From the Department of Medicine, Medical College of Virginia Campus of
Virginia Commonwealth University, Richmond (E.P.W., H.A.K.), and Department of
Anesthesiology, University of Alabama at Birmingham (J.S.B.).
Correspondence to Hermes A. Kontos, MD, PhD, Medical College of Virginia Campus of Virginia Commonwealth University, PO Box 980549, Richmond, VA 23298-0549. E-mail hakontos{at}vcu.edu
Methods—We investigated the effect of three
antioxidants—dimethylsulfoxide (DMSO), salicylate, and
L-cysteine—on the cerebral arteriolar dilation caused by
topical application of hydrogen peroxide and peroxynitrite in
anesthetized cats equipped with cranial windows. We also tested
the effect of these antioxidants on the vasodilation caused by
pinacidil and cromakalim, two known openers of ATP-sensitive potassium
channels.
Results—DMSO was more effective in inhibiting dilation from
hydrogen peroxide, whereas salicylate and L-cysteine were
more effective in inhibiting dilation from peroxynitrite. All three
antioxidants inhibited dilation in concentrations that were remarkably
low (<1 mmol/L). All three antioxidants inhibited vasodilation
from two known potassium channel openers, pinacidil and cromakalim.
Their effect was specific because they did not affect dilation from
adenosine or nitroprusside.
Conclusions—The findings show that antioxidants block
ATP-sensitive potassium channels in cerebral arterioles. This appears
to be the mechanism by which antioxidants inhibit the dilation from
hydrogen peroxide and peroxynitrite and not through scavenging of a
common intermediate, ie, hydroxyl radical. The differences between
effectiveness in inhibiting dilation from hydrogen peroxide and
peroxynitrite by various antioxidants suggest that hydrogen peroxide
and peroxynitrite act at two different sites, one in a water-soluble
environment and the other in a lipid-soluble environment.
In the present experiments we explored the possibility that we
may be able to distinguish whether vascular responses are due to
hydrogen peroxide or to peroxynitrite by establishing differences in
the inhibitory effects of various hydroxyl radical
scavengers on their vascular actions. These experiments led to the
unexpected finding that antioxidants block ATP-sensitive potassium
channels.
The cerebral microcirculation of the parietal cortex was visualized
through an acutely implanted cranial window, as described in detail
previously.5 The space under the cranial window
was filled with artificial cerebrospinal fluid (CSF) identical in
composition to that of cats. One port of the window was connected to a
pressure transducer for continuous monitoring of intracranial pressure.
The intracranial pressure was maintained at 5 mm Hg by
connecting another outlet of the window to a coiled plastic tube whose
free end was placed at the appropriate height to give the desired
pressure. Two ports of the cranial window were used as inlet and
outlet, allowing topical application of various solutions by
superfusion. Pial arteriolar diameter was measured with an
image-splitting device attached to a microscope. In each animal,
several arterioles were observed covering a wide range of vessel
caliber. The responses of small and large arterioles (smaller and
larger than 100 µm in diameter, respectively) were
analyzed separately to identify any size-dependent differences
in responses.
Dimethylsulfoxide (DMSO), pinacidil, cromakalim,
L-cysteine, sodium salicylate, adenosine, and
sodium nitroprusside were obtained from Sigma Chemical Co. Hydrogen
peroxide was obtained from Baker Chemical Co. Peroxynitrite was
synthesized as described previously.4 It was kept
as 100 mmol/L solution in 1 mol/L sodium hydroxide at pH 14. Under
these conditions, peroxynitrite is stable indefinitely. The
concentration of peroxynitrite was monitored spectrophotometrically by
determining absorbance at 302 nm.6 Because
peroxynitrite when dissolved in aqueous solution decomposes very
rapidly, we used the following technique for its application on the
brain surface.2 We calculated the buffer
composition of the CSF required to maintain pH at 7.3 after the
addition of the appropriate amounts of peroxynitrite. After mixing the
solutions rapidly, we filled the space under the window with the
solution. This procedure required less than 3 seconds. To control the
effects of sodium hydroxide, we added the amount of sodium hydroxide
equivalent to the highest dose of peroxynitrite to the appropriate CSF
volume with a buffer composition design to maintain pH at 7.3 and
placed it under the window in the same fashion as used for the
peroxynitrite solutions. All other solutions were prepared directly in
artificial CSF immediately before use and were equilibrated at 37°C
in a water bath immediately before application.
The experimental design was as follows: The appropriate solutions of
hydrogen peroxide or peroxynitrite were used to fill the space under
the cranial window, and vessel diameters were measured between 2 and 4
minutes after application. This allowed sufficient time for the vessels
to reach a new steady state. Responses were expressed as percent
changes from the baseline diameters. Responses to different
concentrations were examined in cumulative fashion.
We tested the responses to hydrogen peroxide and peroxynitrite before
and after pretreatment with DMSO, salicylate, or
L-cysteine, three hydroxyl radical scavengers. We chose
DMSO, salicylate, and L-cysteine because they are commonly
used antioxidants and they have high reactivity with hydroxyl radical.
The reaction constant with hydroxyl radical for salicylate is
2x1010, for DMSO 7.1x109,
and for L-cysteine 1.3x109
mol/L-1
s-1.7 DMSO is lipid
soluble, whereas the other two antioxidants are water soluble. In
addition, L-cysteine, like other sulfhydryl-containing
compounds, reacts rapidly and directly with
peroxynitrite.8 These differences might be
expected to maximize our ability to differentiate between the two
sources of hydroxyl radical.
In preliminary experiments we identified the lowest dose of DMSO,
salicylate, or L-cysteine that inhibited the vasodilator
action of hydrogen peroxide or peroxynitrite. Systematic studies were
done in the presence of 10 µmol/L DMSO, 100 µmol/L
salicylate, and 250 µmol/L L-cysteine. If these did
not have inhibitory effects, we retested in another series
of experiments responses in the presence of 1000 µmol/L DMSO,
250 to 500 µmol/L salicylate, or 500 µmol/L
L-cysteine. The space under the cranial window was filled
with the appropriate solution of a hydroxyl radical scavenger and left
in place for 15 minutes. The responses to hydrogen peroxide or
peroxynitrite were tested before and immediately after the application
of the scavenger. No attempt was made to replace the hydroxyl radical
scavenger between applications of different concentrations of hydrogen
peroxide and peroxynitrite.
In another series of experiments we tested the effects of DMSO,
salicylate, and L-cysteine on the vasodilator responses to
the ATP-sensitive potassium channel openers pinacidil and cromakalim.
The responses to these agents were tested before as well as after the
application of the scavengers as described above. To establish the
specificity of the action of antioxidant, we also tested responses to
adenosine and nitroprusside before and after DMSO, salicylate,
and L-cysteine.
Statistical analysis of the results was done with ANOVA
followed by t tests modified for multiple comparisons.
Fig 1
Fig 2
Fig 3
Figs 4
Figs 6 through 8
Our initial hypothesis was that the hydroxyl radical scavengers
inhibited the vasodilation from hydrogen peroxide and peroxynitrite by
scavenging hydroxyl radical. Both agents are known to generate hydroxyl
radical. Hydrogen peroxide generates hydroxyl radical by the
Haber-Weiss reaction via catalysis by transition
metal,3 while peroxynitrite in aqueous solution
produces peroxynitrous acid, which decomposes spontaneously to generate
hydroxyl radical.4 In addition, we found earlier
that the vasodilation in response to hydrogen peroxide is eliminated by
scavenging iron with deferoxamine, suggesting that hydroxyl
radical is the mediator of the vasodilation from hydrogen
peroxide.2 As explained below, there are,
however, strong reasons why the hypothesis that the inhibition of the
vasodilation by hydrogen peroxide and peroxynitrite in the presence of
antioxidants is due to their scavenging hydroxyl radical is not
tenable.
Cells are well defended against the superoxide by superoxide dismutase
and against hydrogen peroxide by catalase and glutathione peroxidase.
These enzymes are specific and highly effective. As a result, the
steady state concentrations of superoxide and hydrogen peroxide are
kept at a very low range, insufficient to cause cellular damage. In
contrast, there is no natural endogenous scavenger of
hydroxyl radical because this radical is extremely reactive. Hydroxyl
radical reacts with most common organic and biological molecules at
near diffusion-limited rates.7 The compounds used
to scavenge hydroxyl radical are useful because they can be tolerated
by cells in remarkably high concentrations and not because they are
more reactive with hydroxyl radical than potential biological targets.
Commonly used hydroxyl radical scavengers, like the ones we used in the
present experiments, must be used in 10 to 1000 mmol/L
concentrations to effectively scavenge hydroxyl radical in
vitro.4 9
While antioxidants such as DMSO and salicylate are widely used to
scavenge hydroxyl radical in brain and other organs, the minimum
effective concentration for this action has rarely been investigated.
The surprisingly low concentrations of DMSO and salicylate that were
effective in inhibiting vasodilation from hydrogen peroxide and
peroxynitrite reported above are at least 1000-fold lower than required
to block the action of hydroxyl radical in
vitro.4 9 This finding strongly suggests that the
mechanism of action of these agents is different.
A second reason for seeking a different mechanism of action for the
antioxidants was that they were not equally effective in preventing the
vasodilation from hydrogen peroxide and peroxynitrite, suggesting that
hydroxyl radical was not the common intermediate. While DMSO and
salicylate do not directly affect the rate of peroxynitrite
decomposition, L-cysteine reacts directly with
peroxynitrite at rates that are considerably faster than with hydrogen
peroxide.8 It is possible, therefore, that the
difference in the effectiveness of L-cysteine against
peroxynitrite and hydrogen peroxide may be due in part to its direct
reaction with peroxynitrite.
For the reasons outlined above, we sought other mechanisms for
the inhibition of the vasodilation by hydrogen peroxide and
peroxynitrite. In earlier studies we found that these two agents opened
ATP-sensitive potassium channels based on the finding that glyburide, a
known inhibitor of these channels, abolished the
vasodilation they caused in cerebral arterioles.2
It was therefore logical to examine whether the hydroxyl radical
scavengers we used might be inactivating ATP-sensitive potassium
channels rather than scavenging hydroxyl radical directly. The
experiments reported above show that indeed this is the case, because
all three scavengers eliminated the vasodilation from pinacidil or
cromakalim, two known ATP-sensitive potassium channel openers.
Therefore, the present experiments and our earlier
findings2 suggest that in cerebral arterioles of
cats, oxidants open ATP-sensitive potassium channels and antioxidants
inhibit these channels.
Understanding of the structure of ATP-sensitive potassium channels has
improved considerably as a result of successful attempts to clone these
channels.10 11 12 13 14 As a result of these studies it
now appears that they consist of two components: an inward rectifying
potassium channel and a sulfonylurea receptor. The sulfonylurea
receptor from pancreas of rats and hamsters has recently been
cloned.10 It consists of 1582 amino acids, has
two ATP-binding sites, and belongs to the ATP-binding cassette family
of proteins. It appears to function by modulating the activity of the
potassium pore protein.11 Sulfonylurea
receptor–like proteins have recently been identified in
extrapancreatic tissues.12 The sulfonylurea
receptor is the binding site for sulfonylureas such as glyburide, which
inhibit the ATP-sensitive potassium channels.15
The mechanisms by which agents like pinacidil and cromakalim open
ATP-sensitive potassium channels are incompletely understood and appear
to be complex. Some evidence, mainly based on the fact that these
compounds reversibly and competitively antagonize the action of
glyburide, suggests that they act on the same binding site as
glyburide, namely on the sulfonylurea receptor.15
It is therefore likely that the antioxidants we used are acting on the
same receptor. The findings that DMSO was more effective against
hydrogen peroxide while the two water-soluble antioxidants were more
effective in inhibiting the action of peroxynitrite suggest the
possibility that there may be two separate sites of action, one
accessible to lipid-soluble agents and the other accessible to
water-soluble ones. The pattern of blockade of the effect of pinacidil
and cromakalim by the three antioxidants is consistent with
this view. Both of these agents are lipid soluble. Their effects were
blocked completely by very-low-dose (10 µmol/L) DMSO. On the
other hand, the two water-soluble antioxidants, L-cysteine
and salicylate, blocked the effects of pinacidil and cromakalim
completely at high dose, a pattern similar to what was seen with
hydrogen peroxide but unlike that seen with peroxynitrite.
In a recent study Sobey et al16 found that
hydrogen peroxide dilated cerebral arteries in rats by opening
calcium-activated potassium channels. An obvious difference
between these experiments and ours is that Sobey et
al16 used 10 to 100 µmol/L hydrogen
peroxide, ie, 100 to 1000 times the largest concentration we used. At
these high concentrations hydrogen peroxide inhibits membrane and
sarcolemmal calcium pumps17 and would be expected
to raise intracellular calcium ion concentration. Such an increase in
calcium ion concentration may be responsible for activating
calcium-activated potassium channels. In earlier
studies18 we found that very high concentrations
of hydrogen peroxide induced histologically
demonstrable damage to the vascular wall associated with sustained
arteriolar vasodilation. Lower concentrations of hydrogen peroxide (1
to 3 µmol/L) caused reversible arteriolar dilation but also
caused inhibition of release of endothelium-derived
relaxing factor by acetylcholine as well as guanylate
cyclase inhibition, which outlasted the vascular
effects.1 The low concentrations of hydrogen
peroxide and peroxynitrite we used in the present experiments had
vascular effects that were readily reversible. It is evident,
therefore, that large concentrations of oxidants cause tissue damage,
while lower concentrations have effects that are reversible and may be
involved in physiological regulation.
Received November 4, 1997;
revision received January 15, 1998;
accepted January 21, 1998.
2.
Wei EP, Kontos HA, Beckman JS. Mechanisms of cerebral
vasodilation by superoxide, hydrogen peroxide, and peroxynitrite.
Am J Physiol. 1996;271(Heart Circ Physiol
40):H1262–H1266.
3.
McCord JM, Day DE Jr. Superoxide-dependent
production of hydroxyl radical catalyzed by iron-EDTA complex.
FEBS Lett. 1978;86:139–142.[Medline]
[Order article via Infotrieve]
4.
Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman
BA. Apparent hydroxyl radical production by peroxynitrite:
implications for endothelial injury from nitric oxide
and superoxide. Proc Natl Acad Sci U S A. 1990;87:1620–1624.
5.
Levasseur JE, Wei EP, Raper AJ, Kontos HA, Patterson
JL. Detailed description of a cranial window technique for acute and
chronic experiments. Stroke. 1975;6:308–317.
6.
Hughes MN, Nicklin HG. The chemistry of pernitrites,
I: kinetics of decomposition of pernitrous acid. J Chem
Soc. 1968;1968:450–452.
7.
Dorfman LM, Adams GE. Reactivity of the Hydroxyl
Radical in Aqueous Solutions. Washington, DC: US Dept of Commerce,
National Bureau of Standards; 1973.
8.
Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitrite
oxidation of sulfhydryls: the cytotoxic potential of superoxide and
nitric oxide. J Biol Chem. 1991;266:4244–4250.
9.
Winterbourn CC. The ability of scavengers to
distinguish OH· production in the iron-catalyzed Haber-Weiss
reaction: comparison of four assays for OH·. J Free Radic Biol
Med. 1987;3:33–39.
10.
Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JP
IV, Boyd AE III, González G, Herrera-Sosa H, Nguy K, Bryan J,
Nelson D. Cloning of the ß cell high-affinity sulfonylurea
receptor: a regulator of insulin secretion. Science. 1995;268:423–426.
11.
Ammala C, Moorhouse A, Gribble F, Ashfield R, Proks P,
Smith PA, Sakura H, Coles B, Ashcroft SJ, Ashcroft FM. Promiscuous
coupling between the sulfonylurea receptor and inwardly rectifying
potassium channels. Nature. 1996;379:545–548.[Medline]
[Order article via Infotrieve]
12.
Chutkow WA, Simon MC, Le Beau MM, Burant CF. Cloning,
tissue expression, and chromosomal localization of SUR2, the putative
drug-binding subunit of cardiac, skeletal muscle, and vascular
KATP channels. Diabetes. 1996;45:1439–1445.[Abstract]
13.
Inagaki N, Gonoi T, Clement JP, Namba N, Inazawa
J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J. Reconstitution of
IKATP: an inward rectifier subunit plus the
sulfonylurea receptor. Science.. 1995;270:1166–1170.
14.
Inagaki N, Tsuura Y, Namba N, Masuda K, Gonoi T, Horie
M, Mizuta M, Seino S. Cloning and functional characterization of a
novel ATP-sensitive potassium channel ubiquitously expressed in rat
tissues, including pancreatic islets, pituitary, skeletal muscle, and
heart. J Biol Chem. 1995;270:5691–5694.
15.
Quale JM, Nelson MT, Standen NB. ATP-sensitive
and inwardly rectifying potassium channels in smooth muscle.
Physiol Rev. 1997;77:1165–1232.
16.
Sobey CG, Heistad DD, Faraci FM. Mechanisms of
bradykinin-induced cerebral vasodilation in rats. Stroke. 1997;28:2290–2295.
17.
Grover AK, Samson SE, and Fomin VP. Peroxide
inactivates calcium pumps in pig coronary artery.
Am J Physiol. 1992;263(Heart Circ Physiol
32):H537–H543.
18.
Wei EP, Christman CW, Kontos HA, Povlishock JT. Effects
of oxygen radicals on cerebral arterioles. Am J
Physiol. 1985;248(Heart Circ Physiol
17):H157–H162.
Department
of Internal Medicine,
Cardiovascular Division,
University of Iowa College of Medicine,
Iowa City, Iowa
The KATP channel is a complex of proteins consisting of a
pore-forming subunit and the sulfonylurea receptor.1 The
latter component is responsible for glibenclamide sensitivity of this
channel and is probably the site that confers sensitivity to
KATP channel openers such as pinacidil.1
The present study provides pharmacological evidence that antioxidants
inhibit KATP channels in blood vessels. This conclusion is
based on the finding that three antioxidants (dimethylsulfoxide,
salicylate, and L-cysteine) inhibited cerebral
vasodilatation in response to KATP channel openers
(pinacidil and cromakalim) and exogenously applied hydrogen peroxide
and peroxynitrite (which produce glibenclamide-sensitive vasodilatation
in this model).
How would antioxidants inhibit activation of ATP-sensitive potassium
channels? Glibenclamide binds to the sulfonylurea receptor and is the
most commonly used inhibitor of KATP channels in studies of
blood vessesl.1 2 It might seem logical to assume that the
site of action of other inhibitors of KATP channels would
also be the sulfonylurea receptor. However, recent evidence suggests
that inhibition of KATP channels by phentolamine (a
nonsulfonylurea) is not mediated by effects on the sulfonylurea
receptor. Rathger, phentolamine inhibits channel activity by effects on
the pore-forming subunit or other proteins that regulate activity of
this channel.5 Thus, the site of action of antioxidants may
or may not be the sulfonylurea receptor. One finding of this study was
that the three antioxidants exhibited differential efficacy with
respect to inhibition of responses to hydrogen peroxide and
peroxynitrite. Thus, the mechanisms and sites of action of these
antioxidants may differ.
Received November 4, 1997;
revision received January 15, 1998;
accepted January 21, 1998.
2.
Faraci FM, Heistad DD. Regulation of the cerebral circulation:
role of endothelium and potassium channels. Physiol
Rev.1998;78:53–97.
3.
Wei EP, Kontos HA, Beckman JS. Mechanisms of cerebral
vasodilation by superoxide, hydrogen peroxide, and peroxynitrite.
Am J Physiol.. 1996;271:H1262–H1266.
4.
Sobey CG, Heistad DD, Faraci FM. Mechanisms of
bradykinin-induced cerebral vasodilatation: evidence that reactive
oxygen species activate K+ channels. Stroke.. 1997;28:2290–2295.
5.
Proks P, Ashcroft FM. Phentolamine block of KATP
channels is mediated by Kir6.2. Proc Natl Acad Sci U S A.. 1997;94:11716–11720.
© 1998 American Heart Association, Inc.
Original Contributions
Antioxidants Inhibit ATP-Sensitive Potassium Channels in Cerebral Arterioles
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Background and Purpose—Hydrogen
peroxide and peroxynitrite are capable of generating hydroxyl radical
and are commonly suspected as sources of this radical in tissues. It
would be useful to distinguish the source of hydroxyl radical in
pathophysiological conditions and to clarify the
mechanisms by which antioxidants modify vascular actions of
oxidants.
Key Words: hydrogen peroxide • hydroxyl radical • microcirculation • peroxynitrite • vascular regulation
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Hydrogen peroxide and
peroxynitrite cause pronounced cerebral arteriolar dilation in
sufficiently low concentrations to merit consideration as mediators of
cerebral vascular responses.1 2 Since hydrogen
peroxide and peroxynitrite can generate hydroxyl
radical,3 4 it is not surprising that both have
been suggested as sources of this radical and hence have been
implicated as mediators of cellular injury. We recently found that
hydrogen peroxide and peroxynitrite dilate cerebral arterioles by the
same mechanism, ie, by opening ATP-sensitive potassium
channels.2
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Experiments were performed in cats anesthetized with
sodium pentobarbital (30 mg/kg IV). Additional doses of anesthetic were
given as required to maintain surgical anesthesia based on
testing of corneal reflexes and on responses to tail pinch. The animals
were subjected to tracheostomy and ventilated with a positive-pressure
respirator. The end-expiratory CO2 of the animals
was continuously monitored with a CO2
analyzer and was maintained at a constant level of
approximately 30 mm Hg. Arterial blood pressure
was measured with a pressure transducer connected to a cannula
introduced into the aorta through the femoral artery.
Arterial blood samples were collected for determination of
arterial blood oxygen, CO2 partial
pressures, and pH at appropriate intervals during the experiment. Blood
gas tensions and pH were measured with oxygen and
CO2 electrodes and a pH meter. The rectal
temperature of the animal was monitored continuously and was kept
constant with the aid of a heating blanket.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
None of the hydroxyl radical scavengers in any of the doses used
had any significant effect on pial arteriolar caliber. 
shows that DMSO 10 µmol/L
inhibited significantly and to a pronounced extent the vasodilation
from hydrogen peroxide, but it had no significant effect on the
responses to peroxynitrite. DMSO 1000 µmol/L inhibited responses
to peroxynitrite completely.
View larger version (31K):
[in a new window]
Figure 1. Effect of pretreatment with DMSO on cerebral
arteriolar responses to hydrogen peroxide (left panel) and to
peroxynitrite (two right panels). Values are mean+SE of the percent
changes in diameter. Baseline values from which the percent changes
were calculated are given in micrometers in the inset.
Values were obtained from 9 small and 17 large arterioles in 5 cats in
the left panel, from 17 small and 12 large arterioles in 4 cats in the
middle panel, and from 19 small and 19 large arterioles in 6 cats in
the right panel. Note that DMSO 10 µmol/L significantly
inhibited responses to hydrogen peroxide, but it had no effect on
responses to peroxynitrite. DMSO at the higher dose significantly and
completely inhibited responses to peroxynitrite. shows that salicylate 100
µmol/L inhibited to a pronounced degree the vasodilation from
peroxynitrite. This dose of salicylate, on the other hand, had no
significant effect on the vasodilation from hydrogen peroxide. At a
concentration of 250 µmol/L, salicylate partially inhibited the
responses to hydrogen peroxide, and the effect was more pronounced at
500 µmol/L.
View larger version (37K):
[in a new window]
Figure 2. Effect of pretreatment with salicylate on cerebral
arteriolar responses to peroxynitrite (left panel) and to hydrogen
peroxide (three right panels). Values are mean+SE of the percent
changes in diameter. Baseline diameters from which the percent changes
were calculated are given in micrometers in the insets.
Values were obtained from 20 small and 17 large arterioles in 5 cats in
the left panel, from 20 small and 17 large arterioles in 5 cats for the
100-µmol/L salicylate panel, from 19 small and 17 large arterioles in
5 cats for the 250-µmol/L salicylate panel, and from 20 small and 17
large arterioles in 5 cats in the 500-µmol/L salicylate panel in the
hydrogen peroxide data. Note that salicylate at 100 µmol/L
significantly inhibited responses to peroxynitrite, but it had no
effect on responses to hydrogen peroxide. Salicylate at the two higher
doses induced dose-dependent, significant reductions in the responses
to hydrogen peroxide. shows that L-cysteine
250 µmol/L completely inhibited the responses to peroxynitrite,
while responses to hydrogen peroxide were not affected.
L-Cysteine 500 µmol/L partially inhibited the
responses to hydrogen peroxide.
View larger version (36K):
[in a new window]
Figure 3. Effect of pretreatment with L-cysteine
on cerebral arteriolar artery responses to peroxynitrite and hydrogen
peroxide. Values are mean+SE of the percent changes in diameter.
Baseline diameters from which the percent changes were calculated are
given in micrometers in the insets. Values were obtained
from 24 small and 15 large arterioles in 5 cats in the 250-µmol/L
L-cysteine panel and from 16 small and 15 large arterioles
in 5 cats for the 500-µmol/L L-cysteine panel in the
peroxynitrite data, and from 24 small and 15 large arterioles in 5 cats
in the 250-µmol/L L-cysteine panel and 16 small and 15
large arterioles in 5 cats for the 500-µmol/L L-cysteine
panel in the hydrogen peroxide data. Note that L-cysteine
at both doses significantly and completely inhibited responses to
peroxynitrite, while L-cysteine at 250 µmol/L had no
effect on responses to hydrogen peroxide but had a significant
inhibitory effect at the higher dose. and 5 show that DMSO 10 µmol/L,
L-cysteine 250 to 500 µmol/L, and salicylate 100 to
250 µmol/L inhibited responses to either pinacidil or
cromakalim.
View larger version (34K):
[in a new window]
Figure 4. Effect of pretreatment with DMSO, salicylate, and
L-cysteine on cerebral arteriolar responses to pinacidil.
Values are mean+SE of the percent changes in diameter. Baseline
diameters from which the percent changes were calculated are given in
micrometers in the insets. Values were obtained from 24
small and 17 large arterioles in 5 cats in the left panel, from 20
small and 14 large arterioles in 5 cats in the middle panel, and from
18 small and 15 large arterioles in 5 cats in the right panel. Note
that all antioxidants, at all doses used, significantly inhibited
responses to pinacidil.
View larger version (33K):
[in a new window]
Figure 5. Effect of pretreatment with DMSO,
L-cysteine, and salicylate on cerebral arteriolar responses
to cromakalim. Values are mean+SE of the percent changes in diameter.
Baseline values from which the percent changes were calculated are
given in the insets. Values were obtained from 20 small and 19 large
arterioles in 5 cats in the left panel, from 18 small and 16 large
arterioles in 5 cats in the middle panel, and from 19 small and 17
large arterioles in 5 cats in the right panel. Note that all
antioxidants, at all doses used, significantly inhibited responses to
cromakalim.
show that vasodilator responses to adenosine and nitroprusside
were unaffected by DMSO, L-cysteine, or salicylate.
View larger version (24K):
[in a new window]
Figure 6. Effect of pretreatment with DMSO on cerebral
arteriolar responses to nitroprusside (SNP) and to adenosine
(ADO). Values are mean+SE of the percent changes in diameter. Baseline
diameters from which the percent changes were calculated are given in
micrometers in the insets. Values are from 12 small and 12
large arterioles in 4 cats. Note that DMSO had no significant effect on
responses to either SNP or ADO.
View larger version (24K):
[in a new window]
Figure 7. Effect of pretreatment with L-cysteine
on cerebral arteriolar responses to nitroprusside (SNP) or
adenosine (ADO). Values are mean+SE of the percent changes in
diameter. Baseline diameters from which the percent changes were
calculated are given in the insets. Values are from 12 small and 12
large arterioles in 4 cats. Note that L-cysteine had no
significant effect on responses to either SNP or ADO.
View larger version (24K):
[in a new window]
Figure 8. Effect of pretreatment with salicylate on cerebral
arteriolar responses to nitroprusside (SNP) or adenosine (ADO).
Values are percent changes in mean+SE of the percent changes in
diameter. Baseline diameters from which the percent changes were
calculated are given in micrometers in the insets. Values
are from 12 small and 11 large arterioles in 4 cats. Note that
salicylate did not affect responses to either SNP or ADO.
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
The results reported above show that the three hydroxyl radical
scavengers we used were effective in inhibiting the vasodilation
induced by hydrogen peroxide and peroxynitrite. The two water-soluble
agents, salicylate and L-cysteine, were more effective in
inhibiting the vasodilation from peroxynitrite than that from hydrogen
peroxide, while the reverse was true for the lipid-soluble DMSO. This
suggests that the sites of action of hydrogen peroxide and
peroxynitrite may be different. The potency of the various
inhibitors against the vasodilation of hydrogen peroxide
and peroxynitrite differed sufficiently to render a distinction between
the two feasible.
Acknowledgments
This study was supported by grant NS 19316.
Footnotes
Reviews of this article were directed by Richard J. Traystman, PhD. To avoid possible conflict of interest, Dr Hermes Kontos was not involved in the review process.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
1.
Wei EP, Kontos HA.
H2O2 and
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implications for the identity of endothelium-derived
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Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Activity of potassium channels in smooth muscle has a major
influence on vascular tone.1 2 Reactive oxygen species and
peroxynitrite produce dilatation of cerebral arterioles, and recent
evidence suggests that a key mechanism that mediates this response is
activation of potassium channels in vascular muscle. For example,
exogenously applied hydrogen peroxide produces vasodilation that can be
largely blocked using inhibitors of ATP-sensitive (KATP) or
calcium-activated potassium channels.3 4 Importantly,
potassium channels have also been implicated as mediators of dilatation
of cerebral arterioles in response to endogenously produced reactive
oxygen species.4
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
1.
Quayle JM, Nelson MT, Standen NB. ATP-sensitive and
inwardly rectifying potassium channels in smooth muscle. Physiol
Rev.. 1997;77:1165–1232.
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