295
Oxygen Radicals Mediate the Cerebral Arteriolar
Dilation from Arachidonate and Bradykinin in Cats
Hermes A. Kontos, Enoch P. Wei, John T. Povlishock, and Carole W. Christman
From the Departments of Medicine and Anatomy, Medical College of Virginia, Richmond, Virginia
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SUMMARY. Topical application of sodium arachidonate (50-200 /ig/ml) or bradykinin (0.1-10
fjg/ml) on the brain surface of anesthetized cats caused dose-dependent cerebral arteriolar dilation.
This dilation was blocked by 67-100% in the presence of superoxide dismutase and catalase.
These enzymes did not affect the changes in arteriolar diameter caused by alterations in arterial
blood Pco2/ or the arteriolar dilation from topical acetylcholine. Enzymes inactivated by heat had
no effect on the vasodilation from arachidonate or bradykinin. Superoxide dismutase alone or
catalase alone reduced the dilation during application of 200 Mg/ml of arachidonate for 15
minutes; they also completely prevented the residual dilation seen 1 hour after washout, as well
as the reduction in the vasoconstrictive effects of arterial hypocapnia observed at this time. The
results show that superoxide anion radical and hydrogen peroxide, or radicals derived from them,
such as the hydroxyl radical, are mediators of the cerebral arteriolar dilation from sodium
arachidonate or bradykinin. These radicals are not the endothelium-derived relaxant factor
released by acetylcholine. The presence of both superoxide anionradical and hydrogen peroxide
is required for the production of the vascular damage seen during prolonged application of high
concentrations of sodium arachidonate. (Circ Res 55: 295-303, 1984)
TOPICAL application of sodium arachidonate (Wei
et al., 1980b; Busija and Heistad, 1983) or bradykinin (Wahl et al., 1983) to the brain surface induces
cerebral arteriolar dilation. Since the response to
arachidonate is blocked by cyclooxygenase inhibitors (Wei et al., 1980b; Busija and Heistad, 1983), it
was ascribed to the production of vasodilative prostaglandins. It is known that bradykinin stimulates
prostaglandin synthesis (Blumberg et al., 1977;
Needleman et al., 1975; McGiff et al., 1972); in other
vascular beds, the vasodilative response to bradykinin is at least partially blocked by cyclooxygenase
inhibitors (Blumberg et al., 1977; Needleman et al.,
1975), suggesting that it is mediated in part by
prostaglandins.
Recently, we found that, in response to topical
application of high concentrations of sodium arachidonate (200 Mg/ml) or bradykinin (20 Mg/ml), superoxide anion radical appeared in the cerebral cortical extracellular space (Kontos and Wei, 1983).
Since superoxide anion radical and other oxygen
radicals derived from it, such as hydrogen peroxide
or the free hydroxyl radical, dilate cerebral vessels
(Kontos et al., 1983; Rosenblum, 1983), it appeared
probable that the vasodilation in response to sodium
arachidonate or to bradykinin might be due in part
to the action of oxygen radicals. In this study, we
investigated the effect of superoxide dismutase, an
enzyme which destorys superoxide anion radical,
and catalase, which destroys hydrogen peroxide, on
the cerebral arteriolar dilation caused by topical
application of arachidonate or bradykinin.
Methods
Experiments were carried out in cats anesthetized with
sodium pentobarbital (30 mg/kg, iv). After completion of
tracheostomy, each animal was ventilated with a positive
pressure respirator and received 5 mg/kg of gallamine
triethiodide for skeletal muscle paralysis. The end-expiratory CO2 of the animal was monitored continuously
with a Beckman infrared CO2 analyzer and was maintained at a constant level of about 30 mm Hg by adjusting
the respirator rate and volume. Arterial blood pressure
was measured with a Statham transducer connected to a
cannula introduced into the aorta via the femoral artery.
Arterial blood samples were collected periodically for the
determination of Po2, Pco2, pH, and hematocrit. Blood
gases and pH were measured with Radiometer electrodes;
hematocrit was measured by a micromethod.
Pial precapillary vessels were visualized through an
acutely implanted cranial window, as described in detail
previously (Levasseur et al., 1975). The window was implanted over the parietal cortex to allow visualization of
the vessels in the marginal, suprasylvian, and ectosylvian
gyri. The cranial window was equipped with three openings. Two openings were used as inlet and outlet for filling
the space under the window with various solutions. The
third opening was connected to a Statham pressure transducer for monitoring intracranial pressure. The outlet of
the window was connected to plastic tubing arranged in
a loop, the open end of which was placed at a fixed level
to give a constant intracranial pressure of 5 mm Hg
throughout the experiment. The space under the window
was initially filled with artificial cerebrospinal fluid (CSF)
having a composition identical to that of cats (Levasseur
et al., 1975). Pial arterioles were visualized via a Leitz
Ultropak microscope equipped with a Vickers image-split-
296
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ting device for measuring vessel diameter. Usually, four
to six vessels covering a wide range of vessel caliber were
studied in each animal.
We used the following materials: bradykinin acetate,
acetylcholine chloride, superoxide dismutase (SOD) from
bovine blood (3000 U/mg protein), and catalase from
bovine liver (2000 U/mg protein) all obtained from Sigma;
arachidonic acid obtained from NuChek. A 10 mg/ml
sodium arachidonate stock solution was prepared by dissolving three parts of arachidonic acid and one part sodium carbonate (wt/wt) in distilled water. From this sodium arachidonate stock solution, appropriate volumes
were added to artificial CSF to give the desired final
concentrations. All solutions were prepared in artificial
CSF shortly before the experiment and were placed in a
water bath at 37°C until use. The solutions were used to
fill the space under the cranial window.
The experimental design was as follows: first, the response of cerebral arterioles to arterial hypocapnia was
determined. Only animals whose vessels had normal reactivity were used. After restoration of the Pco2 to normal,
and return of vessel caliber to the control level, doseresponse curves to arachidonate, in doses of 50-200 /xg/
ml, or to bradykinin, in doses of 0.1-10 Mg/ml, were
obtained in the absence of other agents, as well as in the
presence of SOD (60 U/ml) plus catalase (40 U/ml), and
in the presence of these same enzymes after they had
been inactivated by heat. The order of testing was as
follows: in the case of arachidonate, we alternated the
testing of the control responses and of the responses in
the presence of inactive enzymes and tested the response
to the active enzymes last. The reason for this was we
suspected that, after application of active enzymes, washout might be incomplete. In the case of bradykinin, in one
group of animals, we tested first control responses and
then responses in the presence of active SOD and catalase.
In another series, we alternated the control responses and
responses in the presence of inactivated enzymes followed
by testing of responses in the presence of active enzymes.
This was done to exclude the possibility that tachyphylaxis
to bradykinin might influence the results and complicate
interpretation. Measurements of vessel caliber were made
2-4 minutes after application of each solution. This allowed ample time for the establishment of a new steady
state. We arbitrarily divided vessels into small arterioles
with resting diameters <100 ^m and large arterioles with
resting diameters >100 ^m, because the responses of these
two groups of vessels to a variety of influences differ
quantitatively (Kontos, 1981).
In two other groups of cats, we studied the effect of
topically applied acetylcholine and the effect of changes
in arterial blood CO2 on vessel caliber with and without
topically applied SOD and catalase. The responses to
acetylcholine were studied because the relaxant effect of
bradykinin and of arachidonic acid in some cases have
been reported to be endothelium dependent (Furchgott,
1983). The responses to CO2 were studied to verify that
the effect of SOD and catalase was relatively specific.
Acetylcholine chloride was dissolved in artificial CSF to
give concentrations equal to 10~" to 10~7 M, and the
resultant solutions were used to fill the space under the
cranial window. Measurements of vascular caliber were
made between 1 and 3 minutes. Arterial hypocapnia was
induced by mechanical hyperventilation. Hypercapnia
was induced by ventilation with gases containing 3 and
5% CO2. Each level of CO2 was maintained for approxi-
Circulation Research/Vo/. 55, No. 3, September 1984
mately 10 minutes to assure the establishment of a steady
state.
In another series of experiments, we evaluated the effect
of SOD and catalase separately and in combination on the
cerebral arteriolar dilation induced by high concentration
of topical arachidonate (200 Mg/ml), both during its application as well as 1 hour following washout. We had found
earlier that this dose of arachidonic acid, when applied
for 15 minutes, caused residual dilation long after washout, accompanied by morphological abnormalities of the
endothelium and vascular smooth muscle, as well as reduced reactivity to the vasoconstrictive effects of arterial
hypocapnia (Kontos et al., 1980). Cats were assigned to
one of several groups which received by topical application the following agents: (1) arachidonate 200 Mg/ml; (2)
arachidonate 200 Mg/ml, plus SOD 60 U/ml; (3) arachidonate 200 Mg/ml, plus catalase 40 U/ml; and (4) arachidonate 200 Mg/ml, plus SOD 60 U/ml, plus catalase 40
U/ml. These agents were applied under the cranial window for 15 minutes. Vessel diameters were measured
again, and then the space under the window was washed
with fresh CSF. Vessel diameters and responsiveness to
arterial hypocapnia were examined again 1 hour later.
The morphology of cerebral arterioles was studied after
the functional studies were completed in the animals
which received arachidonate and in the animals which
received arachidonate plus SOD plus catalase, as described
in detail before (Dietrich et al., 1980). The animal's head
was perfused at a pressure equal to his own arterial blood
pressure, first with 0.9% sodium chloride solution and
then with a fixative consisting of 2% glutaraldehyde and
2.5% paraformaldehyde in 0.1 M phosphate buffer. When
fixation was completed, the arachnoid membrane beneath
the cranial window, together with the pial vessels, was
removed according to a technique which permits the
identification of the same vessels that had been studied
previously from the functional standpoint (Dietrich et al.,
1980). The pial arterioles were then processed for electron
microscopic examination. Scanning electron microscopy
was used to determine quantitatively the number of lesions in the endothelium. This was achieved by counting
the number of lesions in five randomly selected microscopic fields, each measuring 2100 Mm2. The average
number of lesions per microscopic field was used as a
measure of the density of these lesions.
The statistical significance of the results were evaluated
by analysis of variance and, if significant differences were
found by this analysis, by f-tests modified for multiple
comparisons (Wallenstein et al., 1980).
Results
Figure 1 shows that the combination of SOD and
catalase inhibited markedly the vasodilative response to topical application of arachidonate. SOD
and catalase inactivated by heat had no significant
effect on the response. Figures 2 and 3 show that
SOD and catalase inhibited the vasodilative response to topical application of bradykinin. Figure
3 shows that SOD and catalase inactivated by heat
had no significant effect on the response. Figure 4
shows that the cerebral arteriolar response to
changes in arterial blood Pco2 was not affected by
the combination of SOD and catalase. Figure 5
shows that the vasodilative effect of topical acetyl-
Koittos et al./Oxygen Radicals from Arachidonate and Bradykinin
SMALL VESSELS
LARGE VESSELS
•
•
Control
53 + 2.9 M-m(l5)
o Inoctivoted SOD a Catalase
52 ± 3.0 jj.m(l5)
A Active SOD a Catalase
4 9 + 4.8 p-m(l5)
160
o
297
Control
163 ± 8 . 2 M-m(l8)
o Inactivated SOD a Catalase
FIGURE 1. Effect of arachidonic acid on
cerebral arteriolar caliber under control
conditions (solid circles), in the presence
of heat-inactivated SOD and catalase
(open circles), and in the presence of active
SOD and catalase (open triangles). Mean
values ± SE of initial vessel diameters in
nm for each group are shown. Number in
parentheses after SE is the number of vessels studied. Data are mean ± sEfrom five
cats. Analysis of variance showed that
responses with active SOD plus catalase
were significantly lower than in the other
two groups.
161 ± 8.0 M-mO8)
A Active SOD 8 Catalase
165 ± 8.8 M-m(l8)
140
o
rr
I-
I
1
120
I
100
100
200
0
ARACHIDONIC
100
SMALL VESSELS
160 -
•
Control
-
•
A Active SOD 8 Catalase
Control
178 ±10.8 |im(l3)
FIGURE 2. Effect of bradykinin
on cerebral arteriolar caliber
under control conditions (solid
circles) and in the presence of
active SOD and catalase (open
triangles). Note that the bradykinin concentration on the
abscissa is on a logarithmic
scale. Mean values ± SE of the
initial vessel diameters in pm
for each group are shown.
Number in parentheses after SE
IS the number of vessels studied. Data are mean ± SE from
six cats. Analysis of variance
showed that responses with active SOD plus catalase were
significantly lower than in the
control group.
A Active SOD 8 Catalase
181 ± 9.7 iun(l3)
55 ±3.1 iLm(l8)
c
arterioles during its application, that this dilation
persisted 1 hour after washout, and that it was
accompanied by reduction in the vasoconstrictive
response to hypocapnia. SOD alone or catalase alone
reduced the vasodilation during the application of
arachidonate and eliminated the residual dilation
after washout, as well as the decrease in responsiveness to hypocapnia. The combination of SOD and
catalase was most effective; it eliminated the vasodilation during application and converted the residual dilation to a small vasoconstriction. Scanning
LARGE VESSELS
57 ± 2 . 6 jirndS)
o
200
ACID - u.g/ml
choline was not affected by the combination of SOD
and catalase.
Tables 1-4 summarize the effects of arachidonate
with and without SOD and catalase on cerebral
arteriolar caliber during the exposure, and 1 hour
after washout, as well as the effect on the response
to arterial hypocapnia. Table 5 contains a comparison of the vasodilation during and after application
of arachidonate with and without SOD and catalase
each by itself and in combination. It is seen that
arachidonate induced dilation of small and large
o
o
140 \
1
Jj
1
120
xl
DIAfV
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i
2
*
i
i
100
}
-5
i
O.I
I
i
I
i
i i i 11
I
i
i
•
i
i
• •••
i
•
10 O.I
BRADYKININ
-
i
I
I
I
I
I
I
I
i
i
I
I
I
I
I
I
I
10
Circulation Research/Vo/. 55, No. 3, September 1984
298
-
SMALL VESSELS
160
•
•
Control
LARGE- VESSELS
Control
205 +9.0 u.m(5)
•
58 ±2.7 M-m(6)
o Inactivated SOD 8 Catalase
o
208 ±7.1 nm(5)
55 ±3.1 tim(6)
c
o
A Active SOD 8 Catalase
A Active SOD 8 Catalase
o
o
o Inactivated SOD 8 Catalase
54 ±2.8 n.m(6)
140
'
204 ± 8 . 6 iim(5)
1
or
120
DiAr
UJ
i
UJ
"^
i
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*
100
I
I
* -I
I
O.I
S
1
1
1 1 1 1 1 1
1
1
-
SMALL
•
VESSELS
.
Control
M-m(l5)
cantly lower; in 10 vessels, the density of these
lesions was 2.6 ± 0.8 lesions/2100 ^m2.
Discussion
The important finding of the experiments described above is that the combination of SOD and
catalase inhibited the cerebral arteriolar dilation induced by topical application of either arachidonate
or bradykinin. This result could be explained by one
of two mechanisms. First, it is possible that the
VESSELS
Control
h
o SOD and Catalase
159 ± 7 . 5 M-m (13)
o
I
120 -
I
2
LJ
IUJ
•o
•o
100 -
i
80
30
11
154 ± 7.3 n-m(l3)
o SOD and Catalase
59±4.0
•
i
^g/ml
LARGE
61 ± 4.0 n.m (15)
I I I
10
electron microscopy of the luminal surface of arterioles from animals treated with arachidonate disclosed focal endothelial lesions in the form of craters
or dome-like protrusions into the lumen of the vessels, identical to those described previously (Dietrich
et al., 1980; Kontos et al., 1980, 1981; Wei et al.,
1980a, 1981). The density of these lesions in 15
vessels was equal to 18.3 ± 2.3 lesions/2100 /im2.
The number of lesions in the animals treated with
arachidonate plus SOD plus catalase was signifi-
c
o
1
10 O.I
BRADYKININ
140 -.
2
FIGURE 3. Effect of bradykinin
on cerebral arteriolar caliber
under control conditions (solid
circles), in the presence of heatinactivated SOD and catalase
(open circles), and in the presence of active SOD and catalase (open triangles). Mean values ± SE of initial vessel diameters in \im are shown.
Number in parentheses after SE
is the number of vessels studied. Data are mean ± SE from
two cats. Analysis of variance
showed that responses with active SOD plus catalase were
significantly lower than in the
other two groups.
60 0
PaC0 2 - mmHg
30
60
FIGURE 4. Effect of changes in Pacc>2 on
cerebral arteriolar caliber under control
conditions (solid circles) and in the presence of active SOD and catalase (open
circles). Mean values ± SE of initial vessel
diameters at control Pacoi are shown for
each group. Number in parentheses after
SB is the number of vessels studied. Data
are mean ± SEfrom five cats. Analysis of
variance showed no differences in the responses between the two groups.
Kontos et al./Oxygen Radicals from Arachidonate and Bradykinin
SMALL
•
o
o
O
140
VESSELS
299
LARGE VESSELS
•
Control
57 ± 2.7 n.m(l8)
SOD and Catalase
59 ± 2.7 M.m(l8)
o
-
Control
177+ 16.2 M-m(7)
SOD and Catalase
175 + ll.9n.m(7)
I
I
?
a
I
1
,
I
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I
100
L
,
1
1
1
IxlO -9
I x 10-n
7
IxlO"
5
I
f
S—
I
IxlO""
IxlO -9
I x l O ,-7
ACETYLCHOLINE - M
FIGURE 5. Effect of acetylcholine on cerebral arteriolar caliber under control conditions (solid circles) and in the presence of active SOD and
catalase (open circles). Mean values ± SE of initial vessel diameters in urn for each group are shown. Number in parentheses after SE is the
number of vessels studied. Data are mean ± SEfrom five cats. Analysis of variance showed that responses in the presence of SOD plus catalase
were not significantly different from those seen under control conditions.
combination of SOD and catalase inhibited prostaglandin synthesis. This is unlikely for the following
reasons: Both SOD and catalase are large molecules
and would not be expected to enter cells rapidly.
Thus, a pronounced effect on prostaglandin synthesis would not be anticipated in the short time following application we used in the present experiments.
Second, prostaglandin synthesis by bovine microsomal fractions was not influenced by SOD (Marnett
et al., 1975). Finally, Ellis and Cockrell (1984) found
that the combination of SOD and catalase did not
inhibit prostaglandin synthesis from exogenous la-
beled arachidonate by either intact platelets or fragmented platelets. A more likely explanation for the
observed inhibition is that the vasodilation caused
by topical application of arachidonic acid or bradykinin is mediated via the generation of superoxide
anion radical and hydrogen peroxide. As noted in
the introduction, we found that, in response to
topical application of high concentrations of arachidonic acid and bradykinin, superoxide anion radical
appears in the extracellular space of the brain (Kontos and Wei, 1983). Superoxide anion radical could
then give rise to hydrogen peroxide by spontaneous
TABLE 1
Effects of Arachidonic Acid (200 Mg/ml) on Cerebral Arterioles
Before
Diameters of small arterioles (jim)
N
H
N
N
H
59 ± 3.8
50 ± 3.5
87 ± 6.8
85 ± 8.4
79 ± 9.0
Decrease in diameter
% of normocapnic value
Diameter of large arterioles (jim)
Paco2 (mm Hg)
8 ± 2.6*
15 ±1.2
186 + 9.6
Decrease in diameter
% of normocapnic value
Mean arterial blood pressure (mm Hg)
After
During
163 ± 7.7
242 ± 12.6
233 ± 14.5
12 ±0.8
212 ± 17.4
7 ± 2.7*
149 ± 10.0
127 ± 10.
143 ± 15.8
141 ± 16.9
131 ± 19.9
31 ±1.5
16 ±0.8
30 ±1.8
29 ±1.5
16 ±0.8
Values are mean ± SE from 14 small and 14 large arterioles in five cats. N = normocapnia; H = hypocapnia.
* Significantly (P < 0.05) different from corresponding value before application.
300
Circulation Research/Vol. 55, No. 3, September 1984
TABLE 2
Effect of Arachidonic Acid (200 /ig/ml) Plus SOD (60 U/ml) on Cerebral Arterioles
Before
Diameter of small arterioles (/im)
H
N
N
H
64 ± 3.4
56 + 3.1
75 ± 4.5
66 ± 3.6
57 ± 3.2
13 + 1.1
147 + 10.8
163 ± 11.4
Paco2 (mm Hg)
13 ±1.2
175 ± 12.8
171 ± 11.5
156 ± 11.5
10
± 0.9
128 ± 5.6
121
± 7.0
133 ± 2.9
130 ±3.3
119 ±5.8
33 ± 0.8
18
± 1.3
33 ±0.7
33 ± 0.7
19 ± 1.2
Decrease in diameter
% of normocapnic value
Mean arterial blood pressure (mm Hg)
After
N
Decrease in diameter
% of normocapnic value
Diameter of large arterioles (jim)
During
9 ± 1.0
Values are mean ± SE from 17 small and 13 large arterioles in five cats. N = normocapnia; H = hypocapnia.
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TABLE 3
Effect of Arachidonic Acid (200 jig/ml) Plus Catalase (40 U/ml) on Cerebral Arterioles
During
Before
N
Diameter of small arterioles (^m)
H
63 ± 3.3
Decrease in diameter
% of normocapnic value
Diameter of large arterioles (jim)
Paco2 (mm Hg)
± 2.8
12
± 0.9
143 + 8.5
164 ± 10.0
Decrease in diameter
% of normocapnic value
Mean arterial blood pressure (mm Hg)
55
12
117 ± 2.8
31 ± 1.0
N
N
H
79 ± 4.6
63 ± 3.5
56 ± 3.5
12 ± 0.9
200 ± 13.1
169 + 10.6
153 ± 10.0
± 0.8
9 ±0.8
114 + 5.1
128 ± 7.2
± 0.9
31 ± 1.0
18
After
128 + 3.7
114 ±4.3
± 0.9
18 ± 0.9
31
Values are mean ± SE from 17 small and 17 large arterioles in five cats. N = normocapnia; H = hypocapnia.
TABLE 4
Effect of Arachidonic Acid (200 ng/ml), Plus SOD (60 U/ml), Plus Catalase (40 U/ml) on Cerebral Arterioles
Before
Diameter of small arterioles (nm)
N
H
N
N
H
71 ± 2.1
62 ±2.1
74 ± 3.3
66 ± 3.6
59 ± 3.9
Decrease in diameter
% of normocapnic value
Diameter of large arterioles (/im)
Paco2 (mm Hg)
11 ±1.6
12 ±1.1
176 ± 11.2
Decrease in diameter
% of normocapnic value
Mean arterial blood pressure (mm Hg)
After
During
155 ±9.8
180 ± 11.7
167 ± 11.4
157 ± 11.2
8 ± 1.2
11 ±0.9
127 ± 3.1
110 ±4.2
127 ± 3.9
124 ± 4.3
107 ± 7.4
31 ± 1.3
20 ± 1.4
31 ± 1.1
31 ± 1.5
19 ± 1.4
Values are mean ± SE from 25 small and 25 large arterioles in six cats. N = normocapnic; H = hypocapnic.
Kontos et al./Oxygen Radicals from Arachidonate and Bradykinin
301
TABLE 5
Comparison of PercentiU Changes in Arteriolar Caliber Caused by Arachidonic Acid with and
without SOD and Catalase
Change in diameter (So of control)
Small arterioles
Large arterioles
During
After
During
After
Arachidonic acid
50 ± 10.3
45 ± 10.6
30 ± 3.5
26 ± 6.2
Arachidonic acid plus catalase
25 ± 5.6*
1 ± 1.6*
22 ±2.8*
3+1.4*
Arachidonic acid plus SOD
19 ±5.2*
3 ± 2.8*
8 ± 3.5*
5 ± 2.8*
4 ± 3.1*
- 7 ± 3.9*
3 ± 1.9*
- 5 ± 3.4*
Arachidonic acid plus SOD plus catalase
Values are mean ± SE.
* Significantly (P < 0.05) different from corresponding arachidonic acid value, based on analysis of
variance and Dunnett's (-test.
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dismutation. The interaction of the latter with superoxide anion radical may give rise to the highly
destructive free hydroxyl radical by means of the
catalyzed Haber-Weiss reaction (McCord and Day,
1978). We believe that this is the most likely explanation for our findings.
It is important to note that the inhibition of the
vasodilation via SOD and catalase was substantial,
amounting to 78-83% of the dilation of small arterioles and 67-75% of the dilation of large arterioles
caused by arachidonic acid, and 67-75% of the
dilation of small arterioles and 79-100% of the
dilation of large arterioles in response to bradykinin.
Hence, the oxygen radicals are the major mechanism
by which arachidonate and bradykinin cause cerebral arteriolar dilation.
The relaxant effect of several agents on vascular
smooth muscle of isolated arteries depends upon the
presence of intact endothelium. The mechanism of
this action involves the generation of an endothelium-derived relaxant factor which then reaches the
vascular smooth muscle by diffusion. The nature of
this relaxant factor is not known with certainty, but
it is under intensive investigation. This subject has
been very recently reviewed by Furchgott (1983).
Acetylcholine causes vascular smooth muscle relaxation which is dependent upon an intact endothelium in all arteries tested (Furchgott, 1983) including
large cerebral arteries from cats (Lee, 1982). Bradykinin causes endothelium-dependent vascular
smooth muscle relaxation of canine arteries from
vascular beds other than the brain (Cherry et al.,
1981; Chand and Alrura, 1981), but not in arteries
from rabbits or cats (Cherry et al., 1981). The dependence of the relaxant effect of bradykinin on
cerebral arteries on endothelium has not been tested.
Similarly, arachidonate causes relaxation of vascular
smooth muscle of canine and rabbit arteries largely
by an endothelium dependent mechanism (DeMey
et al., 1982; DeMey and Vanhoutte, 1982; Furchgott,
1983; Singer and Peach, 1983). The endothelium
dependence of the response to arachidonate in cerebral arteries or arteries of cats in general has not
been tested. The present findings show that oxygen
radicals are not the endothelium-derived relaxant
factor generated by acetylcholine. If the relaxant
effect of arachidonic acid on cerebral arterioles is
endothelium dependent, one may infer that more
than one endothelium-derived relaxant agent exists.
Singer and Peach (1983) found that in the isolated
rabbit aorta the relaxant effect of arachidonic acid
was blocked by lipoxygenase inhibitors and was
potentiated by cyclooxygenase inhibitors. This contrasts with the blockade of the relaxant effect of
arachidonate on cerebral arterioles by cyclooxygenase inhibitors. The effect of oxygen radical scavengers on the arachidonate-induced relaxation in isolated vessels has not been tested. The possibility
exists that an oxygen radical derived from arachidonate metabolism by lipoxygenase might be involved. These differences emphasize the strong possibility of differences between in vivo and in vitro
behavior, species differences, and, possibly, differences between large and small vessels.
In earlier experiments, we found that in response
to topical applications of arachidonic acid or PGG2
(Kontos et al., 1980), or in response to severe hypertension (Kontos et al., 1981) or experimental brain
injury (Wei et al., 1980a, 1981), cerebral arterioles
displayed sustained dilation long after these interventions were terminated. This vasodilation was
associated with reduced responsiveness to the
vasoconstrictive effects of arterial hypocapnia and
with the presence of morphological evidence of
damage to vascular smooth muscle and to the endothelium. These results suggest strongly that the
sustained vasodilation observed 1 hour after arachidonate washout and the associated changes in responsiveness can be used as an indicator of the
presence of vascular wall damage. This is supported
by the limited electron microscopic observations reported here. With these considerations in mind, our
Circulation Research/Vol. 55, No. 3, September 1984
302
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results concerning the effects of SOD and catalase
separately or in combination on the response to
topical application of 200 jtg/ml of arachidonic acid
support the following conclusions. First, the injury
to the vascular wall requires the presence of both
superoxide anion radical and hydrogen peroxide for
its production. The elimination of one of these species by either SOD or catalase separately inhibited
the vascular injury. A second conclusion is that
superoxide anion radical and hydrogen peroxide,
each, by itself, is capable of producing reversible
arteriolar dilation. This is supported by the observation that the vasodilation during application of
arachidonic acid was only partially inhibited by SOD
or catalase separately, but was completely eliminated by the two enzymes in combination. The fact
that both of these radical species are required for
the production of vascular injury suggests strongly
that the immediate cause of vascular injury is the
hydroxyl radical generated from the interaction of
the superoxide anion radical and hydrogen peroxide.
The present experiments suggest that it might be
advantageous to examine the possibility that responses which were previously ascribed to increased
production of stable prostaglandins, such as the
vasodilative responses to arachidonic acid or bradykinin in other vascular beds, might in actuality be
due to the associated production of oxygen radicals.
The evidence on which the involvement of stable
prostaglandins is suspected depends on inhibition
of responses by pretreatment with cyclooxygenase
inhibitors and by demonstration of increased production of prostaglandins. Both of these findings are
also consistent with the view that the observed
responses are mediated by oxygen radicals generated in association with accelerated arachidonate
metabolism via the cyclooxygenase pathway. Furthermore, since the inhibition of the responses was
seen at low concentrations of arachidonic acid and
bradykinin, which ordinarily lead to reversible
changes without any residual manifestations of vascular damage, it is possible that these radicals may
actually have a role in the mediation of physiological
responses. The same view has been expressed by
Rosenblum (1983), based on the finding of reversible
cerebral arteriolar dilation from oxygen radicals generated via the xanthine oxidase reaction.
Supported by Grants HL 21821, NS 19316, and NS 12587 from
the National Institutes of Health.
Address for reprints: H.A. Kontos, Box 282 MCV Station, Medical
College of Virginia, Richmond, Virginia 23298.
Received February 1, 1984; accepted for publication June 20, 1984.
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INDEX TERMS: Superoxide anion radical • Hydroxyl radical •
Hydrogen peroxide • Microcirculation • Vascular damage • Superoxide dismutase • Catalase • Acetylcholine • Endotheliumderived relaxant factor
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Oxygen radicals mediate the cerebral arteriolar dilation from arachidonate and bradykinin
in cats.
H A Kontos, E P Wei, J T Povlishock and C W Christman
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Circ Res. 1984;55:295-303
doi: 10.1161/01.RES.55.3.295
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