© 1997 American Heart Association, Inc.
Articles |
Vascular Superoxide Dismutase Deficiency Impairs Endothelial Vasodilator Function Through Direct Inactivation of Nitric Oxide and Increased Lipid Peroxidation
From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts (S.M.L., B.F., A.X., T.J., R.R., J.A.V., J.F.K.), the Departments of Pharmacology and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee (J.D.M., L.J.R.), and the United States Department of Agriculture, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota (L.M.K.).
Correspondence to John F. Keaney, Jr., Whitaker Cardiovascular Institute, Boston University School of Medicine, 80 East Concord Street, Room W507, Boston, Massachusetts, 02118. E-mail jkeaney{at}acs.bu.edu
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Abstract |
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Abstract Nitric oxide (NO) and superoxide are both constitutive products of the endothelium. Because NO is readily inactivated by superoxide, the bioactivity of endothelium-derived NO (EDNO) is dependent on local activity of superoxide dismutase (SOD). We examined the effects of chronic inhibition of copper-zinc SOD (CuZnSOD) using a rat model of dietary copper restriction. Male weanling Sprague-Dawley rats were fed a Cu-deficient diet and received either no Cu replacement (Cu-deficient) or Cu in the drinking water (Cu-sufficient). Compared with Cu-sufficient animals, Cu-deficiency was associated with a 68% reduction in CuZnSOD activity and a 58% increase in vascular superoxide as estimated by lucigenin chemiluminescence (both P<.05). Compared with Cu-sufficient animals, arterial relaxation in the thoracic aorta from Cu-deficient animals was 10-fold less sensitive to acetylcholine, a receptor-dependent EDNO agonist, but only 1.5-fold less sensitive to A23187, a receptor-independent EDNO agonist, and only 1.25-fold less sensitive to authentic NO (all P<.05). In contrast, acute inhibition of CuZnSOD with 10 mM diethyldithiocarbamate produced a more uniform reduction in sensitivity to acetylcholine (8-fold), A23187 (10-fold), and NO (4-fold; all P<.001). Cu-deficient animals demonstrated a 2.5-fold increase in plasma-esterified F2-isoprostanes, a stable marker of lipid peroxidation, that correlated inversely with arterial relaxation to acetylcholine (R=-.83; P<.0009) but not A23187 or authentic NO. From these findings, we conclude that chronic inhibition of CuZnSOD inhibits EDNO-mediated arterial relaxation through two mechanisms, one being direct inactivation of NO and the other being lipid peroxidation that preferentially interrupts receptor-mediated stimulation of EDNO.
Key Words: superoxide • nitric oxide • oxidation
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Introduction |
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Normal vascular homeostasis is dependent on the function of the endothelium. Endothelial elaboration of paracrine factors prevents both platelet adhesion to the endothelial surface and inappropriate vasospasm (for review, see Reference 11 ). One such endothelial product responsible for these functions is endothelium-derived nitric oxide (EDNO).1 Nitric oxide is produced constitutively by the vascular endothelium, and abnormalities in EDNO action that develop in association with vascular disease have been implicated in the development of clinically significant vascular events.2
In vivo, NO is subject to rapid inactivation by superoxide anion,3 4 5 an obligate byproduct of normal oxidative metabolism.6 Because endothelial cells constitutively produce both superoxide7 and nitric oxide,8 the effective release of NO from the vascular endothelium is dependent on the relative concentrations of these two species. Inhibition of endothelial cell copper-zinc superoxide dismutase (CuZnSOD) impairs effective release of NO from endothelial cells.9 10 Intact CuZnSOD function is also required for smooth muscle cell relaxation in response to nitrovasodilators.9 Thus, intact CuZnSOD function in the vascular wall appears necessary for EDNO bioactivity, most likely as a consequence of reducing the availability of superoxide for EDNO inactivation.
In addition to direct inactivation of EDNO, superoxide may interfere with normal EDNO action indirectly. In the presence of redox-active iron, resident vascular cell superoxide production may be associated with lipid peroxidation and the formation of oxidized LDL in the subendothelial space of the arterial wall.11 12 13 Oxidatively modified LDL may then interfere with vascular homeostasis by direct inactivation of EDNO.14 In addition, ox-LDL impairs receptor-mediated stimulation of EDNO production through disruption of G-protein-dependent signal transduction by lysophosphatidylcholine15 formed within the LDL particle as a product of lipid peroxidation.16
The relative contribution of the direct and indirect effects of superoxide on EDNO-mediated control of vascular tone is not known. To date, most studies examining the interaction between superoxide and endothelium-dependent arterial relaxation have involved acute exposure to superoxide4 17 or acute inhibition of CuZnSOD.9 10 18 In this study, we sought to determine the effects of chronic CuZnSOD deficiency on endothelium-dependent arterial relaxation using a rat model of dietary copper restriction.
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Materials and Methods |
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Materials
Sodium pentobarbital was purchased from Anthony Products Co. (Arcadia, Calif), and xanthine oxidase was obtained from Boehringer Mannheim Biochemicals (Indianapolis, Ind). Acetylcholine hydrochloride, calcium ionophore A23187, phenylephrine, diethylenetriamine pentaacetic acid, and all other compounds were purchased from Sigma Chemical Co. (St. Louis, Mo).
Physiologic salt solution (PSS) contained 118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CACl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 11.1 mM glucose, 10 µM indomethacin, and .026 mM Na2EDTA. PBS consisted of 10 mM NaH2PO4, .15M NaCl, pH 7.4. A23187 was prepared and diluted in 2% dimethyl sulfoxide, whereas all other reagents were prepared with distilled water. Solutions of authentic NO were prepared as described by Pagano and colleagues19 in the presence of Bio-Rad AG-I-X8 resin (200-400 mesh) to remove nitrite formed during NO preparation.
Animal Subjects
We modulated CuZnSOD activity through the production
of dietary copper deficiency. A total of 32 male, weanling
Sprague-Dawley (Harlan Sprague-Dawley, Madison, Wis) rats were fed ad
libitum purified diets that were deficient in copper and zinc as
previously described.20 21 22 All animals were provided with
water ad libitum that contained zinc (10 µg/mL as acetate) and
maintained at the Grand Forks Human Nutrition Research Center in
quarters at 22-24°C with a 12:12 hour light:dark cycle. In
control animals (n=16), adequate copper status was maintained with 4
µg/mL copper sulfate added to the drinking water. After
approximately 5 weeks of dietary treatment, copper deficiency developed
(as indicated by increased serum
cholesterol20 ), and animals were transferred
to the Whitaker Cardiovascular Institute in Boston,
Mass, placed into similar cages, maintained on the same diets and
drinking solutions, and used within 1 week. Animals were killed with
intraperitoneal pentobarbital (150 mg/kg),
and samples of plasma, serum, heart, and liver were obtained. Plasma,
serum, and tissue samples were frozen at -70°C until
analysis. Serum ceruloplasmin activity and liver content of
copper and iron were measured as previously described.23
Thoracic aorta was collected and used immediately for assessment of
vascular function.
In Vitro Assay of Vascular Function
The thoracic aorta was excised, and the perivascular tissue was
removed carefully with the vessel in ice-cold PSS. Each animal yielded
three to four vessel segments that were suspended in organ chambers
essentially as described24 except that vessels were
stretched to 3g of passive tension. Vessels were contracted
with phenylephrine (1 µM), and relaxation was assayed in
response to acetylcholine, A23187, or authentic NO. Sensitivity to the
effect of acetylcholine, A23187, or NO was determined by the
concentration required to produce half-maximal relaxation
(EC50) calculated from the dose-response curve using a
commercially available graphics program (Origin 4.0, Microcal Software,
Northampton, Mass). In some studies, vessels were treated for 30
minutes with 10 mM diethyldithiocarbamate (DDC, an
inhibitor of CuZnSOD25 ) before the assessment
of vascular function.
Vascular Superoxide
We used lucigenin chemiluminescence to estimate the steady-state
rate of superoxide generation in segments of thoracic aorta. For assay
of superoxide production in the presence of intact CuZnSOD
function (i.e., "net" vascular superoxide production),
vessels were incubated with PSS at 37°C and gently bubbled with 95%
O2:5% CO2. After 30 minutes, vessels were
transferred to 5-mL polyethylene tubes containing HEPES-buffered PSS
(PSS containing 20 mM HEPES) with 0.25 mM lucigenin
(bis-N-methylacridium nitrate) and incubated for 10 minutes.
After equilibration with lucigenin, net superoxide levels were
estimated from chemiluminescence recorded with a Turner Designs
Model 20e luminometer at 37°C. The integral of the chemiluminescence
signal was recorded at 30-second intervals for 5 minutes, and the
integral readings were combined and expressed as arbitrary
chemiluminescence units over 5 minutes. Background chemiluminescence
was determined from identically processed vessel-free incubations and
subtracted from the chemiluminescence determined with vessels.
Superoxide production was also determined in the absence of
intact CuZnSOD function (i.e., "total" superoxide
production) by incubating vessels for 30 minutes with 10 mM
DDC. Chemiluminescence was inhibited 
95% by pretreatment of vessels
with Tiron (4,5 dihydroxy-1,3-benzene sulfonic acid), a scavenger of
superoxide anion,26 or EUK-8, a Mn-SOD
mimic,27 and all assays were performed in a dark,
light-sealed room.
Tissue SOD Activity
Segments of thoracic aorta did not yield enough tissue for SOD
activity assays; therefore, we estimated tissue SOD activity from liver
specimens. Frozen samples (.5g) were incubated at 37°C
in .5 mL of PBS with or without 10 mM DDC. Samples were
homogenized using a ground-glass
homogenizer (Glas-Col, Terre Haute, Ind), and the
homogenate was diluted 100-fold with PBS. The SOD activity
was determined in untreated and DDC-treated samples as previously
described.28 The CuZn-independent SOD activity was derived
from the DDC-treated samples, and the CuZn-dependent SOD activity was
derived from the difference in SOD activity in the presence and absence
of DDC. SOD activity was expressed as international units per milligram
of protein using bovine erythrocyte CuZnSOD (Sigma) as a standard.
Measurement of Plasma F2-Isoprostanes
The plasma content of esterified F2-isoprostanes was
determined as previously described using gas chromotography/negative
ion chemical ionization mass spectrometry that used stable isotope
dilution techniques.29
Measurement of Plasma Antioxidant Levels
For plasma 
- and 
-tocopherol content, plasma
(.25 mL) was extracted with .25 mL of methanol and 2.5 mL of hexane.
The hexane phase (2 mL) was dried under N2, resuspended in
.2 mL of absolute ethanol, and analyzed using reverse-phase
HPLC with an LC18 column (8.3 cmx4.6 mm I.D., Waters Associates
Inc., Milford, Mass) and a mobile phase containing 1% water in
methanol with 20 mM lithium perchlorate at a flow rate of 1.4
mL/minutes. Quantification of - and -tocopherol was
performed using a computerized HPLC system (series 1050 Chemstation,
Hewlett-Packard Co., Palo Alto, Calif) coupled to electrochemical
detection (model 1049, Hewlett-Packard Co.) at an applied potential of
0.6 V. By using this system, - and -tocopherol eluted
as single, baseline-separated peaks with retention times of 5.4 and 4.6
minutes, respectively.
Data Analysis
All values are presented as mean±SEM. The vascular
responses to acetylcholine, A23187, and NO are reported as the percent
reduction in tension (relaxation) compared with the contraction
produced by 1 µM phenylephrine. Dose responses to
acetylcholine, A23187, and NO were compared within dietary groups with
repeated measures ANOVA, and responses among dietary groups were
compared with two-way ANOVA. Other variables were compared between
the two treatment groups using a Student's t test or
Mann-Whitney U test as appropriate for parametric or
nonparametric variables, respectively. Correlations
between variables were performed using the Pearson's
product-moment or Spearman's rank-order as appropriate.
Statistical significance was accepted if the null hypothesis was
rejected with a P<.05.
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Results |
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Characteristics of Animal Subjects and Copper Status
The characteristics of control and copper-deficient subjects are contained in Table 1
. Animals fed the
copper-deficient diet were characterized by common features of copper
deficiency, including a reduced liver content of copper, a reduced
serum ceruloplasmin activity, an increased heart-to-body weight ratio,
lower body weight, and a mildly elevated serum cholesterol
level (Table 1). We found no differences in plasma levels of vitamin E
or vitamin C in association with the dietary manipulation (data not
shown).
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Copper Status and Tissue SOD Activity
To confirm that the restriction of dietary copper resulted in
impaired tissue SOD activity, we determined the CuZn-dependent and
-independent SOD activity in liver samples of our study animals. As
shown in Table 2, tissue derived from
copper-deficient rats demonstrated only 32% of the CuZnSOD activity
found in the control animals. The CuZn-independent SOD activity in the
two treatment groups was similar, thus the difference in total SOD
activity between the two groups was entirely attributable to the
decreased CuZnSOD activity in the copper-deficient animals.
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Copper Status and Vascular Reactivity
To assess the implications of chronically reduced CuZnSOD activity
on vascular function, we harvested the aorta from these animals and
assessed the vascular responses. The contractile responses of aortic
vessels from the control and copper-deficient animals were similar.
Vessels from the control and copper-deficient animals incubated with 80
mM KCl demonstrated contractions of 4.6±.1g and
4.2±.2g, respectively (P=NS). The contractile
response to phenylephrine was also similar in the control
and copper-deficient animals (2.4±.2g and
2.5±.1g, respectively; P=NS).
Vessels derived from the control animals demonstrated dose-dependent
arterial relaxation in response to the receptor-dependent
EDNO agonist, acetylcholine (P<.001), with an
EC50 of 11±1 nM (Fig 1A). In
contrast, vessels derived from the copper-deficient animals were
10-fold less sensitive to acetylcholine with an EC50 of
110±1 nM (P<.001 by two-way ANOVA). Control vessels also
demonstrated significant dose-dependent relaxation in response to the
receptor-independent EDNO agonist, A23187 (P<.001), with an
EC50 of 11±1 nM. As shown in Fig 1B, vessels derived from
copper-deficient animals demonstrated only a .6-fold reduction in
sensitivity to A23187 compared with control vessels (EC50
of 17±1 nM; P<.001 versus control by two-way ANOVA).
Vessels from copper-deficient animals were only .36-fold less sensitive
to arterial relaxation in response to authentic NO compared
with control animals (EC50 of 351±1 nM versus 258±1 nM,
respectively), and this difference was statistically significant
(P=.050 by two-way ANOVA).
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) or copper-adequate (
)
for 6 weeks. Vessels were prepared and contracted with 1 µM
phenylephrine as described under "Methods" and exposed
to the indicated concentrations of (A) acetylcholine (ACH), (B) A23187,
or (C) NO. Values are plotted as mean±SEM and are derived from six to
seven animals in each group. *P<.001 and 
P=.05
versus control group by two-way ANOVA.
Acute inhibition of CuZnSOD produced a more uniform inhibition of
vascular relaxation in response to acetylcholine, A23187, and authentic
NO (Fig 2A-C). Treatment of control vessels with DDC produced a
reduction in the sensitivity of vessels to acetylcholine, A23187, and
NO of approximately 10-fold, 10-fold, and 4-fold, respectively (all
P<.001 by two-way ANOVA). Arterial relaxation
to forskolin was not materially different with either chronic or acute
inhibition of CuZnSOD (data not shown).
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Vascular Superoxide
The release of intact EDNO also appears to depend on
endothelial cell SOD activity.9 10 To
determine if excess vascular superoxide production was
responsible for the impairment of endothelium-dependent
arterial relaxation that we observed in copper-deficient
animals, we estimated both net and total vascular superoxide
production using lucigenin chemiluminescence28 30
in the thoracic aorta from both treatment groups. As shown in Table 3, without inhibition of CuZnSOD, net
vascular superoxide production in the copper-deficient animals
was 58% greater than that in the control animals. In contrast, vessels
derived from copper-deficient and control animals demonstrated similar
total vascular superoxide production rates (Table 3). Thus,
dietary copper deficiency is associated with increased net
vascular superoxide production without an accompanying increase
in the underlying total vascular superoxide production.
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Vascular Reactivity and Vascular Superoxide Production
To gain insight into the mechanism(s) responsible for abnormal
vascular dysfunction with chronically reduced CuZnSOD activity, we
examined the relation between arterial relaxation and
vascular superoxide production. As shown Fig 3A, arterial relaxation to
acetylcholine correlated inversely with net vascular superoxide
production (R=-.76; P=.004). Similarly,
arterial relaxation in response to A23187 also inversely
correlated with net vascular superoxide production, although
the correlation was not as strong (R=-.61;
P=.046; Fig 3B). Arterial relaxation in response
to authentic NO did not correlate well with net vascular
production of superoxide (R=.39; P=.19;
Fig 3C). Thus, EDNO-mediated relaxation in response to acetylcholine
appears particularly sensitive to a chronic increase in net vascular
superoxide production.
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Vascular Reactivity and Lipid Peroxidation
Arterial relaxation in response to acetylcholine is
dependent on intact receptor function and signal transduction across
the cell membrane.31 Superoxide generation, in the
presence of transition metals, can initiate lipid
peroxidation,32 and the accumulation of lipid peroxidation
products in the vascular wall is associated with an impairment of
acetylcholine-mediated arterial relaxation.28
Therefore, we measured the plasma content of esterified
F2-isoprostanes, stable products of
arachidonic acid autoxidation.33 In
control animals, plasma esterified F2-isoprostane levels
were 263±40 pg/mL, whereas the levels in copper-deficient
animals were 2.5-fold greater (665±156 pg/mL; P<.05
versus control). Esterified F2-isoprostane levels were
strongly inversely correlated to arterial relaxation in
response to acetylcholine (R=-.83; P=.0009; Fig 4A) but not significantly correlated with
arterial relaxation to either A23187 (R=34;
P=.29; Fig 4B) or authentic NO (R=-.13;
P=.71; Fig 4C).
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Discussion |
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The data presented here demonstrate that chronic impairment of CuZnSOD activity, through dietary copper restriction, results in a significant loss of endothelium-dependent arterial relaxation. This defect in endothelium-dependent arterial relaxation was associated with a 68% reduction in tissue CuZnSOD activity and a concomitant 58% increase in net vascular superoxide production. Vascular superoxide production correlated with the magnitude of impairment in acetylcholine- and A23187-mediated endothelium-dependent arterial relaxation. However, we did not observe a close association between vascular superoxide production and arterial relaxation in response to authentic NO. Furthermore, the impairment in acetylcholine-mediated arterial relaxation was closely linked to the plasma content of esterified F2-isoprostanes, a stable marker of in vivo lipid peroxidation. In contrast, A23187 and NO-mediated arterial relaxation did not correlate with F2-isoprostane levels.
These data are consistent with previous reports describing impaired endothelium-dependent arterial relaxation in copper-deficient rats.34 35 In those studies, Saari found that rats consuming a copper-deficient diet demonstrated less endothelium-dependent arterial relaxation to the EDNO agonists acetylcholine and histamine. In addition, arterial relaxation in response sodium nitroprusside was inhibited, although the dilatory response to papaverine, a nonspecific phosphodiesterase inhibitor, remained intact.35 It was concluded that the interaction of the endothelium with the underlying smooth muscle was interrupted by dietary copper deficiency. Dietary copper deficiency in rats is associated with a substantial reduction of CuZnSOD activity,36 and in the present study, we did find decreased tissue CuZnSOD activity and increased net vascular superoxide production. Because EDNO is inactivated by superoxide,3 4 it is attractive to speculate that the impairment of endothelium-dependent arterial relaxation reported here, as in the studies by Schuschke et al34 and Saari,35 is a consequence of impaired vascular CuZnSOD activity.
There is considerable evidence to suggest that intact CuZnSOD activity is required for EDNO action. Superoxide and nitric oxide combine readily with a bimolecular rate constant of 6.9x109 mol/Lxsecond.5 Because endothelial cells produce both nitric oxide37 and superoxide7 constitutively, the relative production of either species should modulate the biologic activity of EDNO. Several investigators have demonstrated that increased SOD activity augments EDNO-mediated arterial relaxation in unstimulated cells3 4 17 and in response to acetylcholine,3 suggesting that basal endothelial superoxide production modulates EDNO action. As a result, vascular SOD activity exerts considerable influence over the local activity of EDNO9 10 and NO-dependent vasodilators.9 Cultured endothelial cells exposed to the CuZnSOD inhibitor, DDC, produce less active EDNO, although the production of inactive nitrogen oxides remains similar to cells not exposed to DDC, indicating that endogenous SOD prevents inactivation of vasoactive NO.10 Similarly, isolated bovine coronary arteries exposed to DDC fail to relax normally to the EDNO agonist, acetylcholine, or NO-dependent vasodilators such as nitroprusside or nitroglycerin.9 Taken together, these results suggest that vascular SOD activity is required for both the effective release of EDNO from endothelial cells and for the smooth muscle cell response to NO.
In the present study, we did find that EDNO-mediated
arterial relaxation was impaired in the copper-deficient
animals in response to both acetylcholine and A23187 (Fig 1
). We also
found a close relation between the impairment of
endothelium-dependent arterial relaxation
and net vascular superoxide production (Fig 3), suggesting that
the defect in EDNO-mediated vasodilation resulted from the increased
availability of superoxide. However, the magnitude of impairment in
vascular relaxation was considerably greater for acetylcholine than for
A23187 or even authentic NO, an unexpected finding if
superoxide-mediated NO inactivation is the sole mechanism of impaired
relaxation in this model.
A chronic excess of superoxide within the vascular wall may produce a number of local effects. The most obvious effect is the aforementioned inactivation of NO. The product of the reaction between NO and superoxide is peroxynitrite, a potent oxidant that is known to initiate lipid peroxidation and oxidize intracellular thiols.38 With respect to the present study, one must consider that the increase in available superoxide associated with copper deficiency may have resulted in the formation of peroxynitrite and, thus, an increase in lipid peroxidation within the vascular wall. Our observations that copper deficiency is associated with increased plasma levels of esterified F2-isoprostanes are entirely consistent with this contention. Moreover, we have found that infusion of SOD (bovine, 8 mg over 15 minutes) to three individuals with severe liver injury from acetaminophen overdose significantly suppressed circulating isoprostane levels (J. Morrow, unpublished observation).
Superoxide may also produce lipid peroxidation within the vascular wall
independent of peroxynitrite formation. Superoxide is a reducing agent
and is itself incapable of initiating lipid peroxidation at physiologic
pH.32 However, in the presence of iron, superoxide may be
converted to the hydroxyl radical, a species known to initiate lipid
peroxidation.39 This process involves the dismutation of
superoxide to H2O2 followed by the reduction of
H2O2 to the hydroxyl radical (·OH) via the
Fenton reaction, as shown in Reactions 1 and 2 below.
 | (1) |
 | (2) |
 | (3) |
The aforementioned mechanisms through which superoxide may induce lipid
peroxidation have particular relevance for endothelial
function. Lysophosphatidylcholine, a lipid peroxidation by-product,
is an amphipathic compound that is known to interrupt
G-protein-dependent signal transduction.31 In fact, the
treatment of endothelial cells or intact blood vessels
with lysophosphatidylcholine produces an impairment in
endothelial function that is reminiscent of that
depicted in Fig 1. Specifically, receptor-mediated stimulation of EDNO
release is impaired to a greater extent than receptor-independent
stimulation.41 Thus, increased net vascular superoxide
production may also impair vascular function through lipid
peroxidation that interrupts receptor-mediated signaling at the level
of the cell membrane, rendering these cells less sensitive to
acetylcholine.
The results presented in this study should be interpreted with some caution. We were not able to directly measure CuZnSOD activity in blood vessels, and the possibility remains that our measurements in the liver do not reflect the events in the blood vessel wall. However, previous studies with animal models of copper-deficiency indicate that vascular CuZnSOD activity is impaired.42 Similarly, we measured plasma esterified F2-isoprostanes as a marker of lipid peroxidation rather than vascular levels of these compounds. In this regard, we can only infer that vascular lipid peroxidation was increased in this model, although we cannot identify any rationale for selective lipid peroxidation that would exclude the vasculature. Finally, recent evidence indicates that lucigenin chemiluminescence, although sensitive, is not specific for superoxide.43 This assay is better suited for the assessment of SOD activity, which is still consistent with the data presented here.43
In summary, chronic inhibition of CuZnSOD is associated with an impairment of endothelial control of vascular tone. This impairment appears to result from both direct and indirect actions of superoxide. Superoxide directly inactivates EDNO-producing inhibition of the vascular responses to the EDNO agonists acetylcholine and A23187 as well as to authentic NO. The responses to acetylcholine are particularly sensitive to inhibition of CuZnSOD, and this effect may result from additional indirect actions of superoxide resulting in vascular lipid peroxidation. These results suggest that copper, and CuZnSOD activity in particular, is important in the control of vascular tone through both the protection of NO against inactivation and the prevention of lipid peroxidative damage to endothelial cells.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The authors thank Dale Christopherson, Jason Lambrechtt, Denice Schafer, James Lindlauf, Timi Mannion, and the vivarium staff of both the Grand Forks Human Nutrition Research Center and Boston University School of Medicine for excellent technical assistance. This work was supported by grants from the United States Department of Agriculture (L.M.K.), the American Heart Association (J.F.K), the Council for Tobacco Research-USA (J.F.K.), and the National Institutes of Health (HL49954 to B.F., GM42056 and DK48831 to L.J.R. and J.D.M.). Joseph A. Vita is an Established Investigator of The American Heart Association, and John F. Keaney, Jr., is the recipient of a Clinical Investigator Development Award (HLO3195) from the National Institutes of Health.
Received February 19, 1997; accepted April 4, 1997.
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