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Departments of Internal Medicine, Pharmacology, and Physiology, Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa 52242; and Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We examined the
hypotheses that responses to acetylcholine are impaired and responses
to NO are enhanced in carotid artery from mice made deficient in
endothelial nitric oxide synthase (eNOS) by gene targeting
(eNOS-deficient mice). We also tested the hypothesis that deletion of
one copy of the eNOS gene is sufficient to alter vascular responses.
Vessels were studied in vitro from heterozygous (+/
) and
homozygous (/) eNOS-deficient mice as well as wild-type
[eNOS(+/+)] littermates. After precontraction with
prostaglandin F2
, acetylcholine
produced marked relaxation of carotid arteries in eNOS(+/+) mice, with
impaired vasorelaxation in eNOS(+/) mice. For example, 1 µM
acetylcholine relaxed carotid arteries by 55 ± 5% (mean ± SE)
in eNOS(+/) mice (n = 13)
compared with 83 ± 3% in eNOS(+/+) mice
(n = 14, P < 0.001 vs. +/). In contrast, acetylcholine caused no relaxation in carotid arteries from
eNOS(/) mice (P < 0.001 vs. +/+ and +/). Relaxation of the carotid artery in
response to nitroprusside [a nitric oxide (NO) donor] was
enhanced (P < 0.001) in
eNOS-deficient mice. For example, in response to 10 nM nitroprusside,
the carotid artery relaxed by 18 ± 2% in eNOS(+/+) mice
(n = 14), 33 ± 2% in
eNOS(+/) mice (n = 13), and 47 ± 4% in eNOS(/) mice
(n = 5). Thus relaxation of the
carotid artery is impaired with acetylcholine and enhanced with the NO
donor nitroprusside in eNOS-deficient mice. Enhanced responses to NO
may represent a compensatory response expressed in the absence of eNOS.
The findings that vascular responses to acetylcholine and NO are
altered in eNOS(+/) mice compared with those observed in
eNOS(+/+) mice suggest a "gene-dosing" effect.
gene targeting; nitric oxide; acetylcholine; nitroprusside; soluble guanylate cyclase; NG-nitro-L-arginine; endothelial nitric oxide synthase
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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PRODUCTION AND RELEASE of nitric oxide (NO) by NO synthase (NOS) in endothelium are thought to play a major role in regulation of vascular tone (8, 14). Three isoforms of NOS, which are products of separate genes, have been identified and are designated as neuronal, inducible, and endothelial (eNOS) NOS (9, 14). We and others have used pharmacological inhibitors of NOS to examine the role of different isoforms of NOS in regulation of vascular tone (see Ref. 8 for review). A common approach is to use analogs of L-arginine, including NG-nitro-L-arginine (L-NNA), to inhibit activity of NOS. Although inhibitors of NOS are very useful in examination of the role of NO in vascular responses, a major limitation is that most agents nonselectively inhibit all isoforms of NOS (20, 25). There are no known selective inhibitors of eNOS.
Mice with targeted disruption of the gene encoding eNOS (11, 22) provide an opportunity to examine the role of eNOS in complex physiological systems. Through studies performed in eNOS-deficient mice, the role of the endothelial isoform of NOS can be defined without relying on pharmacological inhibitors that lack specificity for NOS isoforms.
Recent studies suggest that, in the presence of inhibition or impairment of eNOS (19, 23), compensatory mechanisms may regulate vascular tone. In mice that are deficient for the eNOS gene, dilatation of cerebral arterioles in response to acetylcholine has been reported to be normal in magnitude but mediated by non-eNOS-dependent mechanisms (16). In contrast to cerebral arterioles, relaxation of the aorta in response to acetylcholine is absent in eNOS-deficient mice (11).
The first goal of the present study was to examine the hypothesis that responses of the carotid artery to acetylcholine are absent in mice lacking the gene for eNOS. Because the previous study used only aorta, this represents the first study of a large muscular artery from eNOS-deficient mice. The study was performed in vitro, which allows examination of mechanisms without the potential influence of NOS in surrounding tissue. As part of these experiments, we also examined mechanisms that mediate responses to acetylcholine in carotid arteries from normal [wild-type, eNOS(+/+)] mice. Because mechanisms that mediate endothelium-dependent responses in mouse carotid arteries have not been studied previously, it was important to exclude the possibility that non-NO-dependent mechanisms may mediate relaxation to acetycholine.
Relaxation of blood vessels in response to NO or NO donors is typically enhanced after acute inhibition of NOS (1, 6, 7, 17, 23). Thus the second goal of the present study was to examine the hypothesis that relaxation of the carotid artery in response to nitroprusside (an NO donor) is enhanced in mice lacking the gene for eNOS.
To our knowledge, previous studies have not examined vascular responses
in any heterozygous gene-targeted animals. Thus our third goal was to
examine the hypothesis that deletion of one copy of the eNOS gene was
sufficient to alter vascular responses. Altered vascular responses in
eNOS(+/) mice compared with eNOS(+/+) mice would suggest that a
"gene-dosing" effect was present for eNOS gene expression.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animals. eNOS-deficient mice were
produced as described previously (22). We interbred eNOS(+/)
mice to generate eNOS(+/+), eNOS(+/), and eNOS(/)
mice within the same litter. This approach allowed us to use eNOS(+/+)
littermates as controls. For all studies in which we compared responses
of eNOS(+/+) with eNOS(+/) and eNOS(/) mice, we
used littermates as the wild-type control. For these studies, each
mouse was genotyped. For some studies of mechanisms of vasorelaxation
in wild-type mice, C57BL/6J mice were obtained from Jackson
Laboratories.
Genotyping of the animals was performed by Southern blotting DNA from
tail biopsies. High-molecular-weight genomic DNA was isolated from tail
biopsies as previously described (5, 10). The identification of
eNOS(+/+), eNOS(+/), and eNOS(/) mice was
essentially as described previously (22). Briefly, 10-µg samples were
digested with BamH I, separated on
0.8% agarose gels, and then transferred to nylon-supported
nitrocellulose. The blots were then hybridized using a random
primer-labeled 1.4-kb eNOS cDNA probe that was described previously
(22). A 5.3-kb fragment was diagnostic of the endogenous eNOS locus,
and a 6.4-kb fragment was diagnostic of the targeted allele.
Vascular ring preparation. Mice were anesthetized with pentobarbital sodium (75-100 mg/kg ip), and both carotid arteries were quickly removed and placed in Krebs buffer with the following ionic composition (mmol/l): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose. Loose connective tissue in the adventitia was removed, and each carotid artery was cut into two rings (3-4 mm in length). Vascular rings were suspended in an organ bath containing 25 ml Krebs solution maintained at 37°C. The rings were connected to a force transducer to measure isometric tension (contraction and relaxation). Resting tension was increased stepwise to reach the final tension of 0.2-0.25 g, and the rings were allowed to equilibrate for at least 60 min. Preliminary studies indicated that this amount of resting tension was optimal for contraction in these arteries.
Protocols. Vessels were contracted
submaximally (30-55% of maximum) using prostaglandin
F2
(PGF2). In initial studies, we found that PGF2 produces
stable and reproducible contraction of carotid arteries. After reaching
a stable contraction plateau, dose-response curves were obtained for
acetylcholine or sodium nitroprusside. Dose-response curves were
obtained for each agonist. Acetylcholine was used to assess endothelial
function, and nitroprusside was used as an NO donor to examine direct
effects on smooth muscle. We have used these techniques for studies of
mouse vessels in vitro previously (3).
We examined mechanisms that mediate relaxation to acetylcholine and
nitroprusside in carotid arteries from eNOS(+/+) mice. For
these studies, responses to acetylcholine and nitroprusside were
obtained in the absence and presence of L-NNA (100 µM),
an inhibitor of NOS, or 1H-(1,2,4)oxadiazolo(4,3,-
)quinoxalin-1-one (ODQ, 10 µM), an inhibitor of soluble guanylate cyclase (18, 24).
Between each concentration-response curve, the vessels were washed at
least three times with fresh Krebs buffer and allowed to reequilibrate.
Vasoconstrictor responses to
PGF2 tended to be enhanced in
arteries taken from eNOS(+/+) mice and treated with L-NNA
or ODQ. Thus higher concentrations of
PGF2 were sometimes used in
untreated vessels to produce similar contraction to that seen in
vessels treated with either inhibitor. Similarly, vasoconstrictor
responses to PGF2 tended to be
enhanced in arteries taken from eNOS(+/) and
eNOS(/) mice compared with arteries from eNOS(+/+) mice,
and higher concentrations of
PGF2 were sometimes used in
vessels from eNOS(+/+) mice to produce contraction that was similar to
that seen in vessels from eNOS(+/) and eNOS(/)
mice.
At the end of each experiment, we examined responses of carotid
arteries to the thromboxane analog
9,11-dideoxy-11, 9-epoxy-methanoprostaglandin F2 (U-46619) to determine
maximal contractile responses. In preliminary experiments, we found
that U-46619 produces greater maximal contraction of mouse carotid
arteries than high concentrations of KCl. Maximum contraction was
similar in carotid arteries from eNOS(+/+), eNOS(+/), and
eNOS(/) mice (0.66 ± 0.06, 0.65 ± 0.05, and 0.71 ± 0.10 g, respectively).
Statistical analysis. All data are
expressed as means ± SE. Relaxation responses to acetylcholine and
nitroprusside were expressed as the percent relaxation from the amount
of precontraction produced by
PGF2. Comparisons were made
using analysis of variance followed by Bonferroni's multiple
comparison test. Statistical significance was accepted at
P < 0.05.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Vascular responses in eNOS(+/+) mice. In vessels from normal mice, acetylcholine produced concentration-dependent relaxation (Figs. 1 and 2). In normal mice, relaxation of the carotid artery in response to acetylcholine was markedly attenuated by L-NNA (100 µM; Figs. 1 and 2) or ODQ (Figs. 3 and 4). For example, maximum vasorelaxation in response to acetylcholine was 85 ± 2 and 10 ± 3% in the absence and presence of ODQ, respectively (Fig. 4). These findings suggest that relaxation in response to acetylcholine is mediated almost exclusively by NO and activation of soluble guanylate cyclase in the mouse carotid artery.
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Relaxation of the carotid artery in response to nitroprusside was enhanced in the presence of L-NNA (Fig. 2). In contrast to L-NNA, ODQ produced marked inhibition of vasorelaxation in response to nitroprusside (Fig. 4). For example, relaxation in response to 3 µM nitroprusside was 80 ± 4 and 5 ± 3% in the absence and presence of ODQ, respectively (Fig. 4). These findings suggest that relaxation of the mouse carotid artery in response to nitroprusside is mediated by activation of soluble guanylate cyclase.
Vascular responses to acetylcholine in
eNOS(+/) mice. In eNOS(+/) mice,
the lower concentrations of acetylcholine produced relaxation of the
carotid artery that was similar to that observed in
eNOS(+/+) mice (Figs. 5 and
6). However, higher concentrations of acetylcholine
produced less relaxation in eNOS(+/) mice than in eNOS(+/+)
mice. For example, 1 µM acetylcholine produced 55 ± 5 and 83 ± 3% relaxation in eNOS(+/) and eNOS(+/+) mice,
respectively [P < 0.001 vs.
eNOS(+/+); Fig. 6]. Furthermore, higher concentrations of
acetylcholine often produced contraction of the carotid artery in
eNOS(+/) mice (Fig. 5). In these studies of carotid arteries, contraction to higher concentrations of acetylcholine was observed in
only 1 out of 14 eNOS(+/+) mice but was seen in 9 out of 13 eNOS(+/) mice. Altered responses to acetylcholine suggest that endothelial function is altered in eNOS(+/) mice.
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Vascular responses to acetylcholine in
eNOS(/) mice. In contrast to eNOS(+/+)
mice and eNOS(+/) mice, acetylcholine did not produce relaxation
of the carotid artery from eNOS(/) mice (Fig. 6). In
eNOS(/) mice, higher concentrations of acetylcholine often produced contraction of the carotid artery (see Fig.
7 for example). Thus relaxation of the carotid artery to
acetylcholine is absent in eNOS(/) mice.
Vascular responses to sodium nitroprusside.
Sodium nitroprusside caused marked relaxation of the
carotid artery from eNOS(+/+), eNOS(+/), and
eNOS(/) mice (Fig. 8). Relaxation of the
carotid artery in response to nitroprusside was augmented in
eNOS-deficient mice, particularly in eNOS(/) mice (Fig.
8). For example, in response to 10 nM nitroprusside, the carotid artery
relaxed by 18 ± 2% in eNOS(+/+) mice, 33 ± 2% in
eNOS(+/) mice, and 47 ± 4% in eNOS(/) mice.
In contrast to responses to acetylcholine in eNOS(+/) mice,
transient contractions of the carotid artery were not observed during
application of nitroprusside in any group of mice. Maximum relaxation
to 10 µM nitroprusside was similar in eNOS(+/+), eNOS(+/), and
eNOS(/) mice (90 ± 6, 89 ± 3, and 93 ± 4%,
respectively). Thus relaxation of the carotid artery to low
concentrations of the NO donor nitroprusside is increased in
eNOS(+/) and eNOS(/)
mice.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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There are several major new findings in the present study. First, the
pharmacological data obtained using L-NNA and ODQ suggest that relaxation of the mouse carotid artery to acetylcholine is mediated normally by NO and activation of soluble guanylate cyclase. Absence of relaxation to acetylcholine in eNOS(/) mice
provides direct evidence that relaxation of the carotid artery in
response to acetylcholine is mediated by eNOS. Second, altered
responses to acetylcholine suggest that altered endothelial function is present in eNOS(+/) mice. Thus deletion of one copy of the eNOS gene appears to be sufficient to alter responses to acetylcholine in
carotid arteries. Third, data obtained with ODQ suggest that relaxation
of the mouse carotid artery to nitroprusside is mediated by activation
of soluble guanylate cyclase. Interestingly, vasorelaxation in response
to nitroprusside is enhanced in both eNOS(+/) and eNOS(/) mice. The finding that responses to nitroprusside
are increased in eNOS(+/) mice also suggests that deletion of
one copy of the eNOS gene is sufficient to alter vascular responses. Thus responses to both an endothelium-dependent and -independent agonist are altered in eNOS(+/) mice.
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To our knowledge, the only study that examined responses of isolated
vessels from eNOS-deficient mice [eNOS(/)] is
that of Huang et al. (11), who studied the aorta in vitro. Our findings confirm and extend results obtained previously using aorta and indicate
that acetylcholine does not produce relaxation in the carotid artery (a
large muscular artery) from eNOS(/) mice. In addition, we
obtained two major new findings in vessels from eNOS-deficient mice.
The first finding is that deletion of one copy of the eNOS gene is
sufficient to alter vascular responses to the endothelium-dependent
agonist acetylcholine. The second finding is that relaxation of the
carotid artery to NO is enhanced in eNOS-deficient mice and that
deletion of one copy of the eNOS gene is sufficient to alter vascular
responses to NO.
Relaxation of carotid artery to acetylcholine in eNOS-deficient mice. Although the use of pharmacological inhibitors of NOS is useful in examination of the role of NO in blood vessels, a major limitation is that these analogs of L-arginine nonselectively inhibit all isoforms of NOS (14, 20, 25). Importantly, there are no selective inhibitors of eNOS. Studies of vessels in eNOS-deficient mice allow the definition of the contribution of the endothelial isoform of NOS in producing relaxation in response to vasoactive stimuli, without relying exclusively on inhibitors that lack specificity for NOS isoforms.
In the present study, we found that relaxation of the carotid artery in
response to acetylcholine is absent in eNOS(/) mice. This
response is in marked contrast to that observed in eNOS(+/+) mice. In
eNOS(/) and eNOS(+/) mice, acetylcholine often
produced contraction of the carotid artery. This latter effect could be the result of direct contractile effects of acetylcholine on vascular muscle or possible release of an endothelium-derived contracting factor. These findings provide direct evidence that relaxation of the
carotid artery to acetylcholine is mediated by eNOS.
Few studies have examined endothelium-dependent relaxation of mouse
blood vessels in vitro. In the aorta from mice, we (3) and others (11,
13, 21) have shown that relaxation in response to acetylcholine is
attenuated substantially by inhibitors of NOS, suggesting that the
response is mediated by NO (endothelium-derived relaxing factor). All
previous studies have examined responses in aorta, and this is the
first study to examine responses of carotid or other muscular arteries
from mice. In normal mice, acetylcholine produced relaxation of the
carotid artery, which was markedly attenuated by the NOS inhibitor
L-NNA. Thus genetic evidence obtained with
eNOS(/) mice is consistent with pharmacological data
obtained using L-NNA in eNOS(+/+) mice. In addition,
vasorelaxation in response to acetylcholine was almost completely
inhibited by ODQ, indicating that relaxation of normal carotid artery
in response to endogenously produced NO (endothelium-derived relaxing
factor) is mediated by activation of soluble guanylate cyclase.
In eNOS(+/) mice, low concentrations of acetylcholine produced
relaxation of the carotid artery that was similar to that observed in
eNOS(+/+) mice. Higher concentrations of acetylcholine produced
contraction of the carotid artery. Thus higher concentrations of
acetylcholine produced less relaxation in eNOS(+/) mice than in
eNOS(+/+) mice. The data suggest that deletion of one copy of the eNOS
gene is sufficient to alter endothelial function in the carotid artery.
Differences in vascular responses to acetylcholine in the
eNOS(+/) and eNOS(+/+) mice suggest a gene-dosing effect. Previous studies have provided evidence for gene dosing in relation to
regulation of blood pressure (12, 15). To our knowledge, the present
study provides the first evidence to suggest that gene dosing may also
be present for vascular responses.
Relaxation of carotid artery to nitroprusside in eNOS-deficient mice. NO can potentially produce relaxation of vascular muscle by guanylate cyclase-dependent or by guanylate cyclase-independent mechanisms. In the present study, vasorelaxation to nitroprusside was attenuated markedly by ODQ, a recently developed inhibitor of soluble guanylate cyclase that appears to be very specific (18, 24). For example, ODQ inhibits vasorelaxation in response to acetylcholine and nitroprusside but not adenosine or an analog of guanosine 3',5'-cyclic monophosphate (24). The efficacy of ODQ suggests that relaxation of mouse carotid artery in response to nitroprusside is mediated essentially entirely by activation of soluble guanylate cyclase.
Relaxation of the carotid artery in response to low concentrations of
nitroprusside was augmented in eNOS(+/) and
eNOS(/) mice. Thus relaxation to the NO donor
nitroprusside is augmented in eNOS-deficient mice, perhaps as a
compensatory response to reductions in the amount of eNOS in blood
vessels. The finding that vasorelaxation in response to nitroprusside
is enhanced in eNOS-deficient mice is similar to results published by
us (6, 7, 17) and others (1, 23) in which relaxation of cerebral vessels in response to NO or NO donors is enhanced after acute pharmacological inhibition of NOS with agents such as
L-NNA. In the present study in eNOS(+/+) mice, we also
observed enhancement of responses of the carotid artery to
nitroprusside after treatment with L-NNA. Thus genetic
evidence obtained with eNOS-deficient mice is consistent with
pharmacological evidence obtained using L-NNA in eNOS(+/+)
mice. Similarities in findings suggest that enhanced vasorelaxation in
response to NO donors obtained in previous studies (1, 6, 7, 17, 23)
was not due to a nonselective effect of pharmacological inhibitors of
NOS. The finding that relaxation of the carotid artery to nitroprusside
was enhanced in eNOS(+/) mice compared with eNOS(+/+) mice is
additional evidence that deletion of one copy of the eNOS gene is
sufficient to alter vascular responses.
Compensatory mechanisms in eNOS-deficient
mice. Both our genetic and pharmacological data
indicate that relaxation of the carotid artery in response to
acetylcholine is mediated by eNOS. The finding that acetylcholine does
not produce relaxation in eNOS(/) mice indicates that
formation of a non-NO endothelium-derived hyperpolarizing factor does
not contribute to endothelium-dependent relaxation. After selective
deletion of a gene, it is not uncommon for such gene-targeted animals
to display no or minimal differences in phenotype (26). Such findings
may suggest the presence of redundant or compensatory mechanisms after
selective gene deletion. In pial arterioles of eNOS(/)
mice, responses to acetylcholine are thought to be mediated by neuronal
NOS (16).
Although expression of compensatory mechanisms can occur in gene-deficient mice, we found no evidence for such compensation in the carotid artery with regard to responses to acetylcholine. Because NO is thought to inhibit production or release of endothelium-derived hyperpolarizing factor in the carotid artery (2), eNOS-deficient mice seemed to be an ideal model in which to test for the presence of non-NO endothelium-dependent relaxation (such as vasorelaxation mediated by endothelium-derived hyperpolarizing factor). We found, however, no evidence for a non-NO endothelium-derived relaxing factor in mouse carotid artery. Our data, which suggest that responses of the carotid artery to acetylcholine are mediated exclusively by eNOS and soluble guanylate cyclase, are consistent with a recent study using rabbit carotid artery (4). Nevertheless, our findings do not exclude the possibility that NO itself may function as an endothelium-derived hyperpolarizing factor in the carotid artery, as suggested previously (4).
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ACKNOWLEDGEMENTS |
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We thank Dr. Kathryn Lamping for critical evaluation of the manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-38901, NS-24621, HL-16066, HL-55006, and HL-14388. F. M. Faraci and C. D. Sigmund are Established Investigators of the American Heart Association.
Address for reprint requests: F. M. Faraci, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242-1081.
Received 15 September 1997; accepted in final form 14 October 1997.
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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P < 0.05 vs.
eNOS(+/+) mice.
