601
Hydroxyl Radical Mediates the
Endothel ium-Dependent Relaxation Produced by
Bradykinin in Mouse Cerebral Arterioles
William I. Rosenblum
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Bradykinin relaxes arterioles on the brain's surface. This response is endothelium-dependent. The
data presented here confirm the hypothesis that hydroxyl free radical mediates this response and may
be the endothelium-dependent relaxing factor for bradykinin in this microvascular bed. The response
to a locally applied bolus of bradykinin (80 ng/mY) was monitored by intravital TV microscopy. The
response was significantly inhibited or totally blocked by the presence of superoxide dismutase 60U/ml,
catalase 46U/ml, or deferoxamine 0.1 or 0.2 mM. The superoxide dismutase scavenges superoxide
radical, which is known to enter the subarachnoid space as a consequence of cyclooxygenase activation.
Cyclooxygenase is activated by bradykinin. The superoxide can form H2O2, scavenged by catalase,
and the two together generate hydroxyl. The formation of hydroxyl radical is catalyzed by iron.
Deferoxamine O.lmM scavenges the iron, blocking the generation of hydroxyl. Deferoxamine 0.2mM
also directly scavenges the hydroxyl. None of the pharmacologic probes had an effect on arteriolar
diameter when locally applied without bradykinin. Since the dilation produced by bradykinin was
inhibited or totally blocked by probes that prevented hydroxyl formation or directly scavenged
hydroxyl radical, that radical is either an essential mediator of the arteriolar relaxation, or is the
endothelium-dependent relaxing factor for bradykinin in pial arterioles. (Circulation Research
1987;61:601-603)
ecently,12 endothelium-dependent relaxation
has been demonstrated in the microcirculation
in vivo. This demonstration depended on the
selective elimination of the relaxation produced by
acetylcholine and bradykinin, following injury of
endothelium in situ, and the vessels were arterioles on
the surface of the brain (pial arterioles).
In pial arterioles, the relaxations produced by
acetylcholine and bradykinin have thus far been shown
to have different mediators. The response to acetylcholine is eliminated by an oxygen-centered free
radical and restored by radical scavengers. In contrast,
the response to bradykinin is eliminated by catalase and
superoxide dismutase. w
The inhibitory effect of scavengers on the response
to bradykinin, suggested that the hydroxyl radical was
either the endothelium-dependent relaxing factor
(EDRF) for bradykinin or that the production or action
of that EDRF was dependent on the hydroxyl radical.3
The suggestion that free radicals modulate endothelium-dependent relaxation or other physiologic vascular responses is relatively new and important.36'7
Oxygen-centered radicals are generally considered to
be destructive rather than essential for the action of
vasoactive agents. Consequently, it is essential to
replicate the report that an oxygen-centered radical is
involved in the response to bradykinin.3 The following
R
From the Department of Pathology (Neuropathology), Medical
College of Virginia, Virginia Commonwealth University, Richmond, Va.
Supported by National Institutes of Health gTant HL 35935.
Address for correspondence and reprints: William I. Rosenblum,
MD, Neuropathology, Medical College of Virginia, Box 17, MCV
Station, Richmond, VA 23298 Received October 27, 1986; accepted May 8, 1987.
data confirm that report by extending the findings to
a different species and uses an additional pharmacologic probe that implicates the hydroxyl radical as the
essential mediator.
Materials and Methods
Male mice (ICR strain, Dominion Labs, Va.), were
anesthetized with urethane and the pial vessels exposed
by craniotomy as previously described.8"10 Body temperature was maintained at 37° C. The cerebral surface
was continuously suffused at 1 ml/minute with artificial
cerebrospinal fluid at pH 7 . 3 5 . ' " An arteriole 30-50
fj.m in diameter was arbitrarily selected for observation
through a Leitz microscope. Whenever possible, a
segment exactly 35 fxm in diameter was used because
it simplified calibration of the chart paper in the
recorder used to monitor the diameters. This recorder
was linked to a TV image splitter, which in turn sheared
the image of the vessel on a TV camera as described
by Baez.12 The drugs used were administered topically
at pH 7.35 in a bolus of artificial cerebrospinal fluid.
The duration of the bolus was 60 seconds. The diameter
was monitored continuously before, during, and for 5
minutes after the bolus was delivered. The maximal
change in diameter produced by the bolus was used to
calculate a drug's effect. This change was expressed as
a percent of baseline diameter. Thus a 10% dilation was
expressed as 110% of baseline.
The drugs used were bradykinin triacetate (Sigma
Chemical Co., St. Louis, Mo.); acetylcholine chloride
(Sigma); superoxide dismutase (SOD), 2,000 U/mg
(Sigma); catalase 11,500 U/mg, thymol free (Sigma);
deferoxamine mesylate (Desferal, Ciba, Medfield,
Mass.). All stock solutions were freshly made in
deionized water on the day of use and kept on ice. Final
602
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dilution was made by adding no more than 5 /LX.1 to 1
ml of artificial CSF.
Preliminary experiments confirmed the observation3
that heat inactivated SOD and catalase had no effect on
the response to acetylcholine or bradykinin. Thereafter,
experiments were performed as described below.
Four separate experiments were performed with
bradykinin triacetate, 80 /ig/ml. This dose was selected
after experiments showing dose dependent reduction of
responses at lower doses. In each experiment responses
to bradykinin triacetate plus scavenger vehicle (i.e., 5
/xl or less of deionized water) were compared with
responses to bradykinin triacetate plus scavenger,
mixed together in the 1 ml bolus of artificial CSF. The
responses were obtained 15 minutes apart to allow for
complete washoff of the bolus and restoration of
baseline diameter. Thus, each mouse served as its own
control, permitting analysis of the results by paired t
test after log transformation of the data to normalize the
distribution. Fifteen minutes after the final test of
bradykinin's action, the scavenger in question was
tested by itself. The first experiment tested the effect of
60 U/ml SOD on the response to bradykinin in 10 mice.
SOD is a scavenger of superoxide radical, 3 and the
dose chosen was used by Kontos et al to inhibit
bradykinin. The second study tested the effect of 46
U/ml catalase in 10 mice. Catalase destroys hydrogen
peroxide and the dose was similar to that used by
Kontos et al to inhibit bradykinin. Superoxide plus
hydrogen peroxide can generate the hydroxyl radical.13
This reaction is catalyzed by free iron in amounts
known to exist in the cerebrospinal fluid bathing the pial
vessels. Therefore, a third set of experiments was
performed on 10 mice, using deferoxamine, a chelator
of this free iron.
High concentrations of deferoxamine not only inhibit formation of hydroxyl radical but
also directly scavenge hydroxyl.
Therefore, the
fourth experiment employed a higher concentration of
deferoxamine in 5 mice.
After these experiments were completed the effect of
SOD on the relaxation produced by acetylcholine
chloride (80 /Ag/ml) was also tested.
At the end of each experiment blood gas determinations were made from 100 /AI of blood from the
carotid artery. These values were similar from study to
study. The mean values ( ± SD) were as follows and
will not be referred to again: CO2, 36 ± 3 mm Hg; O2,
106± 10 mm Hg; pH 7.34±0.04.
Results
The results are shown in Table 1. Bradykinin
triacetate, 80 /xg/ml, produced relaxation that was
significantly inhibited by each of the tested drugs,
SOD, catalase, deferoxamine (at 2 dose levels). The
inhibition was most complete by SOD or 2 mM
deferoxamine. Indeed, some small constrictions were
seen when bradykinin plus SOD were applied. In each
experiment, scavengers by themselves failed to change
the diameter. This data is not shown in the table.
SOD failed to alter the dilation produced by acetylcholine (baseline diameter 3 6 ± 2 fim, n= 10, di-
Circulation Research
Vol 61, No 4, October 1987
Table 1. Inhibition of Bradykinin Relaxation by Agents Interfering With Production of Hydroxyl Radical
Study No.
1 (n=10)
BK + 60 U/ml SOD
BK alone
Resting
diameter
(/xm,
mean ± SD)
Response to
bradykinin
(80 ng/ml)
(Diameter as %
(mean + SD)
resting
diameter)
34±3
96 + 7*
113 + 7
BK + 46 U/ml catalase
BK alone
3 (n=10)
BK + 0.1 mM deferoxamine
BK alone
34±1
106 + 2*
110 + 3
34±4
107 + 3*
113 + 5
BK + 0.2 mM deferoxamine
BK alone
31+5
101+2*
113+6
•Dilation significantly impaired compared to bradykinin alone;
p<0.01, paired t test after log transformation to normalize the
distribution.
ameter increased to 110 ± 3% (mean ± SD) of baseline
in presence of 60 U/ml SOD, and to 1 1 2 ± 3 % of
baseline in absence of SOD; p>0.2, paired t test.)
Discussion
Our data confirm the inhibitory effects of SOD and
catalase on the response of pial arterioles to bradykinin.3 Kontos et al3 suggested that these data implicated the hydroxyl radical that is formed when superoxide and H2O2 interact in an iron catalyzed
reaction.1314 We extended the research design testing a
role for hydroxyl radical by using deferoxamine, which
chelates iron and would, therefore, inhibit hydroxyl
production.14 A dose of 0.1 mM significantly inhibited
bradykinin, and a dose of 0.2 mM had an even greater
effect. The higher dose not only chelates iron but also
begins to have significant ability to directly scavenge
hydroxyl.1617
None of the inhibitors had any effect on the diameter
of pial arterioles when used alone. Thus, inhibition of
the response to bradykinin was not caused by an
opposing constricting influence.
The selectivity of the effect of SOD was indicated
by failure of this scavenger to significantly impair
dilation produced by acetylcholine. This agrees with
the findings of Kontos et al3 who failed, also, to find
an action of catalase on acetylcholine.
Our data establishes in mice what the data of Kontos
et al suggested in cats. Mutually confirmatory data
coming from two species is extremely important
because others have reported no effect of SOD on the
response to bradykinin in rat cremaster.18 The negative
report using rats left open the possibility of species
differences. Such differences are common when endo-
Rosenblum
Hydroxyl Radical and Endothelium Dependent Dilation
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thelium-dependent relaxations, like that to bradykinin,
are investigated." 20 However, our data makes less
likely interspecies differences as an explanation for
failure of others to inhibit bradykinin with SOD.
Perhaps there are different mechanisms for bradykinin's action in different vascular beds. For example,
since the production of a dilating prostaglandin2' occurs
as a result of cyclooxygenase stimulation by bradykinin, in some beds this prostaglandin could conceivably
dilate even in the absence of a concomitantly produced
oxygen-centered radical.
The mechanism by which bradykinin may initiate
production of superoxide has been discussed by Kontos
et al. 3 They point out that bradykinin is known to
stimulate prostaglandin synthesis by blood vessels.
They have shown that cyclooxygenase, the enzyme
responsible for prostaglandin synthesis, generates superoxide radical from pial arterioles during prostaglandin synthesis and that the radical is released into the
subarachnoid cerebrospinal fluid.22
Left unresolved is the question of whether hydroxyl
radical is a sufficient, or merely a necessary, mediator
of the relaxation of pial arterioles produced by bradykinin. In other words, is the hydroxyl radical the EDRF
for bradykinin in these vessels or merely necessary for
its action? This question will not be resolved unless
another EDRF for bradykinin's action on pial arterioles
can be identified and isolated. If so, one might attempt
to block its action with the scavengers and the chelator
used here. That would demonstrate the necessity of the
radical for the function of the true EDRF. Until such
a time, we can tentatively offer the hypothesis that the
hydroxyl radical is the EDRF for bradykinin in pial
arterioles.
References
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KEY WORDS
•
bradykinin
endothelium-dependent relaxation
brain microcirculation
Hydroxyl radical mediates the endothelium-dependent relaxation produced by bradykinin
in mouse cerebral arterioles.
W I Rosenblum
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Circ Res. 1987;61:601-603
doi: 10.1161/01.RES.61.4.601
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1987 American Heart Association, Inc. All rights reserved.
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