© 2001 American Heart Association, Inc.
Vascular Biology |
Role of Sex Differences and Effects of Endothelial NO Synthase Deficiency in Responses of Carotid Arteries to Serotonin
From the Departments of Internal Medicine and Pharmacology, The Cardiovascular Center, The University of Iowa, and Veterans Administration Medical Center, Iowa City, Iowa.
Correspondence to Kathryn G. Lamping, PhD, Medical Services (111), VA Medical Center, 601 Highway 6 West, Iowa City, IA 52246. E-mail kathryn-lamping{at}uiowa.edu
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Abstract |
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Abstract—We examined the hypothesis that contraction of the carotid arteries to serotonin is normally inhibited by endothelial NO synthase (eNOS) and is enhanced in mice lacking the gene for eNOS. Because the influence of eNOS may vary with the sex of the mouse, we also tested whether responses to serotonin were dependent on sex. We studied carotid arteries in vitro from littermate control (eNOS+/+) mice, heterozygous (eNOS+/-) mice, and homozygous eNOS-deficient (eNOS-/-) mice (male and female). Contraction to serotonin was greater in male eNOS+/+ mice than in female eNOS+/+ mice. In male mice, contraction to serotonin increased by
40% and 2.5-fold in male
eNOS+/- and
eNOS-/- mice, respectively. Contraction
to serotonin was more than doubled in female
eNOS+/- mice and increased >5-fold in
arteries from eNOS-/- mice. In contrast,
maximum vasoconstriction to U46619 was similar in male and female
eNOS+/+,
eNOS+/-, and
eNOS-/- mice. Relaxation to
acetylcholine was not different in male and female
eNOS+/+ or
eNOS+/- mice but was absent in
eNOS-/- mice. These findings suggest
that the contraction of carotid arteries to serotonin is
influenced by the sex of the animal. eNOS deficiency in gene-targeted
mice is associated with enhanced contraction to serotonin,
particularly in female mice, providing direct evidence that eNOS is a
major determinant of vascular effects of serotonin. The
results with eNOS+/- mice suggest a
"gene-dosing" effect for vascular responses to
serotonin.
Key Words: NO synthase • acetylcholine • thromboxane • mice • genetically altered mice
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Introduction |
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The endothelium can modulate vascular function by the production and release of a variety of vasodilator and vasoconstrictor agents.1 NO released from the endothelium has been identified as a major endothelium-derived relaxing factor2 that primarily produces relaxation of vascular muscle by the activation of soluble guanylate cyclase and cGMP-dependent protein kinase I.3 In addition, studies that used pharmacological approaches and gene-targeted mice suggest that NO is the primary mediator of endothelium-dependent relaxation in several blood vessels, including coronary, carotid, and cerebral arteries.4 5 6 7 8 9
In addition to mediating vasorelaxation, a second major role for endothelium is to inhibit responses of vascular muscle to vasoconstrictors. For example, contraction of coronary and cerebral arteries to serotonin is augmented after the removal of endothelium.10 11 Mechanisms that may account for increased vasoconstrictor responses to serotonin after endothelial removal may include deficiency of NO or other endothelium-derived relaxing factors that oppose the direct contractile effect of serotonin on vascular muscle. Inhibitors of NO synthase (NOS) increase vasoconstriction and/or decrease vasodilation to serotonin,11 12 13 but limitations related to the specificity of these pharmacological agents may complicate the interpretation of the studies. This is particularly true if one is attempting to define the role of specific isoforms of NOS. For example, in addition to endothelial NOS (eNOS), recent studies suggest that neuronal NOS is expressed in vascular muscle and may also influence responses to vasoconstrictors.14 Thus, the specific role for eNOS in modulating vasoconstrictor responses has not been defined. The first major goal of these studies was to test the hypothesis that in the absence of eNOS, vasoconstriction to serotonin is enhanced.
Studies in animal models and in humans have demonstrated that some vascular responses are affected by the sex of the study subject.15 16 17 18 Mechanisms that account for sex differences in vascular responses are not well defined and may include differences in expression and/or activity of eNOS, neuronal NOS, endothelium-derived hyperpolarizing factor(s), or cyclooxygenase(s).19 20 21 Thus, it is unclear how the influence of eNOS on the regulation of vascular tone varies with sex.
The second goal of the present study was to test the hypothesis that constrictor responses of carotid arteries to serotonin are affected by the sex of the study subject. We also hypothesized that if sex differences in vascular reactivity are solely due to differences in eNOS, then in the absence of eNOS, sex differences will be abolished. To test these hypotheses, we measured the responses of carotid arteries from male and female mice wild-type (eNOS+/+) mice and mice completely deficient in the expression of the gene for eNOS (eNOS-/- mice). To determine whether deletion of a single copy of the gene for eNOS is sufficient to alter vascular responses, we also examined the responses of the carotid arteries from male and female eNOS+/- mice.
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Methods |
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Animals
The animal protocol used in these experiments was reviewed and approved by the University of Iowa Animal Care and Use Committee. Mice for this study (n=75) were derived from the breeding of eNOS+/- mice with eNOS+/- mice to generate eNOS+/+, eNOS+/-, and eNOS-/- mice within the same litter. This approach allowed us to use eNOS+/+ mice as littermate controls. For the present study, all 3 groups of mice were examined: wild-type control mice (eNOS+/+ littermates), eNOS heterozygous (eNOS+/-) mice, and homozygous eNOS-deficient (eNOS-/-) mice. These mice were originally generated as a hybrid of 129xC57BL/6J. Mice used in the present study were derived from 3 or 4 generations of backcross breeding to C57BL/6J mice. Mice were fed regular chow, and water was available ad libitum. The ages of mice in the different groups were similar (8 to 9 months).
Genotyping of mice was performed by Southern blotting DNA from tail biopsies. High-molecular-weight genomic DNA was isolated from tail biopsies, and identification of eNOS+/+, eNOS+/-, and eNOS-/- mice was accomplished as described previously.4
General Preparation
Mice were anesthetized with pentobarbital (75
to 100 mg/kg IP), and both carotid arteries were quickly removed. After
removal, arteries were placed in Krebs’ buffer with the following
ionic composition (mmol/L): NaCl 118.3, KCl 4.7,
CaCl2 2.5, MgSO4 1.2,
KH2PO4 1.2,
NaHCO3 25, and glucose 11. Loose connective
tissue on the adventitia was removed, and each carotid artery was cut
into 2 rings (3 to 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). Resting tension was increased stepwise
to reach the final tension of 0.2 to 0.25 g, and the rings were
allowed to equilibrate for at least 60 minutes. This amount of resting
tension is optimal for contraction of murine carotid arteries. We have
used these methods previously to study responses in carotid arteries
from mice in
vitro.4
Protocols
Contractile responses to serotonin (10
nmol/L to 30 µmol/L) and the thromboxane analogue
9,11-dideoxy-11a,9a-epoxy-methanoprostaglandin
F2
(U46619, 0.03 to 3 µg/mL) were measured
in carotid arteries from eNOS+/+,
eNOS+/-, and
eNOS-/- mice. Relaxation to
acetylcholine (10 and 100 nmol/L) was measured after submaximal
preconstriction of arteries with prostaglandin
F2 (PGF2, 10 to 50
µmol/L). We and others have previously shown by use of
pharmacological approaches and gene-targeted mice that responses of the
carotid artery to acetylcholine are mediated by
eNOS.4 22
Drugs
U46619 was obtained from Biomol Research
Laboratories, Inc, and dissolved in 100% ethanol. Acetylcholine,
serotonin, and PGF2 were obtained
from Sigma Chemical Co and dissolved in distilled water. All
concentrations are final molar concentrations in the organ
chamber.
Statistical Analysis
Contractions are presented as grams of
tension developed and are presented as mean±SEM. Relaxation in
response to acetylcholine is presented as percent change in
tension from the preconstriction. When multiple vessel rings were
studied from 1 mouse, responses were averaged, and n represents
the number of mice per group. Comparisons were made by using a 1-way
ANOVA with repeated measures followed by the Student-Newman-Keuls test
to detect individual differences. A value of
P<0.05 was defined as
significant.
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Results |
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Responses of Carotid Arteries From Wild-Type eNOS+/+ Mice
Serotonin produced concentration-dependent contraction of carotid arteries from male and female eNOS+/+ mice, which was maximal at a concentration of 1 to 3 µmol/L (Figure 1
). Contractions to serotonin were
greater in vessels from male eNOS+/+ mice
compared with female eNOS+/+ mice. For
example, 3 µmol/L serotonin contracted the artery by
0.14±0.01 and 0.05±0.01 g in male and female
eNOS+/+ mice, respectively
(Figure 1). Thus, maximal contractions to
serotonin were almost 3-fold greater in arteries from male
mice compared with arteries from female mice.
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In contrast, although contractions to U46619 at submaximal
concentrations were somewhat greater in carotid arteries from male
compared with female eNOS+/+ mice, the
maximal response to U46619 was similar in the 2 groups
(Figure 2, P>0.05).
For example, contraction of the carotid artery to 0.3 µg/mL was
0.46±0.01 and 0.35±0.01 g in male versus female mice, respectively
(Figure 2, P<0.05).
Acetylcholine produced concentration-dependent relaxation of carotid
arteries precontracted with PGF2
(Figure 3). Relaxation in response to acetylcholine was
similar in carotid arteries from male and female
eNOS+/+ mice
(Figure 3).
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Responses of Carotid Arteries From
eNOS+/- Mice
Deletion of a single copy of the gene for eNOS was
associated with increased contraction to serotonin in
arteries from female mice
(Figures 4 and 5). In contrast, deletion of a single copy of
the gene for eNOS resulted in no significant alteration in contraction
to serotonin in arteries from male mice
(Figure 5). For example, in arteries from female mice,
maximal contractions to serotonin were more than doubled
after deletion of a single copy of the eNOS gene
(Figures 4 and 5). In contrast to the response to
serotonin, contractions to U46619 were similar in carotid
arteries from female and male eNOS+/- mice
(Figure 2
). Deletion of a single copy of the gene for eNOS
had no effect on relaxation to submaximal concentrations of
acetylcholine in carotid arteries from either female or male mice
(Figure 3).
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Responses of Carotid Arteries From
eNOS-/- Mice
Contractions to serotonin were increased
markedly in carotid arteries from male and female
eNOS-/- mice compared with arteries from
eNOS+/+ mice. Maximal contractions to
serotonin were increased >5-fold in arteries from female
eNOS-/- mice compared with arteries from
female eNOS+/+ mice (0.27±0.02 versus
0.05±0.003 g, respectively;
P<0.05;
Figure 5). An example of the greatly augmented response to
serotonin in a female
eNOS-/- mouse is shown in
Figure 4. Maximal contractions to serotonin were
increased 2.6-fold in carotid arteries from male
eNOS-/- compared with
eNOS+/+ mice (0.36±0.01 versus 0.14±0.004
g, P<0.05;
Figure 5).
In contrast to responses to serotonin, maximal
contractions to U46619 were similar in arteries from male and female
eNOS-/- mice
(Figure 2). In the absence of eNOS (in
eNOS-/- mice), the response of carotid
arteries from male and female mice to acetylcholine was completely
abolished
(Figure 3).
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Discussion |
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There are 4 major new findings in the present study. First, vasoconstriction to serotonin and to a thromboxane A2 mimetic was affected by sex differences in eNOS+/+ mice; however, sex had a greater effect on responses to serotonin. Maximal contraction to U46619 was similar in arteries from male and female eNOS+/+ mice, whereas maximal contraction to serotonin was greater in arteries from male mice. Second, in contrast to serotonin and U46619, relaxation of carotid arteries to acetylcholine was similar in male and female eNOS+/+ mice. Third, deletion of a single copy of the gene for eNOS increased contraction to serotonin in carotid arteries from female mice but did not significantly alter contraction in arteries from male mice. Vasorelaxation to acetylcholine and contraction to U46619 were not altered in eNOS+/- mice. Fourth, deletion of both copies of the gene for eNOS abolished relaxation to acetylcholine and increased contraction to serotonin in arteries from male and female mice. The relative effect of complete eNOS deficiency on responses to serotonin was more pronounced in vessels from female mice compared with male mice. Contraction to U46619 was increased only modestly at submaximal concentrations in arteries from eNOS-/- female mice but was not altered in vessels from male mice. Thus, deletion of the gene for eNOS has a greater effect on responses of carotid arteries from female mice compared with male mice, and the effect was most prominent for contractions to serotonin.
Role of eNOS in Vascular Responses to
Serotonin
Serotonin is an important stimulus
that can exert marked effects on vascular tone. Serotonin
released by aggregating platelets produces
vasoconstriction23 and may
contribute to vasospasm under pathophysiological
conditions. Vascular responses to serotonin are complex,
involving a balance of direct contractile effects via activation of
5-hydroxytryptamine2 receptors
on smooth muscle and relaxation due to the release of vasodilator
substances from the
endothelium.24
Endothelial removal augments contraction of the blood
vessels to serotonin, suggesting that the release of
vasodilator
substances12 25 26
from the endothelium inhibits contraction to
serotonin.10 24
Pharmacological inhibition of NOS increases vasoconstrictor responses
to
serotonin12 13
and decreases relaxation to serotonin in coronary
arteries in which the contractile response is blocked with
ketanserin.27 Such an effect
could be due to the activity of eNOS, but because there are no
selective pharmacological inhibitors of eNOS, the role of
eNOS as an inhibitor of vasoconstrictor responses has not
been defined. This is further complicated by recent findings suggesting
that neuronal NOS is expressed in vascular muscle and can modulate
vasoconstrictor responses to
serotonin.14
The present study in eNOS-deficient mice has several unique advantages. First, we were able to examine the role of eNOS in modulating the responses selectively. Second, we were able to examine the role of eNOS without mechanical removal of the endothelium, which may damage smooth muscle and adventitia, particularly in small vessels, such as those from mice. Third, it is difficult to completely inhibit the production of NO by eNOS with pharmacological blockers.22 28 By studying vascular responses in mice completely deficient in the expression of the gene for eNOS, we have avoided this potential limitation. Finally, by studying vessels from mice deficient in a single copy of the eNOS gene, we can examine the effects of "gene-dosing." The present study suggests that eNOS modulates contractions to serotonin, inasmuch as deletion of 1 or both copies of the eNOS gene increased contractions of the carotid arteries. These findings from gene-targeted mice provide direct evidence that eNOS plays a major role in inhibiting vasoconstriction to serotonin.
Effect of Sex Differences on Vascular
Responses
Few studies have examined the influence of sex
differences on responses to serotonin or
thromboxane. Contraction of arteries to
serotonin and U46619 was found to be similar in male and
female rats and
pigs.21 29 30
In the present study, contraction of the carotid artery to
serotonin was greater in carotid arteries from male
compared with female eNOS+/+ mice. In
contrast, contraction to U46619 was only modestly increased at lower
concentrations, whereas the maximal contraction was similar. These
differences in the influence of sex on responses to
serotonin may be due to species or regional differences in
vascular responses. Although mice are increasingly being used in
studies of vascular
responses,6 we are not aware
of any previous studies demonstrating sex differences in
vasoconstrictor responses in mice.
Previous studies have suggested that abnormal vascular function can be detected relatively easily with the use of serotonin. For example, atherosclerosis is associated with hyperresponsiveness to serotonin, which tends to be greater than that observed with other vasoconstrictors.31 32 In previous studies from our laboratory, relaxation of coronary arteries to serotonin was impaired in genetically altered, hypercholesterolemic mice at a time when vasorelaxation to acetylcholine was still normal.27
In the present study, sex differences in vascular responses were more easily detected with the use of serotonin compared with acetylcholine or U46619. In the absence of a single copy of the gene for eNOS in female mice, contraction of the carotid artery to serotonin was more than doubled, whereas relaxation to acetylcholine was normal. Responses to U46619 were only modestly altered and only in vessels from female mice. These data suggest that responses to serotonin are particularly sensitive to the expression of eNOS. It is unclear whether the observed difference between acetylcholine and serotonin is related to the ability of acetylcholine to release greater amounts of NO or other effects within the vessel wall that influence responses to serotonin. The finding that vasoconstrictor responses to serotonin are affected to a greater extent than are responses to U46619 suggests that mechanisms other that the mere inhibition of vasoconstriction by basal NO must be involved. Basal levels of NO produced by eNOS are greater in vessels from females compared with males.33 However, the finding that contractile responses to serotonin were augmented to a greater extent than responses to U46619 may reflect the ability of serotonin to directly stimulate endothelium to produce NO. If the inhibition of vasoconstrictor responses was simply due to effects of basal NO, one might expect responses to serotonin and U46619 to be affected similarly. These data suggest that differences in responses to serotonin versus acetylcholine and U46619 may be related to an effect on endothelium and not smooth muscle; however, we did not test responses after the removal of the endothelium.
Vascular Responses to Acetylcholine: Effects of
Sex and eNOS Deficiency
Results of studies examining the influence of sex
differences on endothelium-dependent relaxation have
been quite variable. Relaxation to acetylcholine and flow-mediated
dilation is greater in female than in male
rats,17 18 but
basal release of NO is greater in aortas from female rabbits, and
agonist-stimulated release is similar in male and female rabbits and
pigs.29 30 33
Variable effects of sex have been described in humans as well in
sex-dependent15 34
and sex-independent responses to acetylcholine and flow-mediated
dilation.35 36 We
are aware of only 1 previous study in normal mice that examined the
influence of sex on endothelium-dependent
responses.37 In that study,
relaxation of the aorta to acetylcholine was similar in males and
females,37 which is
consistent with our finding that relaxation of the carotid
arteries is not affected by the sex of the study subject. Differences
in the effect of sex on responses to
endothelium-dependent agonists may be related to
differences in the mechanisms of agonist-mediated release of NO in
specific vessel types or species differences.
In addition to NO, other endothelium-derived relaxing substances may be involved in mediating responses to acetylcholine in some blood vessels.1 Some studies have suggested that there may be sex differences in the relative role of NO versus endothelium-derived hyperpolarizing factor as mediators of endothelium-dependent responses.20 This conclusion was based on the finding that there are sex differences in the NOS-sensitive component of the vascular response to acetylcholine.20 21 However, it is also possible that there are sex differences in tissue or cellular access of the NOS inhibitor as well as the extent of enzyme inhibition. Recent reports have provided direct evidence that it is very difficult to completely inhibit eNOS with pharmacological inhibitors.22 28
In the present study, vasorelaxation to acetylcholine was absent in male and female mice completely deficient in the gene for eNOS, consistent with previous work by us4 and others.38 These data indicate that eNOS is the mediator for responses of the carotid artery to acetylcholine, regardless of sex. The unique advantage of the present study in eNOS-deficient mice is that we have examined vascular responses in a model in which uncertainties related to the extent and selectivity of pharmacological inhibition of NOS are eliminated. The results also indicate that there is no upregulation or compensation for the lack of eNOS in the carotid artery, which can occur in the face of eNOS deficiency in other vascular beds.5 39
In summary, the present study indicates that vascular responses of the carotid artery to serotonin are influenced by the sex of the mouse. The data from gene-targeted mice provide direct evidence that eNOS plays a major role in influencing vascular responses to serotonin and acetylcholine but a much lesser role in responses to thromboxane. Selective eNOS deficiency produces enhanced contractile responses of the carotid artery to serotonin, particularly in female mice. The results with eNOS+/- mice suggests that a gene-dosing effect is present for vascular responses to serotonin. Finally, endothelium-dependent relaxation of the carotid artery in response to acetylcholine is mediated exclusively by eNOS in male and female mice.
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health (HL-39050, HL-38901, NS-24621, and HL-62984) and a grant from the American Heart Association. K.G.L. and F.M.F. are Established Investigators of the American Heart Association. We thank Kristen Rummelhardt for her valuable technical assistance. We also acknowledge Curt Sigmund, PhD, and the University of Iowa Transgenic Core for genotyping the mice used in these studies.
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Footnotes |
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Guest Editor was Alan M. Fogelman, MD, Department of Medicine, UCLA School of Medicine, Los Angeles, Calif.
Received October 5, 2000; accepted January 11, 2001.
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K. B. Brosnihan, P. Li, J. P. Figueroa, D. Ganten, and C. M. Ferrario Estrogen, nitric oxide, and hypertension differentially modulate agonist-induced contractile responses in female transgenic (mRen2)27 hypertensive rats Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H1995 - H2001. [Abstract] [Full Text] [PDF]
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 A. M. Beyer, G. L. Baumbach, C. M. Halabi, M. L. Modrick, C. M. Lynch, T. D. Gerhold, S. M. Ghoneim, W. J. de Lange, H. L. Keen, Y.-S. Tsai, et al. Interference With PPAR{gamma} Signaling Causes Cerebral Vascular Dysfunction, Hypertrophy, and Remodeling Hypertension, April 1, 2008; 51(4): 867 - 871. [Abstract] [Full Text] [PDF]
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 D. W. Nuno, V. P. Korovkina, S. K. England, and K. G. Lamping RhoA Activation Contributes to Sex Differences in Vascular Contractions Arterioscler Thromb Vasc Biol, September 1, 2007; 27(9): 1934 - 1940. [Abstract] [Full Text] [PDF]
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S. P. Didion, D. A. Kinzenbaw, L. I. Schrader, and F. M. Faraci Heterozygous CuZn Superoxide Dismutase Deficiency Produces a Vascular Phenotype With Aging Hypertension, December 1, 2006; 48(6): 1072 - 1079. [Abstract] [Full Text] [PDF]
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D.-D. Chen and A. F. Chen CuZn Superoxide Dismutase Deficiency: Culprit of Accelerated Vascular Aging Process Hypertension, December 1, 2006; 48(6): 1026 - 1028. [Full Text] [PDF]
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 F. M. Faraci Protecting the Brain With eNOS: Run for Your Life Circ. Res., November 10, 2006; 99(10): 1029 - 1030. [Full Text] [PDF]
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S. P. Didion and F. M. Faraci Ceramide-Induced Impairment of Endothelial Function Is Prevented by CuZn Superoxide Dismutase Overexpression Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 90 - 95. [Abstract] [Full Text] [PDF]
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F. M. Faraci, C. Lynch, and K. G. Lamping Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2871 - H2876. [Abstract] [Full Text] [PDF]
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G. L. Baumbach, C. D. Sigmund, and F. M. Faraci Structure of Cerebral Arterioles in Mice Deficient in Expression of the Gene for Endothelial Nitric Oxide Synthase Circ. Res., October 15, 2004; 95(8): 822 - 829. [Abstract] [Full Text] [PDF]
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J. J. Andresen, F. M. Faraci, and D. D. Heistad Vasomotor responses in MnSOD-deficient mice Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1141 - H1148. [Abstract] [Full Text] [PDF]
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 K. Budzyn, P. D. Marley, and C. G. Sobey Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R342 - R348. [Abstract] [Full Text] [PDF]
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K. G. Lamping, J. Wess, Y. Cui, D. W. Nuno, and F. M. Faraci Muscarinic (M) Receptors in Coronary Circulation: Gene-Targeted Mice Define the Role of M2 and M3 Receptors in Response to Acetylcholine Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1253 - 1258. [Abstract] [Full Text] [PDF]
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P. A. Ortiz and J. L. Garvin Cardiovascular and renal control in NOS-deficient mouse models Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2003; 284(3): R628 - R638. [Abstract] [Full Text] [PDF]
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 F. M. Faraci Vascular Protection Stroke, February 1, 2003; 34(2): 327 - 329. [Full Text] [PDF]
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Q. Fang, H. Sun, and W. G. Mayhan Impairment of nitric oxide synthase-dependent dilatation of cerebral arterioles during infusion of nicotine Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H528 - H534. [Abstract] [Full Text] [PDF]
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S. P. Didion, M. J. Ryan, L. A. Didion, P. E. Fegan, C. D. Sigmund, and F. M. Faraci Increased Superoxide and Vascular Dysfunction in CuZnSOD-Deficient Mice Circ. Res., November 15, 2002; 91(10): 938 - 944. [Abstract] [Full Text] [PDF]
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 L. V d'Uscio, M. Barton, S. Shaw, and T. F Luscher Chronic ETA receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice Cardiovasc Res, February 1, 2002; 53(2): 487 - 495. [Abstract] [Full Text] [PDF]
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 A. Huang, Y. Wu, D. Sun, A. Koller, and G. Kaley Effect of estrogen on flow-induced dilation in NO deficiency: role of prostaglandins and EDHF J Appl Physiol, December 1, 2001; 91(6): 2561 - 2566. [Abstract] [Full Text] [PDF]
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