A true maximal
test was based on standard criteria if two of the following
criteria were met: RER ⬎1.10, heart rate ⱖ (220 ⫺ age),
and a plateau in V̇O2 during the last stage of the test (⬍150
mL·min⫺1 increase in V̇O2) (5). If the criteria were not met,
the subject returned on a separate day to repeat the test.
Forearm blood flow. All studies were conducted between 7:00 and 9:00 a.m. after an overnight fast of 12 h. All
subjects were studied between the 2nd and 7th day of their
menstrual cycle when estrogen levels were at the lowest and
have the least affect on endothelial function. Subjects assumed the supine position for 15 min. After the 15-min rest
period, FBF was measured using venous occlusion plethysmography and a mercury-filled silastic strain gauge placed
around the widest part of the subject’s nondominant forearm. The FBF response was recorded on a strip chart and
calculated using the slope of the strain gauge output. Twenty
seconds before each FBF measurement, the wrist cuff was
inflated to 200 mm Hg to remove hand blood flow from the
FBF measurement. Three successive intermittent FBF measurements were taken at baseline by inflating the arm cuff to
55 mm Hg using an automatic rapid cuff inflator. FBF was
recorded for seven heartbeats. In addition, resting heart rate
and blood pressure were recorded simultaneously in the
contralateral arm.
Reactive hyperemia, the vasodilatory response to transient vascular occlusion, was used to increase FBF. To
induce ischemia and subsequent reactive hyperemia, the
upper arm cuff was inflated to 50 mm Hg above the subject’s resting systolic blood pressure obtained during the
15-min rest period at baseline for 5 min (16). After the upper
arm cuff was deflated, FBF was measured intermittently
each min for 3 min. Studies have shown that tissues appear
to recover from hypoxia within 3 min and that peak blood
flow occurs at approximately 1 min (4). FBF was calculated
as FBF (mL·100 mL⫺1 FAV·min⫺1)⫽ K ⫻ dV/dT where K
is a constant, V is voltage, T is time, and FAV is forearm
volume. FVR was calculated as the mean arterial pressure
(MAP) divided by the corresponding FBF and reported as
arbitrary units (u).
eNOS Genotyping. Genomic DNA was extracted from
the leukocytes of the blood sample utilizing a PureGene kit
(Gentra Systems, Minneapolis, MN). Subjects were genotyped for the eNOS ⫺786 site using polymerase chain
reaction amplification with flanking primers F:5'- CACCCAGGCCCACCCCAACT-3' and R:5'-GCCGCAGGTCGACAGAGA GACT ⫺3'. DNA was denatured for 5 min at
http://www.acsm-msse.org
TABLE 1. Subject characteristics in the genotype ⫻ physical activity level groups.
Variables
Sed TCⴙCC (N ⴝ 13)
End Tr TCⴙCC (N ⴝ 18)
Sed TT (N ⴝ 15)
End Tr TT (N ⴝ 17)
ANOVA P Values
Age (yr)
Height (cm)
Weight (kg)
Body fat (%)
Fat free mass (kg)
V̇O2max (mL 䡠 kg⫺1 䡠 min⫺1)
Baseline HR (beats䡠min⫺1)
Baseline SBP (mm Hg)
Baseline DBP (mm Hg)
Baseline MAP (mm Hg)
24.4 ⫾ 1
161.9 ⫾ 2
60.1 ⫾ 4
25.5 ⫾ 2
44.1 ⫾ 2
34.1 ⫾ 1
66.0 ⫾ 3
109.8 ⫾ 3
72.6 ⫾ 2
85.3 ⫾ 2
25.0 ⫾ 1
162.4 ⫾ 1
60.0 ⫾ 1
20.0 ⫾ 1
47.8 ⫾ 1
44.5 ⫾ 1
57.7 ⫾ 2
110.4 ⫾ 2
68.6 ⫾ 2
82.7 ⫾ 2
23.6 ⫾ 1
165.4 ⫾ 2
66.5 ⫾ 4
26.5 ⫾ 2
48.5 ⫾ 2
33.3 ⫾ 1
64.6 ⫾ 3
109.8 ⫾ 3
71.5 ⫾ 2
84.2 ⫾ 2
26.8 ⫾ 1
165.0 ⫾ 2
59.3 ⫾ 2
18.5 ⫾ 1
48.3 ⫾ 1
46.2 ⫾ 1
56.9 ⫾ 2
115.5 ⫾ 5
72.9 ⫾ 2
87.0 ⫾ 3
0.26
0.91
0.21
0.33
0.20
0.34
0.91
0.45
0.23
0.28
V̇O2max, maximal oxygen consumption; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressures; Sed, sedentary; End Tr, endurance
trained. Values are means ⫾ SE.
95°C followed by 35 cycles of denaturation (30 s, 95°C),
annealing (15 s, 54°C), and extension (30 s, 72°C). The
amplicon was digested overnight at 37°C using 5 ␮M of
MspI followed by electrophoresis for 4 h in a gel composed
of 2% agarose ⫹ 1% Nusieve (FMC, Inc.). The T allele
yields a fragment of 415 bp, and the C allele yields fragments of 370 bp and 45 bp.
Data and statistical analysis. In order to detect a
15% difference in FBF at a power of 0.80 and an alpha level
of 0.05, we calculated that a minimum of 15 subjects were
required in each genotype group. Therefore, based on the
reported C allele frequency, the C allele carriers were
grouped into a combined TC⫹CC genotype group and compared with the TT genotype group (25,33). Descriptive
statistics were calculated for the four genotype ⫻ physical
activity level groups (sedentary TC⫹CC, endurance-trained
TC⫹CC, sedentary TT, and endurance-trained TT) using
two-way ANOVA with eNOS genotype and physical activity level as the independent variables. Because body composition and resting FBF can influence the hemodynamic
response during reactive hyperemia, all subsequent analyses
were covaried for percent body fat and baseline FBF. Timecourse changes in FBF during reactive hyperemia were
compared using a three-way ANOVA (genotype ⫻ physical
activity level ⫻ time) with repeated measures. A two-way
ANOVA with eNOS genotype and physical activity level
used as independent variables was used to assess potential
interactive and main effects between the dependent variables at rest (baseline) and at 1, 2, and 3 min during reactive
hyperemia. The primary dependent variables were FVR and
FBF at baseline and at min 1 (peak vasodilation), 2, and 3
during reactive hyperemia. Additional dependent variables
were the percent changes in FVR and FBF at min 1, 2, and
3. To compare differences between the four genotype ⫻
physical activity level groups, post hoc comparisons were
performed using the Bonferroni procedure. The data was
analyzed using StatView 5.0 (SAS Institute Inc.). All results
are presented as means ⫾ SE. A value of P ⬍ 0.05 was
considered statistically significant.
RESULTS
Subject characteristics. The subject characteristics
of the eNOS T-786C genotype groups and the physical
activity level groups are summarized in Table 1. There were
PHYSICAL ACTIVITY, eNOS GENE, AND BLOOD FLOW
63 women in two eNOS genotype groups (TT genotype, N
⫽ 32; TC⫹CC genotype; N ⫽ 31) and 63 women in two
physical activity level groups (sedentary, N ⫽ 28; and
endurance trained, N ⫽ 35). This resulted in the following
eNOS genotype ⫻ physical activity levels groups (sedentary
TC⫹CC, N ⫽ 13; endurance-trained TC⫹CC, N ⫽ 18;
sedentary TT, N ⫽ 15; and endurance-trained TT, N ⫽ 17).
There were no differences in subject characteristics between
the four genotype ⫻ physical activity levels groups. As
expected, V̇O2max was significantly greater (P ⬍ 0.001), and
percent body fat (P ⬍ 0.001) and resting heart rate (P ⫽
0.002) were significantly lower in the endurance-trained
women than in the sedentary women. There were no significant differences in age, height, weight, and blood pressure between the two physical activity level groups.
In general, the sedentary TC⫹CC group had the greatest
baseline FVR and the lowest baseline FBF compared with
the other three groups (Table 2). Within the entire group of
subjects and within each of the four genotype ⫻ physical
activity level groups, FBF increased significantly (P ⬍
0.0001) during reactive hyperemia compared with the baseline values. After accounting for differences in baseline FBF
and percent body fat, the greatest FBF at 1 min (peak
vasodilation) was demonstrated by the sedentary TT group
and the lowest peak FBF was observed in the sedentary
TC⫹CC group. In all groups, FBF remained significantly
higher than the baseline values at min 2. At min 3, FBF in
all groups except the endurance-trained TT group remained
higher than the baseline values. Systolic blood pressure in
all groups did not change; however, diastolic blood pressure
tended (P ⫽ 0.09) to decrease in all groups during the
reactive hyperemic period.
Interactive effect of the eNOS T-786C gene polymorphism and physical activity level on hemodynamic variables. Table 2 summarizes the interactive effects between eNOS T-786C genotype and physical activity
level on hemodynamic variable at baseline and during reactive hyperemia. All analyses used baseline FBF and percent body fat as covariates, except when analyzing baseline
FBF. In this case, only percent body fat was used as a
covariate. There was a significant interactive effect between
eNOS genotype and physical activity level on baseline FVR
(P ⫽ 0.0003) and baseline FBF (P ⫽ 0.03). This interaction
resulted because within the sedentary group, the TT genotype had the lowest baseline FVR, but within the enduranceMedicine & Science in Sports & Exercise姞
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TABLE 2. Interactive effect of eNOS T-786C gene polymorphism and physical activity on hemodynamic variables.
Hemodynamic Variables
Sed TCⴙCC
(N ⴝ 13)
End Tr TCⴙCC
(N ⴝ 18)
Sed TT
(N ⴝ 15)
End Tr TT
(N ⴝ 17)
Interaction
P Values
Main Effect
Genotype P Values
Main Effect Phys
Act P Values
Baseline FBF (mL䡠100 mL䡠FAV䡠min⫺1)
Baseline FVR (u)
FBF 1 min (Peak) (mL䡠100 mL䡠FAV䡠min⫺1)
FBF 2 min (mL䡠100 mL䡠FAV䡠min⫺1)
FBF 3 min (mL䡠100 mL䡠FAV䡠min⫺1)
FVR 1 min (Peak) (u)
FVR 2 min (u)
FVR 3 min (u)
2.1 ⫾ 0.2
44 ⫾ 4b
5.7 ⫾ 0.7
3.3 ⫾ 0.4
2.5 ⫾ 0.2
19.0 ⫾ 3.4
28.7 ⫾ 2.6
36.7 ⫾ 3.2
3.0 ⫾ 0.3a
32 ⫾ 3
6.2 ⫾ 0.6
3.9 ⫾ 0.3
3.4 ⫾ 0.2
15.5 ⫾ 1.5
22.7 ⫾ 1.7a
24.5 ⫾ 1.2a
2.6 ⫾ 0.3a
39 ⫾ 5b
7.4 ⫾ 0.9a,b
4.5 ⫾ 0.5
3.5 ⫾ 0.4
14.5 ⫾ 2.3
24.9 ⫾ 5.1a
29.3 ⫾ 5.5a
2.7 ⫾ 0.4a
39 ⫾ 4b
6.9 ⫾ 0.6a,b
3.5 ⫾ 0.3
2.7 ⫾ 0.3
13.3 ⫾ 1.0
25.7 ⫾ 2.1
34.7 ⫾ 3.7
0.03
0.0003
0.97
0.35
0.79
0.66
0.04
0.07
0.98
0.34
0.03
0.13
0.54
0.09
0.98
0.45
0.18
0.003
0.84
0.39
0.77
0.24
0.30
0.26
FBF, forearm blood flow; FVR, forearm vascular resistance; RH, reactive hyperemia; Sed, sedentary; End Tr, endurance trained. Covaried for baseline FBF (except baseline FBF) and
%body fat. Values are means ⫾ SE.
a
significantly different (P ⬍ 0.05) from the Sed TC⫹CC group.
b
Significantly different (P ⬍ 0.05) from the End Tr TC⫹CC group.
trained group, the TC⫹CC and the TT genotype groups had
identical baseline FVR values. The differences in FVR were
not related to difference in MAP. Within the sedentary
group, the TT genotype had greater baseline FBF than the
TC⫹CC genotype group; however, within the endurancetrained group, the TC⫹CC genotype group had a greater
baseline FBF than the TT genotype group. There was also a
significant interactive effect between eNOS genotype and
physical activity level on FVR at min 2 (P ⫽ 0.04) and a
tendency for an interactive effect at min 3 (P ⫽ 0.07) during
reactive hyperemia. Within the sedentary group, the TT
genotype group had the lowest FVR, but within the endurance-trained group, the TC⫹CC genotype group had the
lowest FVR at 2 min. There were no interactive effects with
respect to any of the blood pressure variables.
Main effect of the eNOS T-786C gene polymorphism and physical activity level on hemodynamic
variables. Table 2 summarizes the main effects of physical
activity level and eNOS genotype. There was only one main
effect of physical activity level detected. The sedentary subjects had a greater baseline FVR compared with the endurancetrained subjects (41 ⫾ 3 vs 35 ⫾ 2 u, P ⫽ 0.0003). All of the
other main effects identified were related to the eNOS T-786C
genotype, and they were all observed in variables related to
peak vasodilation measured at the first minute after arterial
occlusion. The TT genotype group had a greater peak FBF
compared with the TC⫹CC genotype group (7.0 ⫾ 0.3 vs 5.9
⫾ 0.4 mL·100 mL⫺1 FAV·min⫺1, P ⫽ 0.03). This corresponded to the tendency for the TT genotype group to have
lower FVR at 1 min compared with the TC⫹CC genotype
group (14 ⫾ 1 vs 17 ⫾ 1 u, P ⫽ 0.09).
We calculated the percent change in FBF and FVR from
the baseline values at min 1, 2, and 3 during reactive
hyperemia. There was a tendency for a main effect of eNOS
genotype on both the percent change in FBF and FVR
during peak vasodilation (Fig. 1). The TT genotype group
had a greater percent increase in FBF than the TC⫹CC
genotype group (186 ⫾ 19 vs 139 ⫾ 19%, P ⫽ 0.05), which
corresponded to a greater percent decrease in FVR at min 1
(⫺62 ⫾ 2 vs ⫺51 ⫾ 4%, P ⫽ 0.04). There were no main
effects of eNOS genotype with respect to any of the blood
pressure variables.
DISCUSSION
The present study examined the potential interactive effects between the T-786C eNOS gene polymorphism and
habitual physical activity level on FVR and FBF at rest and
during reactive hyperemia in young Caucasian women who
were participating in a larger study. We found significant
interactive effects on resting FVR and FBF, and a significant main effect of eNOS genotype on peak FBF, and the
percent change in FVR at peak vasodilation. Within the
sedentary group, the TT genotype group had a lower resting
FVR than the TC⫹CC group. However, within the endurance-trained group, the TC⫹CC and the TT genotype
groups had similar resting FVR values. This finding was
reflected in the resting FBF values. After accounting for
FIGURE 1—Main effects of eNOS T-786C genotype on the percent change in forearm blood flow (A) and forearm vascular resistance (B) at 1 min
(peak vasodilation) in the TCⴙCC (N ⴝ 31) and TT (N ⴝ 32) eNOS genotype groups. * P ⴝ 0.05 (A); * P ⴝ 0.04 (B). Values are means ⴞ SE.
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Official Journal of the American College of Sports Medicine
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baseline FBF and percent body fat, peak FBF was significantly greater, and the percent decrease in FVR at min 1 was
significantly lower in TT genotype subjects. This agrees
with the previous finding of reduced transcriptional and
promoter activity in C allele carriers (25). Interestingly, by
minute 3 of reactive hyperemia, FBF returned to resting
levels only in the endurance-trained TT genotype group.
This capacity to restore homeostasis more quickly may
indicate the favorable response, and thus, the endurancetrained TT genotype group may have the best overall peripheral vascular function response.
Currently, there are no studies that have assessed the
interactive effect of habitual physical activity level and
genetics on FVR and FBF at rest and during reactive hyperemia. The sedentary TC⫹CC group clearly demonstrated the greatest resting FVR and the lowest FBF compared with any other group. Even the endurance-trained
group with the TC⫹CC genotype had a significantly lower
baseline FVR and significantly greater resting FBF than the
sedentary TC⫹CC group. However, the sedentary subjects
with the TT genotype and the endurance-trained subjects
with the TT genotype had virtually the same baseline FVR
values. These findings suggest that perhaps individuals can
overcome the reported negative effects associated with having a C allele with chronic endurance exercise. These results
also suggest that in subjects with the TT genotype, those
reported to have “normal” vasomotor and cardiovascular
function, cardiovascular fitness has less of an effect on
resting FVR and FBF.
Aerobic exercise training reduces the risk of cardiovascular disease and all-cause mortality primarily by decreasing cardiovascular disease risk factors (2). It is thought that
the endothelium is an important component of cardiovascular health (22). We found a significant main effect of physical activity level on resting FVR with the sedentary subjects
demonstrating greater FVR than the endurance-trained subjects. However, this effect was primarily due to the sedentary C allele carriers who had the highest average resting
FVR. This agrees with longitudinal studies that have shown
improved endothelial function with aerobic exercise training
(6,9,15). The physical activity groups clearly had different
cardiovascular fitness levels as evidenced by the groups’
average V̇O2max values. The sedentary group had an average
V̇O2max of 33.8 ⫾ 1 mL·kg⫺1·min⫺1, which is in the 40th
percentile for their gender and age (1). In contrast, the
endurance-trained women, had an average V̇O2max of 45.3
⫾ 1 mL·kg⫺1·min⫺1, which is in the 90th percentile (1).
The T-786C polymorphism has potentially important
functional characteristics. The polymorphism is located in
the promoter region (12,31) of the eNOS gene and therefore
potentially affects gene expression levels. Nakayama et al.
(25) found that individuals with a C allele had 50% decreased promoter activity compared with individuals without a C allele, which is consistent with the concept that the
C allele is associated with reduced NO production. Interestingly, one study showed that a shear stress responsive
element is located near the promoter region of the eNOS
gene between nucleotides ⫺1600 and ⫺779 (18). Our rePHYSICAL ACTIVITY, eNOS GENE, AND BLOOD FLOW
sults agree with these previous findings because the TT
genotype individuals had a greater reduction in FVR and a
greater increase in FBF than the C allele carriers during peak
vasodilation. Our results also agree with a recent report by
Rossi et al. (26), who found a blunted percent increase in
FBF to incremental doses of acetylcholine in hypertensive C
allele carriers compared with the TT genotype individuals.
However, Rossi et al. did not report on the physical activity
status or body composition of their subjects, which can
influence blood flow capacity.
In the present study, subjects were categorized into a
combined TC⫹CC genotype group and compared with the
TT genotype group. Based on available data, the frequency
of the CC genotype in the population is between 0.02 and
0.10. Given these frequencies, we would have had to recruit
between 150 and 750 subjects to fill the CC genotype group.
In the present study, six subjects (10%) were CC homozygotes, which agrees with the reported frequency of the CC
genotype. Thus, we did not have adequate statistical power
to include a separate CC genotype group. Furthermore, the
C allele is considered to have deleterious cardiovascular
effects, with CC and TC genotype groups demonstrating
similar responses that are different than groups with the TT
genotype (25,26,34). Thus, it appears appropriate from a
mechanistic perspective to group the C allele carriers into a
combined TC⫹CC genotype group.
Ischemia is an extreme physiological condition and the
response to ischemia is an increase in blood flow to protect
the tissues against hypoperfusion (17). The extent to which
NO is produced during postischemia vasodilation in the
human forearm is debatable; however, several studies that
used NO synthase inhibitors found modest reductions in
peak reactive hyperemic blood flow and/or total hyperemic
response (8 –10,20). Our purpose was not to determine the
role that NO plays in reactive hyperemia, but rather, given
the functional characteristics of the T-786C polymorphism
(12,24), we sought to determine whether there was a genotype-dependent difference in FVR and FBF. Our purpose
was also not to determine whether the eNOS T-786C gene
polymorphism was associated with endothelial function.
This has been demonstrated by Rossi et al. using pharmacologic methods (26).
Our purpose was to determine the overall role of the
eNOS T-786 gene polymorphism during a physiological
stimulus that induces vasodilation. There are several other
local regulators of blood flow, many of which interact with
the NO system to produce postischemia vasodilation
(4,11,13,21). Because eNOS and NO interact with other
regulators of blood flow, it was important that we first
assess the potential interactive effects of the eNOS gene and
physical activity level on the global FVR and FBF response
during reactive hyperemia. Other regulators of local blood
flow that could potentially interact with eNOS to affect
postischemia vasodilation are adenosine (3,27,32), prostaglandin (8,13,21,24,35), and adrenergic mechanisms
(29,35). One study found that another eNOS gene polymorphism was associated with adenosine-induced hyperemia
(23). Kilbom and Wennmalm (13) showed that prostagMedicine & Science in Sports & Exercise姞
1995
landin’s most prominent role was in postischemia and metabolic vasodilation in the human forearm and under certain
metabolic conditions, prostaglandin modulates eNOS gene
expression (24). Finally, studies have shown that NO counteracts sympathetic vasoconstrictor mechanisms (35) and is
involved in the ␣2-adrenergic receptor-mediated endothelial-dependent vasodilation (30).
Multiple genes likely influence the reactive hyperemic
response, with each gene contributing to a portion of the
overall blood flow response. Thus, the influence of any
single gene on the FVR and FBF response during reactive
hyperemia is likely to be small. The eNOS T-786C gene
polymorphism is a particularly attractive candidate because
functional characteristics, namely, altered gene promoter
activity and its proximity to a shear stress responsive element. Hypercholesterolemia impairs endothelial function
and could thereby influence the reactive hyperemic response
(7). The subjects in the present study were young, healthy,
not obese, and none of them indicated the presence of any
diseases or conditions that would impair endothelial function in their health history questionnaire. Therefore, it is
unlikely that differences in cholesterol levels between the
eNOS genotype groups contributed substantially to the differences in FVR and FBF observed in the present study. It
is more likely that there were differences in cholesterol
between the physical activity groups, but the only variable
that was different between these groups was baseline FVR.
In conclusion, the present study found an interactive
effect between physical activity level and the eNOS T-786C
gene polymorphism on resting FVR and FBF. Among the
sedentary subjects, those with the TT genotype and the
lowest baseline FVR, but among the endurance-trained
group, the TC⫹CC and TT genotype groups had identical
baseline FVR values. With respect to FBF, sedentary subjects with the TT genotype had greater baseline FBF than
the TC⫹CC genotype subjects; however, among the endurance-trained group, the TC⫹CC genotype group had a
greater baseline FBF than the TT genotype subjects. Although the present investigation was a cross-sectional study,
these results suggest that young female subjects possessing
a C allele may reduce their resting FVR and increase their
resting FBF by improving their cardiovascular fitness level.
In contrast, young female sedentary TT homozygotes, who
may have normal eNOS gene function, may not improve
their resting FVR with improvements in cardiovascular fitness. Regardless of physical activity level, young females
with the TT genotype had a significantly greater peak FBF
compared with the C allele carriers. Most importantly, we
found that after accounting for baseline FBF and percent
body fat, peak FBF was significantly greater and the percent
decrease in FVR at minute 1 was significantly lower in TT
genotype subjects. These latter results agree with the report
that the C allele is associated with reduced eNOS gene
transcriptional and a promoter activity, which is consistent
with the notion of reduced NO production.
We thank Gloria Sheng for her assistance in creating and managing the database.
REFERENCES
1. AMERICAN COLLEGE OF SPORTS MEDICINE. ACSM’s Guidelines for
Exercise Testing and Prescription. Philadelphia: Lippincott Williams & Wilkins, 2000, pp. 66 & 77.
2. BLAIR, S. N., H. W. R. KOHL, R. S. J. PAFFENBARGER, D. G. CLARK,
K. H. COOPER, and L. W. GIBBONS. Physical fitness and all-cause
mortality: a prospective study of healthy men and women. JAMA
262:2395–2401, 1989.
3. BRYAN, P. T., and J. M. MARSHALL. Cellular mechanisms by which
adenosine induces vasodilation in rat skeletal muscle: significance
for systemic hypoxia. J. Physiol. 514:163–175, 1999.
4. CORRETTI, M. C., G. D. PLOTNICK, and R. A. VOGEL. Technical
aspects of evaluating brachial artery vasodilation using high-frequency ultrasound. Am. J. Physiol. 268:H1397–H1404, 1995.
5. DENGEL, D., J. M. HAGBERG, P. COON, D. DRINKWATER, and A. P.
GOLDBERG. Comparable effects of diet and exercise on body composition and lipoproteins in older men. Med. Sci. Sports Exerc.
26:1307–1315, 1994.
6. DESOUZA, C. A., L. F. SHAPIRO, C. M. CLEVENGER, et al. Regular
aerobic exercise prevents and restores age-related declines in
endothelium-dependent vasodilation in healthy men. Circulation
102:1351–1357, 2000.
7. DREXLER, H., and A. M. ZEIHER. Endothelial function in human
coronary arteries in vivo: focus on hypercholesterolemia. Hypertension 18(Suppl. 4):II90 –II99, 1991.
8. ENGELKE, K. A., J. R. HALLIWILL, D. N. PROCTOR, N. M. DIETZ, and
M. J. JOYNER. Contribution of nitric oxide and prostaglandins to
reactive hyperemia in the human forearm. J. Appl. Physiol. 81:
1807–14, 1996.
9. HIGASHI, Y., S. SASAKI, N. SASAKI, et al. Daily aerobic exercise
improves reactive hyperemia in patients with essential hypertension. Hypertension 33:591–597, 1999.
1996
Official Journal of the American College of Sports Medicine
10. JOANNIDES, R., W. E. HAEFELI, L. LINDER, et al. Nitric oxide is
responsible for flow-dependent dilatation of human peripheral
conduit arteries in vivo. Circulation 91:1314 –1319, 1995.
11. JONES, C. J. H., L. KUO, M. J. DAVIS, D. V. DEFILY, and W. M.
CHILIAN. Role of nitric oxide in the coronary microvascular responses to adenosine and increased metabolic demand. Circulation 91:1807–1813, 1995.
12. KARANTZOULIS-FEGARAS, F., H. ANTONIOU, S. L. LAI, et al. Characterization of the human endothelial nitric-oxide synthase promoter. J. Biol. Chem. 274:3076 –3093, 1999.
13. KILBOM, A., and A. WENNMALM. Endogenous prostaglandins as local
regulators of blood flow in man: effect of indomethacin on reactive and
functional hyperemia. J. Physiol. (Lond.) 257:109–121, 1976.
14. KINGWELL, B. A., B. SHERRARD, G. L. JENNINGS, and A. M. DART.
Four weeks of cycle training increases basal production of nitric
oxide from the forearm. Am. J. Physiol. 272:H1070 –H1077, 1997.
15. KINGWELL, B. A., B. TRAN, J. D. CAMERON, G. L. JENNINGS, and
A. M. DART. Enhanced vasodilation to acetylcholine in athletes is
associated with lower plasma cholesterol. Am. J. Physiol. 270:
H2008 –H2013, 1996.
16. LIEBERMAN, E. H., M. D. GERHARD, A. UEHATA, et al. Flow-induced
vasodilation of the human brachial artery is impaired in patients
⬍40 years of age with coronary artery disease. Am. J. Cardiol.
78:1210 –1214, 1996.
17. LOSCALZO, J., and J. A. VITA. Ischemia, hyperemia, exercise, and
nitric oxide: complex physiology and complex molecular adaptations. Circulation 90:2556 –2559, 1994.
18. MALEK, A. M., L. JIANG, I. LEE, W. C. SESSA, S. IZUMO, and S. L.
ALPER. Induction of nitric oxide synthase mRNA by shear stress
requires intracellular calcium and G-protein signals and is modulated
by PI 3 kinase. Biochem. Biophys. Res. Commun. 254:231–242, 1999.
http://www.acsm-msse.org
19. MARSDEN, P. A., H. H. HENG, S. W. SCHERER, et al. Structure and
chromosomal localization of the human constitutive endothelial
nitric oxide synthase gene. J. Biol. Chem. 23:17478 –17488, 1993.
20. MEREDITH, I. T., K. E. CURRIE, T. J. ANDERSON, M. A. RODDY, P.
GANZ, and M. A. CREAGER. Postischemic vasodilation in human
forearm is dependent on endothelium-derived nitric oxide. Am. J.
Physiol. 270(4 Pt 2):H1435–H1440, 1996.
21. MESSINA, E. J., D. SUN, A. KOLLER, M. WOLIN, and G. KALEY. Role
of endothelium-derived prostaglandins in hypoxia-elicited arteriolar dilation in rat skeletal muscle. Circ. Res. 71:790 –796, 1992.
22. MOMBOULI, J. V., and P. M. VANHOUTTE. Endothelial dysfunction:
from physiology to therapy. J. Mol. Cell. Cardiol. 31:61–74, 1999.
23. NABER, C. K., D. BAUMGART, C. ALTMANN, W. SIFFERT, R. ERBEL,
and G. HEUSCH. eNOS 894T allele and coronary blood flow at rest
and during adenosine-induced hyperemia. Am. J. Physiol. Heart
Circ. Physiol. 281:H1908 –H1912, 2001.
24. NAJARIAN, T., A. M. MARRACHE, I. DUMONT, et al. Prolonged
hypercapnia-evoked cerebral hyperemia via K⫹ channel-and prostaglandin E2 dependent endothelial nitric oxide synthase induction. Circ. Res. 87:1149 –1156, 2000.
25. NAKAYAMA, M., Y. HIROFUMI, M. YOSHIMURA, et al. T-786 –⬎C
mutation in the 5'-flanking region of the endothelial nitric oxide
synthase gene is associated with coronary spasm. Circulation
99:2864 –2870, 1999.
26. ROSSI, G. P., S. TADDEI, A. VIRDIS, et al. The T-786C and
Glu298Asp polymorphisms of the endothelial nitric oxide gene
affect the forearm blood flow responses of caucasian hypertensive
patients. JACC 41:938 –945, 2003.
27. SMITS, P., S. B. WILLIAMS, D. E. LIPSON, P. BANITT, G. A. RONGEN,
and M. A. CREAGER. Endothelial release of nitric oxide contributes
PHYSICAL ACTIVITY, eNOS GENE, AND BLOOD FLOW
28.
29.
30.
31.
32.
33.
34.
35.
to the vasodilator effect of adenosine in humans. Circulation
92:2135–2141, 1995.
TESTA, M., P. V. ENNEZAT, K. L. VIKSTROM, et al. Modulation of
vascular endothelial gene expression by physical training in patients with chronic heart failure. Ital. Heart J. 1:426 – 430, 2000.
THOMAS, G. D., and R. G. VICTOR. Nitric oxide mediates contraction-induced attenuation of sympathetic vasoconstriction in rat
skeletal muscle. J. Physiol. (Lond.) 506:817– 826, 1998.
THORIN, E., P. L. HUANG, M. C. FISHMAN, and J. A. BEVAN. Nitric
oxide inhibits ␣2⫺adrenoceptor-mediated endothelium-dependent
vasodilation. Circ. Res. 82:1323–1329, 1998.
WANG, X. L., and J. WANG. Endothelial nitric oxide synthase gene
sequence variations and vascular disease. Mol. Genet. Metab.
70:241–251, 2000.
WYATT, A. W., J. R. STEINERT, C. P. WHEELER-JONES, et al. Early
activation of the p42/p44MAPK pathway mediates adenosineinduced nitric oxide production in human endothelial cells: a novel
calcium-insensitive mechanism. FASEB J. 16:1584 –1594, 2002.
YOSHIMURA, M., Y. HIROFUMI, M. NAKAYAMA, et al. Genetic risk
factors for coronary artery spasm: significance of endothelial nitric
oxide synthase gene T-786C and missense Glu298Asp variants.
J. Invest. Med. 48:367–374, 2000.
ZANCHI, A., D. K. MOCZULSKI, L. S. HANNA, M. WANTMAN, J. H.
WARRAM, and A. S. KROLEWSKI. Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric
oxide synthase gene polymorphism. Kidney Int. 57:405– 413,
2000.
ZANZINGER, J. Role of nitric oxide in the neural control of cardiovascular function. Cardiovasc. Res. 43:639 – 649, 1999.
Medicine & Science in Sports & Exercise姞
1997