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Departments of Pediatrics and Physiology, Biophysics Research Institute, and the Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Peroxynitrite (ONOO
)
is a contractile agonist of rat middle cerebral arteries. To determine
the mechanism responsible for this component of ONOO
bioactivity, the present study examined the effect of
ONOO on ionic current and channel activity in rat
cerebral arteries. Whole cell recordings of voltage-clamped cells were
made under conditions designed to optimize K+ current. The
effects of iberiotoxin, a selective inhibitor of large-conductance
Ca2+-activated K+ (BK) channels, and
ONOO (10-100 µM) were determined. At a
pipette potential of +50 mV, ONOO inhibited 39% of
iberiotoxin-sensitive current. ONOO was selective
for iberiotoxin-sensitive current, whereas decomposed ONOO had no effect. In excised, inside-out membrane
patches, channel activity was recorded using symmetrical K+
solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK
channels. Internal ONOO dose dependently inhibited
channel activity by decreasing open probability and mean open times.
The inhibitory effect of ONOO could be overcome by
reduced glutathione. Glutathione, added after ONOO,
restored whole cell current amplitude to control levels and reverted
single-channel gating to control behavior. The inhibitory effect of
ONOO on membrane K+ current is
consistent with its contractile effects in isolated cerebral arteries
and single myocytes. Taken together, our data suggest that
ONOO has the potential to alter cerebral vascular
tone by inhibiting BK channel activity.
free radical; glutathione
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE FORMATION OF PEROXYNITRITE
(ONOO) by the reaction of
O2· with NO· is well
recognized. However, the bioactivity of ONOO within
the vasculature is poorly understood. The oxidizing potency of
ONOO (21), in addition to its ability to cause
nitration of tyrosine and tryptophan residues (1, 15), suggests that
ONOO is a molecule that modulates cell signaling.
We previously showed that ONOO causes rat middle
cerebral arteries to contract (10). At 10 µM, ONOO
caused the internal diameter of isolated arteries to decrease by 15%.
Moreover, ONOO caused single, isolated smooth muscle
cells to contract by 30%. This degree of contraction was 60% of the
maximal response to KCl, suggesting that contraction elicited by
ONOO is quantitatively and physiologically significant.
Large-conductance Ca2+-activated K+ (BK)
channels are expressed in vascular smooth muscle cells, where they
modulate membrane potential and influence cell contraction (3). The
large conductance of BK channels means that the activity of relatively
few channels can exert a relatively large effect on membrane potential.
Activation of BK channels leads to membrane hyperpolarization,
inhibition of voltage-gated Ca2+ channels, and ultimately
cell relaxation. By contrast, inhibition of BK channels by a
contractile agonist such as ONOO would be expected
to cause cell contraction. BK channels are comprised of two subunits: a
pore-forming 
-subunit and a regulatory 
-subunit. The channel
protein possesses some 27 cysteine residues, raising the
possibility that thiol groups belonging to these residues are prime
targets for oxidant attack by ONOO (9).
Thus BK channel activity plays an important role in the contractile
state of vascular smooth muscle cells, and the BK channel protein has
the molecular constitution that might render it susceptible to
ONOO. Accordingly, we hypothesized that
ONOO affects vascular smooth muscle cell function
via alterations in the activity of the BK channel. In the present study
we used the patch-clamp technique to investigate whether
ONOO directly influences activity of BK channels in
cerebral artery vascular smooth muscle cells. The data presented here
indicate that ONOO inhibits whole cell
K+ currents, an effect that is limited to
iberiotoxin-sensitive current. Moreover, ONOO dose
dependently inhibits unitary K+ currents, consistent with
inhibition of BK channels.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Reagents. Papain and collagenase (type IV) were purchased from Worthington Biochemical (Freehold, NJ), dithiothreitol, soybean trypsin inhibitor (type II), BSA, and glutathione (GSH) from Sigma Chemical (St. Louis, MO), iberiotoxin from Calbiochem (La Jolla, CA), and dihydrorhodamine 123 from Molecular Probes (Eugene, OR). All buffer salts were of the highest available purity.
ONOO synthesis.
An ice-cold, flowing solution of 1 M NaNO2 was entrained
with an equal volume of acidified H2O2 (1.8 M
H2SO4-0.3 M H2O2), and
the resultant mixture was dripped into a solution of 1.4 M NaOH.
Granular MnO2 was added to catalyze the removal of
H2O2. At the termination of effervescence, the
solution was filtered (no. 2, Whatman, Kent, UK) to remove
MnO2. The solution was subjected to freeze fractionation,
then the uppermost layer containing the yellow ONOO
salt was removed. A concentration of this stock solution was determined
by absorbance spectrophotometry by use of the reported extinction
coefficient for ONOO (1,670 M1 · cm1)
and was typically 150-300 mM. At the time of each experiment, an
aliquot of the stock solution was diluted into a solution of 1 mM NaOH
to achieve a final working concentration of 2 mM
ONOO.
, an aliquot of 2 mM
stock solution was added to the 1-ml bath chamber. Dihydrorhodamine 123 was used to estimate the effective final concentration of
ONOO reaching the cell. Under conditions identical
to those used with cells present, the absorbance of dihydrorhodamine
123 at 500 nm was linearly related to ONOO
concentration up to 100 µM ONOO. By use of this
estimate, the effective concentration of ONOO was
15% of that which was added. The effect of decomposed
ONOO was tested after the solution of 100 µM
ONOO was left at room temperature for 
2 h. The
decay of ONOO was confirmed spectrophotometrically.
In some experiments the effect of ONOO was tested in
the presence of reduced GSH (5 mM).
Cell isolation. Male Wistar rats (250-300 g; Harlan Sprague Dawley, Indianapolis, IN) were anesthetized with pentobarbital sodium via an intraperitoneal injection. After craniotomy, the brain was removed, and circle of Willis cerebral arteries were carefully excised. Dissociation medium (DM) used for enzymatic cell dispersion contained (in mM) 145 NaCl, 4.0 KCl, 0.05 CaCl2, 1.0 MgCl2, 10.0 HEPES, and 10 glucose (pH 7.4 with NaOH). Cerebral arteries were left for 15 min at room temperature in the DM, which additionally contained papain (1.5 mg/ml), dithiothreitol (1.0 mg/ml), and BSA (1.0 mg/ml). Then the arteries were incubated for 17 min at 37°C. Arteries were transferred to DM solution containing collagenase (1.5 mg/ml), trypsin inhibitor (1.0 mg/ml), and BSA (1.0 mg/ml) and incubated for 20 min at 37°C. Smooth muscle cells were obtained by a gentle trituration with a Pasteur pipette. Cells were stored on ice and used within 3 h. A total of 20 animals were used.
Exclusive use of freshly isolated cells precluded the real-time confirmation of smooth muscle identity by use of techniques that require cell fixation. Electrophysiological measurements were carried out only on cells that exhibited morphological features characteristic of vascular smooth muscle cells when observed under phase-contrast microscopy. In addition, parallel observations were made by taking aliquots of cells and confirming a contractile response to KCl. In a parallel series of experiments, smooth muscle cell identity was further confirmed by demonstration of positive staining with mouse IgG1 anti-calponin monoclonal antibody (Sigma Chemical).Electrophysiology.
Membrane currents were recorded using whole cell and excised inside-out
configurations (13). Data acquisition and command potentials were
carried out using an Axopatch 200B amplifier (Axon Instruments, Foster
City, CA) and a Digidata 1200A analog-to-digital converter (Axon
Instruments). Currents were recorded at 10-kHz bandwidth with a
low-pass Bessel filter at 1 kHz and stored on a computer for later
analysis. Data were analyzed using pCLAMP 6.0 software (Axon
Instruments). Patch pipettes were pulled from borosilicate glass (no.
7052, Garner Glass, Claremont, CA) by use of a vertical puller
(Narashige, Tokyo, Japan) and gently heat polished using a microforge
(model MF-83, Narashige). Pipette resistance was 5-10 M
when
filled with intracellular recording solution. The indifferent electrode
was an Ag-AgCl plug connected to a bath chamber via a 140 mM agar
bridge. All experiments were conducted at room temperature. The ionic
composition of bath and pipette solutions, along with voltage-step
protocols, was designed to optimize the measurement of K+ currents.
Measurement of whole cell currents.
For whole cell configuration, extracellular (bath) physiological salt
solution (PSS) contained (mM) 145 NaCl, 5.4 KCl, 1.8 CaCl2,
1 MgCl2, 5 HEPES, and 10 glucose (pH 7.4 adjusted with NaOH). The pipette (high-K+) solution contained (mM) 145 potassium aspartate, 5 NaCl, 1 CaCl2, 2.2 EGTA, 10 HEPES,
and 7.5 glucose (pH 7.4 adjusted with KOH). A pipette potential of
70 mV was used routinely, and currents were recorded in response
to successive voltage pulses of 200-ms duration, increasing in 10-mV
increments from 100 to +50 mV. In whole cell recording, no
correction for capacitative currents was made.
Data analysis and statistics.
Analysis of whole cell and single-channel data was carried out using
pCLAMP software (Axon Instruments). Whole cell data are presented as
means ± SE, where n is the number of experiments. Differences within and between groups were determined using ANOVA for
repeated measurements followed by Duncan's multiple range test. A
Student's t-test was used to evaluate statistical significance of difference between two paired observations. P < 0.05 was considered statistically significant. The most linear portion
of the current-voltage relationship (0 to +40 mV) was used to calculate
mean outward conductance. Unitary currents were characterized using
open-state probability (Po), defined as
(To / Ti)/N,
where N is the number of functional channels in the patch,
To is the open time for the level under
consideration, and Ti is the time interval over
which Po is measured. The mean open-time constant
(
o) and the mean closed-time constant (c)
were calculated from curves fitted to open- and closed-time histograms
by pCLAMP software.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Whole cell currents. Whole cell currents were recorded in response to voltage steps, with PSS in the bath and high-K+ solution in the recording pipette. Under control conditions, cells displayed predominantly outward current that reached an amplitude of 270 ± 4 pA (n = 8) at +50 mV. Mean outward conductance was 3.0 ± 0.3 nS between 0 and +40 mV. Peak current was taken as the current recorded at +50 mV. Addition of iberiotoxin (100 nM), a selective inhibitor of BK channels, decreased peak current to 220 ± 3 pA and mean outward conductance to 2.5 ± 0.1 nS. These data suggest that, in rat cerebral artery smooth muscle cells, BK channels contribute significantly to whole cell current.
To test the effect of ONOO on whole cell current,
two protocols were performed. In the first protocol, whole cell seals
were made on control cells, then 100 µM ONOO was
added to the bath. Under this condition, ONOO
decreased peak current from 250 ± 3 to 190 ± 4 pA
(n = 5, P < 0.05), a change of 24% (Fig.
1). Subsequent addition of 100 nM iberiotoxin decreased peak current further, to 150 ± 5 pA (P < 0.05). Subtraction analysis revealed that ONOO
inhibited iberiotoxin-sensitive current by 39%.
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and iberiotoxin was reversed.
ONOO (100 µM) had no inhibitory effect when added
to iberiotoxin-treated cells (P > 0.05), suggesting
that the effect of ONOO on whole cell current is
selective for iberiotoxin-sensitive current (Fig.
2). On the basis of the selectivity of
iberiotoxin for BK channels, these data further suggest that
ONOO is selective for this channel type.
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was further characterized by
testing the effect of solutions of decomposed ONOO
on whole cell current. Decomposed ONOO had no effect
on whole cell current (P > 0.05; Fig.
3A). NaOH (1 mM), the vehicle for
ONOO, likewise had no effect (P > 0.05; Fig. 3B).
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Characterization of BK unitary currents.
To further test the contribution of BK channels in rat cerebral artery
myocytes, currents were recorded using excised inside-out membrane
patches. BK channels are characteristically activated by increased
[Ca2+]i or by membrane
depolarization (19). The pipette contained high-K+
solution, and the bath contained high-K+ solution with 0.2 µM free Ca2+. Under this condition, channel activity was
minimal (Fig. 4A). Changing the
bath solution to high-K+ solution containing 2.6 µM free
Ca2+ resulted in increased channel activity (Fig.
4B), with Po at a pipette potential of
40 mV increasing from 0.002 ± 0.002 to 0.89 ± 0.03 (n = 4). Open-time histograms were constructed from inside-out
recordings binned every 600 µs. Values for mean open times
(o1 and o2) were calculated as time
constants of biexponential fitting. With
[Ca2+]i of 2.6 µM,
o1 and o2 were 0.9 ± 0.2 and 7.3 ± 0.4 ms, respectively. This enhanced activity of the channel in response
to increases in [Ca2+]i and to
depolarization is similar to those characteristics typical of
Ca2+-activated K+ channels.
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ONOO inhibits BK channel activity.
The effect of ONOO on BK single-channel activity was
investigated in excised inside-out membrane patches. Under control
conditions at 20 mV pipette potential, the channel remained
mostly in the open state (Po = 0.89 ± 0.03; Fig.
5). At 10 µM, ONOO had
little effect on channel activity. In this regard,
Po was 0.81 ± 0.05 at 60 s after addition of
ONOO. Analysis of channel activity revealed
o1 and o2 to be 2.1 ± 0.5 and 7.9 ± 0.5 ms (n = 3), values not different from control.
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resulted in essentially
complete inhibition of channel activity. In 7 of 11 excised patches
exposed to 100 µM ONOO, channel activity was
rarely encountered after addition of the oxidant, such that
Po was 0.003 ± 0.002 (n = 7). Analysis of
the channel current recording shown in Fig. 5 revealed a mean
closed time of 175.1 ms. This value is in contrast to the mean closed times, c1 and c2, of control patches that
equaled 2.1 ± 0.5 and 5.0 ± 0.4 ms (n = 4). In the
remaining four patches, ONOO had no effect, a result
that likely reflects ineffective delivery of ONOO to
the membrane.
A submaximal concentration of 50 µM ONOO resulted
in the channel displaying two gating modalities. In the open modality,
the channel was predominantly in the open state and exhibited only brief sojourns to the closed state. In the open modality, biexponential fitting of the Po histogram revealed
o1 and o2 equal to 1.7 ± 0.5 and 7.5 ± 0.5 ms, respectively (n = 3). Values of c1
and c2 were 1.2 ± 0.5 and 3.1 ± 0.5 ms (n = 3). Periods of channel activity in the open modality were interrupted
by intervals of almost complete inactivity (closed modality) that
lasted several milliseconds (Fig.
6).
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(Fig.
7) demonstrates that the effect of
ONOO is time and dose dependent. Within several
seconds of addition of 100 µM ONOO,
Po had fallen to <0.1.
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Effect of ONOO is reversible and thiol dependent.
Reversibility of ONOO was determined using cells
exposed to the oxidant during recording of whole cell current. Addition
of 100 µM ONOO decreased peak current amplitude
(at +50 mV) from 175 ± 1 to 114 ± 2 pA (n = 3, P < 0.001) and decreased conductance from 2.15 ± 0.3 to 1.2 ± 0.2 nS. Exchange of the bath solution for a simple PSS had
no effect on the ONOO-decreased peak current
amplitude. Exchange of the bath (PSS) to one that contained the
physiological antioxidant GSH (5 mM) restored current amplitude to 179 ± 6 pA and conductance to 2.44 ± 0.2 nS (Fig.
8). To determine whether GSH is protective,
100 µM ONOO was added in the presence of 5 mM GSH.
Under this condition, ONOO failed to exhibit any
effect on whole cell current (Fig. 9).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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It has been appreciated for many years that superoxide
(O2·) "scavenges" NO·.
In this regard, the formation of ONOO from NO·
and O2· is associated with
decreased bioavailability of NO·. ONOO is
a highly reactive chemical species (4, 15, 21). The formation of
ONOO in vivo has not been quantitatively determined;
its bioactivity is best estimated by the flux of molecules through
reaction pathways, rather than by steady-state concentrations. Under in
vitro conditions, addition of a particular ONOO
concentration does not imply that the oxidant exists in vivo at that
concentration in a steady state.
We and others have reported that ONOO causes
arterial vessels to constrict (6, 10). In Wistar rat cerebral arteries, ONOO causes an ~15% decrease in internal vessel
diameter (10). In that study, Elliott et al. (10) found the effect of
ONOO to be dose dependent between 5 and 100 µM.
Moreover, ONOO caused isolated smooth muscle cells
to shorten by 30%, indicating that at least part of the contractile
action of ONOO involves a direct effect on the
myocyte. The degree of cell shortening triggered by
ONOO represented 60% of maximum contraction,
suggesting that the oxidant's contractile activity might be highly
significant in physiological terms. Our present data provide a
mechanism for the direct actions of ONOO in vascular
smooth muscle cells; namely, ONOO reversibly
inhibits the BK channel activity. Our present and previous results (10)
are consistent with a vasoconstrictor action of this oxidant.
ONOO was observed to produce glibenclamide-sensitive
vasodilation of pial arterioles in vivo (25a). Although we cannot
readily account for these differences, because ONOO
can produce myriad effects on a variety of proteins and cells within
the vascular wall, it is difficult to unequivocally draw conclusions
about its cellular actions on the basis of in vivo investigations.
Nonetheless, we can state with conviction that ONOO
affects the BK channel activity in cerebrovascular smooth muscle cells.
In the vascular smooth muscle cell, sarcolemmal permeability to K+ is a major determinant of membrane potential, [Ca2+]i, and vascular tone. Inhibition of K+ channel activity depolarizes the vascular smooth muscle cell, thereby activating voltage-gated Ca2+ channels and causing contraction. The BK channel is one of the most influential K+ channels in vascular smooth muscle cells. BK channels are selectively blocked by external iberiotoxin (11, 16) and activated by depolarization or by increased [Ca2+]i. In the present study, whole cell currents were activated by depolarization and inhibited by the extracellular application of iberiotoxin, suggesting that BK channels contribute to the overall current measured in whole cell configuration. Indeed, in excised inside-out membrane patches, unitary currents were sensitive to [Ca2+]i and depolarization, characteristics typical of BK channels.
We hypothesized that the contractile activity of
ONOO observed in cerebral arteries and their smooth
muscle cells might be due to inhibition of BK channel activity. BK
channels have been previously identified and characterized in cerebral
arteries obtained from the rat and cat (12, 24). Moreover, the
regulatory -subunit of the human BK channel gene product
hSlo contains six cysteine residues (22). We believe that
ONOO oxidizes these residues to affect the function
of the channel. Gating characteristics of hSlo are sensitive to
thiol reduction and oxidation (8). Specifically, dithiothreitol
increases the channel's voltage sensitivity, accelerates the kinetics
of channel activation, and increases single-channel
Po, whereas H2O2 (300 µM)
decreases voltage sensitivity and decreases Po. In
other cell types, such as equine tracheal smooth muscle, the
NPo of Ca2+-activated K+
channels is decreased by as much as 90% by the thiol oxidizing agent
diamide and increased ~10-fold by GSH (25). Together, these findings
suggest that BK channels might be the target for ONOO, itself a potent oxidant of reduced thiols.
ONOO readily oxidizes reduced thiol groups, such
that the rate constant for the ONOO oxidation of
albumin's thiol moiety is three orders of magnitude greater than that
for H2O2 under similar conditions (21). In a
cell system of bovine pulmonary artery endothelial cells,
cytokine-triggered oxidation of GSH is mediated by
ONOO (20). In the present work we hypothesized that
GSH, a soluble thiol that is abundantly and endogenously present in
vascular tissue, might modify the biological effects of
ONOO. In the presence of GSH in the bath,
ONOO had no effect on whole cell current. The most
reasonable explanation for this finding is that GSH prevents the
oxidant from reaching the cell membrane. The ability of GSH in
protecting membrane current from inhibition by ONOO
is likely to account for GSH protection from the contractile effects of
ONOO in vascular smooth muscle cells (10).
Under whole cell conditions in the present study,
ONOO inhibited outward current at +50 mV by 24%. We
aimed to determine the relationship between that component of whole
cell current due to BK channel activity and the inhibitory effect of
ONOO. To this end, a strategy was employed whereby
ONOO and iberiotoxin were added sequentially during
whole cell recording. Subtraction analysis revealed that
ONOO inhibited 39% of iberiotoxin-sensitive
current. Conversely, addition of iberiotoxin followed by
ONOO indicated that the oxidant's effect was
limited primarily to iberiotoxin-sensitive current.
In inside-out membrane patches, ONOO dose
dependently inhibited BK channel activity. At 10 µM,
ONOO had little effect on channel gating, as
measured by NPo. This result is not surprising,
because evidence from the dihydrorhodamine assay suggests that the
effective concentration reaching the cell is 
15% of the added dose.
Spontaneous decomposition of ONOO at physiological
pH results in a half-life of ~1 s (2), and at low doses the method of
addition to the bath might limit delivery of the oxidant to the
membrane patch being studied. Addition of 50 or 100 µM
ONOO resulted in a partial inhibition or elimination
of channel activity, respectively.
For ONOO to be a physiological signaling molecule,
rather than solely a toxic oxidant that causes cell injury, its
bioactivity must be readily reversible. Indeed, inhibition of whole
cell and unitary currents by ONOO is reversed by the
addition of GSH. In whole cell configuration, GSH restores current to
control levels. Likewise, in excised membrane patches, addition of GSH
returns channel gating to control behavior. Although we did not
specifically titrate various doses of ONOO against a
number of different GSH concentrations, these data suggest that BK
channel activity in the cerebral circulation in vivo is modulated by
the chemical balance between ONOO and
GSH. It is known that ONOO is generated during the
reperfusion phase of ischemia-reperfusion injury (14, 23). In
this regard, the contractile agonist property of
ONOO might contribute to the prolonged phase of
vasoconstriction that follows initial reperfusion. The present
data, which document that GSH is protective and restorative,
provoke the hypothesis that exogenously infused GSH might be clinically
useful in the treatment of ischemic stroke.
The data presented here indicate that activity of the BK channel can be
inhibited by external ONOO. Moreover, the ability of
ONOO to inhibit unitary currents in excised
inside-out membrane patches suggests that the oxidant can act
additionally at the internal aspect of the membrane. Because the
production of ONOO occurs inside or outside of
cells, our results may, in part, explain the mechanism of altered
vascular function under conditions of oxidant stress, e.g., diabetes
and ischemia-reperfusion injury.
In conclusion, we report that ONOO reversibly
inhibits BK channel activity in rat cerebral artery smooth muscle
cells. The inhibition occurs via the actions of ONOO
on cysteine residues. These effects of ONOO provide
a mechanism by which this oxidant causes vasoconstriction of cerebral
arteries (10).
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ACKNOWLEDGEMENTS |
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We thank David J. Lacey for valuable technical assistance.
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FOOTNOTES |
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This work was supported by American Heart Association National Office Grant 96013570, National Institute of Neurological Diseases and Stroke Grant NS-38133, National Heart, Lung, and Blood Institute Grant HL-32788, and funding from the Children's Hospital of Wisconsin Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. K. Brzezinska, Cardiovascular Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: brzezins{at}mcw.edu).
Received 30 June 1999; accepted in final form 7 December 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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