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Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus Ohio, 43210, USA. E-mail: Bauer.140{at}osu.edu
1Correspondence: Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus OH, 43210, USA. E-mail: Bauer.140{at}osu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: nitric oxide • vascular • peroxynitrite • endothelium • angiotensin II • oxidation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Early studies of this hormone demonstrated its potent
vasoconstrictive actions, while more recent studies have defined
important effects on vascular cell growth, differentiation, and gene
expression (3
, 4
, 5)
. Recent studies using isolated
vascular cells demonstrate that ANG II promotes the production of
oxygen radicals (particularly superoxide anion) (6
, 7)
,
and this may be an important component of ANG II-mediated
cardiovascular disease (8
, 9)
. Although a role for ANG II
in cardiovascular disease is well established, the mechanisms by which
it participates have not been elucidated. In particular, the early and
initiating events during elevated ANG II levels in vivo are
not well defined.
In the last decade, recognition of the importance of vascular
endothelial cells for maintenance and regulation of vascular health has
emerged (10
, 11)
. Nitric oxide (NO) derived from vascular
endothelial cells has been shown to be a critical modulator of local
vascular tone and thrombus formation; deficient endothelial NO
production has been demonstrated in a wide array of cardiovascular
diseases including hypertension, atherosclerosis, unstable angina, and
congestive heart failure (10
, 11
, 12)
. The chemistry of NO
in biological matrices is very complex, and several biochemical
pathways other than NO production can influence NO actions (13
, 14)
. For example, superoxide anion interacts with NO, reduces
its efficacy as a signal transduction agent, and promotes the formation
of peroxynitrite, a highly reactive intermediate known to nitrate
protein tyrosine residues and cause cellular oxidative damage
(15
, 16)
. The reaction of NO with superoxide anion occurs
at a diffusion-limited reaction rate and NO is the only molecule known
to compete with superoxide dismutase for its substrate in a biological
setting (17
, 18
, 19)
. Thus, excess or uncontrolled
superoxide anion formation can shift the actions of available NO from a
useful cellular signal to peroxynitrite and redox-related toxic
products.
While dysfunction of vascular endothelium and elevated blood concentrations of ANG II are each commonly observed in many forms of cardiovascular disease, few studies have evaluated the relationships between these phenomena. Here we investigated the actions of ANG II in vivo, testing the hypotheses that sub-pressor ANG II causes selective vascular dysfunction and that vascular peroxynitrite formation (and attendant dysregulation of NO control) participates in this phenomenon.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 5)
. ANG II
was kindly supplied by Bachem Biosciences Inc. (King of Prussia, Pa.).
Prior to pump implantation (day 0) and on day 3 of infusion, systemic
blood pressure and heart rate were evaluated in conscious rats using a
tail cuff apparatus (Stoelting Instruments, Wood Dale, Ill.). After
pentobarbital overdose (75 mg/kg i.p.), blood was rapidly collected by
cardiac puncture and vascular tissues were rapidly isolated.
Plasma renin activity
Blood was collected by cardiac puncture at death and
plasma was rapidly collected. Plasma renin activity was measured by a
commercially available radioimmunoassay kit adapted for low-volume
samples (Incstar, Stillwater, Minn.). Activity was determined as
nanogram of angiotensin I produced per ml/h. Intra-day and inter-day
assay variability was less than 10%.
Isolated vascular studies
After animals were killed, the thoracic aorta was rapidly
isolated for functional evaluations using methods similar to those
previously described (20
, 21)
. Vascular segments (2–3 mm)
were mounted on isometric force transducers (Grass Instruments, Quincy,
Mass.) and incubated in 10 ml organ baths containing Krebs’ buffer
bubbled with 95% O2 at 37°C. After 90 min
equilibration (resting tone 1.0 g), maximal contractile force was
determined for each segment using a high potassium concentration
(modified Kreb’s buffer containing 14.4 mM NaCl and 125 mM KCl). This
complete depolarization was used to define maximal contractile response
and was considered 100% contraction. Segments were then consecutively
washed with Krebs’ buffer four times, with two 5 min intervals and two
10 min intervals, and allowed to equilibrate for 30 min. Cumulative
responses to cumulative concentrations of phenylephrine were then
determined. In parallel studies, in vitro responses to ANG
II were evaluated by single exposure of concentrations ranging from 1
nM to 1 µM (thus preventing development of tachyphylaxis).
After precontraction with phenylephrine at 80% of maximum, relaxant
responses to cumulative acetylcholine were assessed. In preliminary
experiments, acetylcholine relaxation was completely blocked by 200
µM L-nitroarginine (a nonspecific NOS inhibitor); thus, the
vasodilatory response is apparently endothelium dependent and NO
mediated in this vascular tissue. Relaxant responses to cumulative
sodium nitroprusside were also determined (an endothelium independent
but NO-mediated response) (22)
. Contractile and relaxant
response data were fit to the 4-parameter logistic equation using
GraphPad Prism Software (GraphPad Software, Inc., San Diego, Calif.)
(20
, 21)
.
Immunohistochemistry
Immunohistochemistry was performed to determine the relative
extent of protein nitration (a stable biomarker of endogenous
peroxynitrite formation), using an antibody raised against
3-nitrotyrosine (3-NT). A section of aorta from each rat, separated
from the isolated vascular tissue studies, was fixed in formalin and
embedded in paraffin. Aorta cross sections were cut into 5 µm
sections, deparaffinized, and rehydrated. Tissues were then exposed to
a 3% hydrogen peroxide/methanol solution for 10 min to block
endogenous peroxidase activity. To restore antigenicity, slides were
immersed for 15 min in a 10 mM citrate buffer (pH 6) preheated to
boiling. Sections were incubated for 30 min in a 10% normal goat
serum/phosphate-buffered saline solution to block nonspecific antibody
binding. Tissue sections were incubated with rabbit anti-mouse
polyclonal antibody raised against 3-NT (1:200 dilution, Upstate
Biotechnology, Lake Placid, N.Y.) for 1 h. Biotinylated secondary
anti-rabbit serum was then applied (1:200), followed by horseradish
peroxidase complex reagent (ABC Elite, Vector Laboratories, Burlingame,
Calif.). Positive immunoreactivity was visualized through the
development of diaminobenzidine (DAB) chromogen. Harris’ modified
hematoxylin was used for nuclear counterstaining. Preliminary
experiments were conducted to verify the specificity of immunostaining
in our laboratory. Preincubation of primary antibody with free 3-NT (1
mM) completely quenched positive tissue staining, whereas tyrosine (1
mM) had no effect. Nonimmune serum (isotypic) controls also showed no
detectable immunoreactivity in any treatment group.
Digital image analysis
Images of immunostained tissue were captured using an Olympus
microscope (BX-40) and a high-resolution digital camera (Pixera, Inc.,
1260x960 pixel resolution). Unmodified images were analyzed using
research-based image analysis software (ImagePro Plus, Media
Cybernetics, Md.). Six endothelial regions of each aortic ring were
systematically captured at 200x, representing ~45% of the total
vascular endothelium circumference. Relative extent of 3-NT
immunoreactivity was assessed using digital color image analysis.
Similar to other reports (23)
, we observed the most
striking difference in ‘positive’ vs. background staining regions in
the blue channel of RGB color profile (i.e., red, green, blue). There
was a well-defined segmentation of the intensity profile such that
brown DAB staining registers between 0 and 100 intensity units, whereas
shades of blue or white background were consistently greater than 100
intensity units, as shown in Fig. 4
. For each vascular image, the
percentage of positive pixels in the captured images was used as an
objective and semi-quantitative measure of immunoreactivity
(24)
, allowing statistical comparisons among groups
(25)
. Intra-observer variability was consistently less
than 12% (coefficients of variation from 3 daily measurements),
whereas inter-observer variability was less than 10% (coefficients of
variation of average measurements between three observers collecting
six images on three different days).
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Statistical analyses
All data are presented as mean ± SE.
Statistical evaluations were performed using Sigmastat software
(Jandel Scientific Inc., San Rafael, Calif.). Comparisons among
treatment groups were performed using Student’s t tests or
analysis of variance where appropriate (26)
. Spearman
nonparametric correlation analysis was used to test for significant
association of functional and immunohistochemical data
(26)
. In all cases, P < 0.05 was deemed
statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows changes in plasma renin activity, hemodynamics, and cardiac mass
after ANG II or vehicle infusion for 3 days. Statistically significant
reductions in plasma renin activity were observed by day 3 after ANG II
administration relative to vehicle infusion, illustrating hormonal
feedback of the renin-angiotensin axis and demonstrating successful
infusion of low-dose ANG II. There was no significant change in plasma
aldosterone concentrations (data not shown). In contrast, ANG II did
not cause significant changes in systolic blood pressure or heart rate
by day 3 (Fig. 1)
. Similarly, no significant change in body weight,
total heart weight, or left ventricular weight (all parameters known to
be affected at higher ANG II exposures) was observed by day 3. Aortic
cross sectional areas were also unaffected by ANG II infusion (data not
shown).
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Isolated vascular contractile responses are shown in Fig. 2
. Maximal vascular contractile response to total depolarization (125 mM
KCl) was not different between ANG II and vehicle-infused groups.
Contractile responses to the 
-receptor agonist phenylephrine were
also unaltered (EC50 49±12 vs. 73±14 nM; Emax
1.09±0.06 vs. 0.99±0.06 g; ANG II vs. control, respectively).
Contractile response to ANG II in vitro was not
statistically affected by 3 day infusion (EC50
280±110 vs. 183±41 nM; Emax 0.41±0.12 vs. 0.34±0.8 g; ANG II
infused vs. control, respectively).
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Despite unaltered vascular contractile properties, significant
alteration in acetylcholine-induced relaxation was observed (Fig. 3
). A statistically significant sixfold reduction in
EC50 (390±12 vs. 64±13 nM, ANG II infused vs.
vehicle infused) was observed, with no change in maximal relaxant
response (Emax 93±4 vs. 109±4). No significant differences in extent
of phenylephrine-induced precontraction were observed among treatment
groups.
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In contrast to diminished acetylcholine responses, no significant
change in vasorelaxant response to the endothelium independent and
‘spontaneous’ NO donor sodium nitroprusside was observed between
treatments (Fig. 3
, EC50 1.11±0.3 nM vs.
0.84±0.2 nM; Emax 117±9 vs. 107±1; ANG II vs. vehicle,
respectively).
Representative photomicrographs of vascular immunostaining for 3-NT are
shown in Fig. 4
. Extensive protein nitration was observed in the vasculature from ANG
II-treated animals and was found nearly exclusively in the intimal
layer. In contrast, little evidence of 3-NT was found in control
(vehicle infusion). Figure 4
also shows the distinct color distribution
patterns for diaminobenzidine (positive signal for 3-NT
immunohistochemistry) and hematoxylin counterstain. This distinction
was used to determine the percentage of the cross sectional area
considered a positive signal using digital image analysis (see
Materials and Methods).
Digital image analysis demonstrated statistically significant increases
in vascular 3-NT immunoprevalence from ANG II-treated animals relative
to vehicle infusion controls (Fig. 4)
. This difference was confined to
the endothelial layer (no significant differences were observed when
regions of exclusively vascular smooth muscle were compared; see
Fig. 5
). In addition to comparing the two treatment groups, controls were used
to evaluate sensitivity and selectivity of the primary 3-NT antibody.
The positive immunostaining signal could be completely abolished by
preincubation of primary antibody with free nitrotyrosine in solution
(1 mM) or by replacing it with preimmune serum. Significant 3-NT
immunoprevalence was observed in the vehicle treatment group when
compared to these staining controls, demonstrating a slight but
detectable protein nitration (and corresponding peroxynitrite
production) under control conditions (Fig. 5
, top panel).
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The relationship between the prevalence of endothelial protein
nitration and endothelium-dependent functional response to
acetylcholine is shown in Fig. 5
(bottom panel). Measurements of 3-NT
immunoprevalence (using image analysis) and acetylcholine
EC50 were related for each animal in the study. A
highly statistically significant inverse correlation was observed
between the extent of intimal protein nitration and
endothelium-dependent acetylcholine functional response
(P<0.02, Spearman nonparametric correlation analysis).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Even though ANG II was first recognized as a
vasoconstrictor, its roles are now known to be diverse, including
stimulation of cardiac and vascular cell growth and division, and
activation of vascular NADH/NADPH oxidase activity (1
2
3
4
5
6)
.
Given these diverse hormonal influences, the long-term value of ANG II
inhibition for cardiovascular disease may be mediated by nonhemodynamic
actions rather than acute vasorelaxant effects per se (2)
.
The vascular endothelium plays a key role in the local regulation of
vasomotor tone and prevention of thrombus formation. Using agents like
acetylcholine or changes in flow to stimulate the release of NO (e.g.,
endothelium-derived relaxing factor, EDRF), clinical studies have
demonstrated the importance of EDRF/NO in both basal and stimulated
control of vascular tone (10
11
12)
. Dysfunction of vascular
endothelium (particularly decreased activity of NO-dependent pathways)
has been associated with a wide array of cardiovascular risk factors
including chronic smoking, hypercholesterolemia, hypertension, and
chronic heart failure (10
11
12
, 27)
. Loss of endothelial
integrity is known to promote vascular remodeling and thrombus
formation, to impair tissue perfusion (particularly during stress), and
to result in vasoconstriction (11)
. Thus, endothelial
dysfunction is associated with a diverse array of cardiovascular
disease states and may be an important initiator of these progressive
conditions. Inhibition of angiotensin-converting enzyme (ACE) has
recently been shown improve endothelial function in patients with
coronary artery disease or its risk factors (TREND; Trial on Reversing
Endothelial Dysfunction). In this large-scale clinical trial, treatment
with quinapril (an ACE inhibitor with affinity for vascular tissue) for
6 months was associated with a significantly improved vasodilator
response to acetylcholine in coronary artery segments
(28)
. Similar improvements were also observed in
microvasculature (29)
. These clinical findings suggest
that angiotensin has important influence on endothelium in
vivo and that some benefit of ACE inhibitor therapy is mediated by
improving endothelial function in vivo, but the mechanisms
involved have not been established.
Recent studies have demonstrated that ANG II is an activator of
superoxide production in several cell types, including fibroblasts
(30)
, mesangial cells (31)
, and endothelial
cells (32)
. This stimulation is apparently mediated by the
AT 1 receptor subtype activation, leading to increased gene expression
of NADH/NADPH oxidase subunits p67phox (33)
, p22phox
(34)
, and perhaps others. The role of this enhanced
oxidase expression in ANG II-mediated actions is not clear but it may
contribute to the other known cellular responses (e.g., proliferation,
hypertrophy, etc.). Whereas isolated endothelial cell studies have
demonstrated the selective presence of AT1 receptors (and an apparent
absence of the AT 2 subtype) (32)
, whether the endothelium
plays a direct role in ANG II-related cardiovascular disease in
vivo has not been established. Given previous reports of ANG II
promotion of vascular superoxide formation and the newly recognized
importance of endothelial NO for vascular health, we hypothesized here
that ANG II in vivo would selectively modify
endothelium-dependent function. Recent investigations have demonstrated
that the biological activities of NO are highly dependent on both
production- and destruction-related pathways (13)
. A
chemical pathway of biological importance appears to be the formation
of peroxynitrite through the interaction of NO with superoxide anion.
This reaction is exceedingly rapid and efficient
(1.9x1010
M-1s-1; see ref 19
),
leading to reduced levels of available NO and formation of a highly
reactive oxidant in vivo (17
18
19)
.
Peroxynitrite and related species aggressively nitrate protein tyrosine
residues, leading to the chemically stable biomarker 3-NT
(16)
. We and others have demonstrated the value of this
marker as evidence of NO dysregulation in disease, including advanced
atherosclerotic lesions (35)
, sepsis-related organ failure
(36)
, renal transplant rejection (37)
, and
others.
The ANG II infusion used in our studies (200
ng·kg-1·min-1) caused
significant inhibition of plasma renin activity but no change in
systemic blood pressure or cardiac mass on day 3. This dosing strategy
appears to be physiologically relevant since it has been shown to
produce steady-state ANG II blood levels similar to those observed in
hypertensive animals and humans (~100 pg/ml) (38)
. This
strategy of a short-term, ‘slow pressor dose’ administration allowed
us to evaluate early vascular changes during ANG II elevations in the
absence of detectable cell growth or hemodynamic influences. No
significant change in vascular response was observed for total
depolarized contraction, -adrenergic stimulation, or ANG II
contractions. Using an identical animal model but more prolonged ANG II
dosing [120 ng/(min·kg of 21 days)], Dowell et al.
(39)
observed significant potentiation of adrenergic
contractile activities, likely due to increased vascular smooth muscle
cell growth over 3 wk. In contrast, we observed significant reduction
in acetylcholine-induced vasorelaxation prior to any changes in
contractile responses and after only 3 days of elevated blood ANG II.
This endothelium-dependent relaxant response is mediated by NO
formation from the constitutively expressed nitric oxide synthase
within endothelial cells (NOS III) (10)
. Once formed,
endothelium-derived NO apparently diffuses to vascular smooth muscle
cells and elicits relaxation via cyclic GMP-dependent pathways
(22)
. In contrast to diminished acetylcholine response, no
change in nitroprusside action was found. Nitroprusside is an exogenous
agent that generates NO through nonenzymatic and enzymatic pathways in
smooth muscle cells and does not require functional endothelium or NOS
enzymes for activity. Since vascular smooth muscle response to an
exogenous NO source (nitroprusside) was not altered, the diminished
response to acetylcholine was apparently related to either decreased
endothelial production of NO or reduced bioavailability to effector
smooth muscle cells.
Consistent with selective endothelium dysfunction, we also observed
intima-specific staining for protein 3-NT residues. Given the extremely
rapid reaction rates for peroxynitrite formation from NO and superoxide
(known to be diffusion rate limited) and from protein nitration, the
staining pattern observed suggests that peroxynitrite was concentrated
in or near the endothelial layer. In addition to serving as a biomarker
of peroxynitrite, nitration of protein tyrosine residues is known to be
an inhibitor of several biochemical pathways including mitochondrial
respiration (40)
, high-energy phosphate utilization
(41)
, prostaglandin synthesis (42)
, and
superoxide dismutase activity (43)
. Peroxynitrite can also
induce DNA strand breakage in human umbilical vein endothelial cells
(44)
. Thus, endothelial formation of peroxynitrite
(through interaction of NO with superoxide) is likely to reduce
availability of NO to vascular smooth muscle as well as have functional
consequences to endothelial cell biochemistry. We recently reported a
distinctly different vascular 3-NT staining pattern during development
of organic nitrate pharmacodynamic tolerance (widespread distribution
throughout smooth muscle) (45)
. Thus, peroxynitrite
formation and protein nitration may exist in a variety of vascular
disorders, but the mechanisms involved and cellular distributions may
be distinct and depend on the stimuli involved. While recent studies
have suggested the existence of other potential biological pathways of
tyrosine nitration, these studies have demonstrated that the
chemistries responsible are dependent on neutrophil infiltration and
activation (46
, 47)
. Since we did not observe immune cell
involvement in our vascular studies, the protein nitration observed
here is most likely derived from ONOO-
formation. In addition, previous studies demonstrate that
cardiovascular tissue homogenates require exogenous addition of
neutrophil myeloperoxidase (5 µM), 1 mM concentrations of nitrite and
hydrogen peroxide, and long incubation times (>1 h) to produce
detectable protein nitration (46)
. In contrast, NO and
superoxide anion are known to form OONO- at a
diffusion-limited rate, even at very low concentrations. Under our
experimental conditions, therefore, the observed protein nitration may
be explained most simply by increased peroxynitrite formation rather
than more complicated or less efficient chemical processes.
In summary, we found that low-dose and short-term administration of ANG II caused a selective reduction in endothelium-dependent (nitric oxide-mediated) relaxant effects. These changes occurred prior to any other established consequences of elevated ANG II in vivo, including enhanced vascular contractile responses, development hypertension, or increase in cardiac or vascular mass. Further evaluations revealed that this endothelial dysfunction was associated with increased production of peroxynitrite in vivo. Protein nitration in the endothelial layer correlated with the extent of functional impairment observed. Thus, selective perturbation of normal endothelial function is an apparent early or initiating event during ANG II exposure in vivo. Furthermore, vascular peroxynitrite formation (and attendant NO dysregulation) may mediate these early events during ANG II-induced vascular dysfunction in vivo. Our findings may help to explain the results of the recently published TREND trial (see above), and suggest that evaluation of selective endothelial therapies for the prevention of disease appears warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F. S. Gragasin, Y. Xu, I. A. Arenas, N. Kainth, and S. T. Davidge Estrogen Reduces Angiotensin II-Induced Nitric Oxide Synthase and NAD(P)H Oxidase Expression in Endothelial Cells Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 38 - 44. [Abstract] [Full Text] [PDF]
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 I. V. Turko and F. Murad Protein Nitration in Cardiovascular Diseases Pharmacol. Rev., December 1, 2002; 54(4): 619 - 634. [Abstract] [Full Text] [PDF]
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D. D. Thomas, M. G. Espey, M. P. Vitek, K. M. Miranda, and D. A. Wink Protein nitration is mediated by heme and free metals through Fenton-type chemistry: An alternative to the NO/O2- reaction PNAS, October 1, 2002; 99(20): 12691 - 12696. [Abstract] [Full Text] [PDF]
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 L. J. Coppey, J. S. Gellett, E. P. Davidson, J. A. Dunlap, D. D. Lund, and M. A. Yorek Effect of Antioxidant Treatment of Streptozotocin-Induced Diabetic Rats on Endoneurial Blood Flow, Motor Nerve Conduction Velocity, and Vascular Reactivity of Epineurial Arterioles of the Sciatic Nerve Diabetes, August 1, 2001; 50(8): 1927 - 1937. [Abstract] [Full Text] [PDF]
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 M. J. Mihm, F. Yu, C. A. Carnes, P. J. Reiser, P. M. McCarthy, D. R. Van Wagoner, and J. A. Bauer Impaired Myofibrillar Energetics and Oxidative Injury During Human Atrial Fibrillation Circulation, July 10, 2001; 104(2): 174 - 180. [Abstract] [Full Text] [PDF]
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E. M. A. Mervaala, Z. J. Cheng, I. Tikkanen, R. Lapatto, K. Nurminen, H. Vapaatalo, D. N. Muller, A. Fiebeler, U. Ganten, D. Ganten, et al. Endothelial Dysfunction and Xanthine Oxidoreductase Activity in Rats With Human Renin and Angiotensinogen Genes Hypertension, February 1, 2001; 37(2): 414 - 418. [Abstract] [Full Text] [PDF]
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A. Denicola, C. Batthyany, E. Lissi, B. A. Freeman, H. Rubbo, and R. Radi Diffusion of Nitric Oxide into Low Density Lipoprotein J. Biol. Chem., January 4, 2002; 277(2): 932 - 936. [Abstract] [Full Text] [PDF]
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 M. J. Mihm, B. L. Schanbacher, B. L. Wallace, L. J. Wallace, N. J. Uretsky, and J. A. Bauer Free 3-Nitrotyrosine Causes Striatal Neurodegeneration In Vivo J. Neurosci., June 1, 2001; 21(11): RC149 - RC149. [Abstract] [Full Text] [PDF]
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P < 0.05 for both treatment groups vs. isotype
staining controls; 
P < 0.05 for ANG II treatment vs.
vehicle, via one way ANOVA with SNK post-hoc. Bottom panel: significant
positive correlation between aortic endothelial 3-NT immunoprevalence
and endothelial-dependent relaxant responses. Data plotted as mean 3-NT
immunoreactivity per rat (n=6 independent
determinations) vs. mean EC50 values per rat
(n=4–6 rings) from ANG II-treated (filled circles) and
vehicle-treated (open circles) rats. P < 0.02 via
Spearman’s nonparametric correlation analysis.