Published online before print July 5, 2001, doi: 10.1161/hh1401.093294
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© 2001 American Heart Association, Inc.
Article |
High Glucose Impairs Voltage-Gated K+ Channel Current in Rat Small Coronary Arteries
From the Departments of Internal Medicine (Y.L., K.T., D.D.G.) and Pharmacology and Toxicology (N.J.R.), Cardiovascular Center, Medical College of Wisconsin, and Zablocki VA Medical Center (Y.L., K.T., D.D.G.), Milwaukee, Wis.
Correspondence to Yanping Liu, MD, PhD, Assistant Professor, Cardiovascular Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail ypliu{at}mcw.edu
Abstract
Abstract—Hyperglycemia is associated with impaired endothelium-dependent dilation that is due to quenching of NO by superoxide (O2 · -). In small coronary arteries (CAs), dilation depends more on smooth muscle hyperpolarization, such as that mediated by voltage-gated K+ (Kv) channels. We determined whether high glucose enhances O2·- production and reduces microvascular Kv channel current and functional responses. CAs from Sprague-Dawley rats were incubated 24 hours in medium containing either normal glucose (NG, 5.5 mmol/L D-glucose), high glucose (HG, 23 mmol/L D-glucose), or L-glucose (LG, 5.5 mmol/L D-glucose and 17 mmol/L L-glucose). O2·- production was increased in HG arteries. Whole-cell patch clamping showed a reduction of 4-aminopyridine (4-AP)–sensitive current (Kv current) from smooth muscle cells of HG CAs versus NG CAs or versus LG CAs (peak density was 9.95±5.3 pA/pF for HG versus 27.8±6.8 pA/pF for NG and 28.5±5.2 pA/pF for LG; P<0.05). O2·- generation (xanthine+xanthine oxidase) decreased K+ current density, with no further reduction by 4-AP. Partial restoration was observed with superoxide dismutase and catalase. Constriction to 3 mmol/L 4-AP was reduced in vessels exposed to HG (13±5%, P<0.05) versus NG (30±7%) or LG (34±4%). Responses to KCl and nifedipine were not different among groups. Superoxide dismutase and catalase increased contraction to 4-AP in HG CAs. This is the first direct evidence that exposure of CAs to HG impairs Kv channel activity. We speculate that this O2·--induced impairment may reduce vasodilator responsiveness in the coronary circulation of subjects with coronary disease or its risk factors.
Key Words: K+ channels • superoxide • coronary circulation • vascular smooth muscle
Cardiovascular disease is the leading cause of death in the Western world, and diabetes mellitus (DM) is a major factor underlying its development. Many detrimental effects of diabetes are linked to elevations in serum glucose that is punctuated by elevations in superoxide (O2·-). Hyperglycemia-induced vascular oxidative stress is mediated by several mechanisms.1 Increased flux of glucose through the polyol pathway reduces antioxidant enzyme activity by depleting NADPH.2 Synthesis of endoperoxides in hyperglycemia leads to free radical formation.3 4 Acute increases in glucose alter Ca2+ homeostasis in vascular smooth muscle cells (VSMCs) through the production of excess O2·-.5 Finally, nonenzymatic glycation products of chronic hyperglycemia may provide oxidant stress, and glucose itself can auto-oxidize to form reactive oxygen species (ROS).6 7
Tesfamariam and Cohen8 have shown that incubating rabbit aortas in high glucose (44 mmol/L) impairs NO-mediated relaxation. Similar impairment in guinea pig aortas exposed to high glucose was mediated by excess O2·-.9 Thus, increased oxidative stress may be a common pathway of vasodilator dysfunction in diabetes.10 11
Although the role of ROS in NO-mediated endothelium-dependent dilation has been studied, little is known about the effect of hyperglycemia on hyperpolarization-mediated dilation. This form of dilation typically involves the opening of K+ channels in VSMC membranes and may play a critical compensatory vasodilator role in disease states in which NO-mediated dilation is impaired.12 13
VSMC membranes express several K+ channel gene families; however, voltage-gated K+ (Kv) channels and high-conductance Ca2+-activated K+ (BKCa) channels normally contribute the majority of K+ current.14 In preliminary studies, we demonstrated that Kv channels contribute to whole-cell K+ current in VSMCs from rat small coronary arteries (CAs).15 The purpose of the present study was to determine the effect of glucose-induced ROS generation on the function of Kv channels in rat small CAs by using a combination of histofluorescence, patch-clamp, and functional vasomotor methods. We examined the hypothesis that elevated glucose enhances the local production of O2·- and reduces Kv channel activity in rat coronary VSMCs.
Materials and Methods
Preparation of CAs
Seven-week-old male Sprague-Dawley rats (Harlan,
Madison, Wis) were anesthetized with sodium
pentobarbital (60 mg/kg IP). CAs (inner diameter 150 to 200 µm) were
dissected from the ventricle and incubated in Dulbecco’s Modified
Eagle’s Medium (DMEM) with 10% fetal bovine serum, 100 U/mL
penicillin G, and 100 µg/mL streptomycin for 24 hours at 37°C. DMEM
was supplemented with either 5.5 mmol/L
D-glucose (normal glucose,
NG), 23 mmol/L
D-glucose (high glucose,
HG), or 5.5 mmol/L
D-glucose plus 17.5
mmol/L L-glucose (LG).
L-Glucose, which is not
metabolized, was used as an osmotic control. All media were filtered
(0.2-µm filter) before use. After incubation, vessels were prepared
for histofluorescence, patch-clamp, or videomicroscopic
studies. Some CAs were used freshly after
dissection.
Fluorescent Detection of
Superoxide
The cell-permeable dye hydroethidine (HE,
Molecular Probes) was used to evaluate the
production of
O2·-. When
exposed to O2·-,
HE is oxidized to ethidium bromide and trapped within the
nucleus.16 Ethidium bromide
fluoresces when excited by light at a 488-nm wavelength with an
emission spectrum of 620 nm. CAs incubated in one of three solutions of
glucose described above were exposed to HE (5 µmol/L) for 10 minutes,
washed, and examined under confocal microscopy equipped with a
krypton/argon laser. Fluorescence was detected with a 585-nm
long-pass filter. A freshly isolated artery was used as a control to
adjust for laser settings for use in all subsequent vessels. The
fluorescence intensity/vascular area ratio of the central
portion of the vessel was normalized to that obtained from the control
vessel by using NIH image software. The ratio of the
fluorescence (experimental vessel/control vessel) was compared
among CAs incubated in each glucose solution.
Detection of Superoxide in Culture
Media
Detection of
O2·-
production in vessel culture media was determined by monitoring
increases in ferricytochrome c
absorbance at 550 nm.17
Ferricytochrome c (final
concentration 50 µmol/L) was added to the cuvette filled with either
NG or HG culture media, and changes in absorbance were followed each
minute for 20 minutes. Rates of
O2·- anion
production were calculated by the molar extinction coefficient
of reduced ferricytochrome c
[
=21 000
cm-1x(mol/L)-1]
and the portion that was inhibited by superoxide dismutase (SOD, 400
U/mL) and were expressed as micromoles of
O2·- anion per
liter of buffer per minute.
Patch-Clamp Recording of
K+ Currents
Enzymatic isolation of single VSMCs was performed
according to published
methods.18 Patch-clamp
recordings were obtained by using standard pulse protocols and
instrumentation previously described for whole-cell and voltage-gated
K+ current
measurements.18 Briefly,
families of K+ currents were generated by
stepwise 10-mV depolarizing pulses (400-ms duration, 5-second
intervals) from a holding potential of -60 mV in cells dialyzed with
10 nmol/L ionized Ca2+. Seal resistance was
2 to 10 G
. Peak current elicited at a single membrane potential was
defined as the average of 500 sample points encompassing the maximal
current point. In a single cell, Kv currents were defined as the
difference between outward current recorded in drug-free bath
solution and after superfusion with 3 mmol/L
4-aminopyridine (4-AP), a Kv channel
blocker.19 Trials were
performed in triplicate and averaged to estimate peak current
amplitudes (picoamperes per picofarad) to normalize for cellular
membrane area.20 For each
cell, 10-mV hyperpolarizing steps were averaged to provide capacitance
and leak compensation values. Whole-cell tail currents were elicited in
symmetrical 145 mmol/L K+ by
depolarizing cells from a constant holding potential of -60 in 10-mV
increments to 60 mV, followed by an immediate repolarizing step back to
-60 mV to generate families of tail
currents.19
Videomicroscopy
Segments of small CAs were placed into an organ
chamber filled with physiological salt solution
(PSS),13 cannulated with
glass micropipettes, and secured with 10-0 nylon suture. Each pipette
was attached to a pressure reservoir. The PSS in the organ chambers was
warmed to 37°C, bubbled with 21% O2, 5%
CO2, and 74% N2, and
continually circulated with a rotary pump. Vessels were allowed to
equilibrate for 60 minutes at an intraluminal pressure of 60
mm Hg. Internal diameters were measured with a calibrated manual
videomicrometer with a resolution of 2 µm attached to an
inverted microscope. Arteries that developed 15% to 25% spontaneous
tone (percent reduction of initial internal diameter) in PSS were used.
At the end of each experiment, vessels were maximally dilated with
sodium nitroprusside (10-4 mol/L). In some
vessels, endothelium was denuded with
air.21 All chemicals were
obtained from Sigma Chemical
Co.
Statistical Analysis
All data are expressed as mean±SEM. Percent
constriction was defined as the percent reduction from control internal
diameter in response to constrictor agents. Percent dilation was
calculated as the percent change from control internal diameter (in the
presence of established myogenic tone) to maximal diameter measured
after sodium nitroprusside. Data from vessels incubated in NG, HG, or
LG and studied by either patch clamp or videomicroscopy were compared
by using a 1-way ANOVA with repeated measures for dose and condition
(glucose exposure). Differences between individual means were
determined by the Newman-Keuls test. The relative fluorescence
(ethidium bromide technique) of arteries exposed to different levels of
glucose was compared by using a 2-way ANOVA. All differences were
judged to be significant at
P<0.05.
Results
Superoxide Production in CAs Incubated
in HG
Figure 1A
compares
O2·- detected by
HE between a freshly isolated (control) artery and arteries incubated
in either NG, LG, or HG for 24 hours. Similar fluorescence
intensities were observed in control, NG, and LG arteries. CAs
incubated in HG showed greater fluorescence. Average
fluorescence ratios, normalized to the control artery, are
summarized in
Figure 1B. Only incubation in HG stimulated the
production of
O2·- in rat small
CAs. O2·-
production in the incubation media alone was examined by using
the ferricytochrome c method
before and after a 24-hour incubation to mimic the conditions of the
experiment. O2·-
production before (0.88±0.02 and 0.5±0.25 µmol/L for 20
minutes in NG and HG media, respectively) and after (0±0.18 and
0±0.16 µmol/L for 20 minutes in NG and HG media, respectively)
incubation was similar, indicating no
O2·- generation
in the incubation media. As a positive control,
O2·- generation
was 16±1.5 µmol/L for 20 minutes with the use of xanthine
(XA)+xanthine oxidase (XO). Thus, the major source of radical
production is likely from the interaction of glucose with
coronary vessels.
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Antioxidant Treatment Reduces Superoxide
Production in Arteries Exposed to HG
The effect of antioxidants on
O2·- generation
in CAs exposed to HG was also examined by the histofluorescence
method. Two CAs were incubated in parallel with HG for 24 hours. After
incubation, one artery was treated with SOD and catalase (CAT) for 15
minutes, and the amount of
O2·- generated
was compared between arteries with and without antioxidant exposure.
SOD and CAT greatly reduced the fluorescence intensity of CAs
exposed to HG
(Figure 2A) with average ratios of intensity of 5.2±1.6 for
arteries exposed to HG and 0.5±0.1 for arteries exposed to HG with
SOD+CAT (P<0.05, n=6)
(Figure 2B). Fluorescence intensities of control and
SOD+CAT–treated vessels were not different
(P>0.05).
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Comparison of Kv Current Densities
Figure 3A shows families of voltage-gated
K+ currents generated by incremental 10-mV
depolarizing steps from -60 to 60 mV in VSMCs isolated from rat CAs
and incubated for 24 hours in DMEM containing NG, LG, or HG.
Figure 3A shows that peak K+
current amplitude was similar between arteries exposed to NG or LG. The
currents were reduced by 3 mmol/L 4-AP, identifying Kv channels as
the primary conducting pathways. Whole-cell Kv current was suppressed
in VSMCs from arteries exposed to HG, and 3 mmol/L 4-AP had less
effect on total K+ current.
Figure 3B compares K+ current
densities between VSMCs from arteries exposed to NG, LG, and HG (n=10
each). Densities were significantly reduced in cells from arteries
incubated in HG (28±6 pA/pF) compared with NG (41±7 pA/pF) and LG
(42±5 pA/pF). In
Figure 3C, it is seen that Kv current was reduced by 63%
and 64% in VSMCs from arteries exposed to HG compared with those
exposed to NG or LG, respectively. Glibenclamide (1 µmol/L), an
ATP-sensitive K+ channel blocker, had little
effect on total K+ currents (data not
shown). Thus, 24 hours of exposure to HG suppresses Kv current in
coronary VSMCs, and this impairment is not secondary to an
osmotic effect.
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Voltage dependence of K+ currents
was determined by measuring the peak amplitude of tail currents
generated at -60 mV as a function of activating prepulses (10-mV
steps) to voltages as positive as 60 mV. Values obtained from four
cells were fit by a Boltzmann function,
Iact=Imax/[1+exp(Vm-V0.5)/k],
where
Imax is
the maximal tail current amplitude at 60 prepulse,
Iact is
the tail current observed at a given prepulse voltage
(Vm),
V0.5 is the voltage for half of
Imax,
and k is the steepness factor
(slope) indicative of voltage sensitivity.
Figure 3D
shows that HG reduced
Imax but
had little effect on the voltage dependence of
K+ channels.
V0.5 and
k values were -3.3 mV and 18,
respectively, in VSMCs exposed to NG and -8 mV and 16, respectively,
in VSMCs exposed to HG.
Effect of Exogenous Generation of Superoxide on
Kv Current
The direct effect of
O2·- on Kv
currents was examined with exogenous
O2·- generated by
the reaction of 0.1 mmol/L XA and 10 mU/mL XO.
Figure 4A shows that the addition of XA had no effect
on whole-cell current, whereas XA+XO suppressed
K+ currents. The further addition of 4-AP
had little effect, which would indicate that Kv current was already
blocked by XA+XO. Notably, the more rapid activation of
K+ current in
Figure 4A compared with that in the earlier traces shown in
Figure 3A was observed in some cells and may reflect the
variable activation kinetics of different Kv channel
subtypes.22 Current-voltage
relationships in
Figure 4B show that K+ current
densities were 32±7 and 21±4 pA/pF in cells treated with XA versus
XA+XO, respectively. The subsequent application of 3 mmol/L 4-AP
in the presence of XA+XO produced no further reduction,
consistent with the idea that
O2·- decreases Kv
current in coronary VSMCs. Similar to the results observed in
cells from HG arteries, the results shown in
Figure 4C demonstrate that
O2·- decreased
Imax but
did not change the voltage dependence of K+
channel activation. V0.5 and
k values were -10 mV and 17,
respectively, in cells treated with XA and -9 mV and 18,
respectively, in cells treated with XA+XO. XA+XO had no effect on the
electrode offset evaluated by comparing electrode voltage (in cell-free
conditions) between baths filled with drug-free solution, XA, or
XA+XO.
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Effect of Antioxidants on Kv Current
To determine whether the reduced Kv current after
exposure to HG is associated with the generation of
O2·-,
K+ current density was compared among cells
from arteries exposed to NG, LG, and HG before and after the
application of SOD (150 U/mL) and CAT (500 U/mL). SOD and CAT partially
restored peak K+ current density of cells
from arteries incubated in HG
(Figure 5C) but had little effect on
K+ currents in cells from arteries exposed
to NG or LG
(Figures 5A and 5B, respectively). Thus,
O2·- may
contribute to the impaired Kv channel activity in small CAs exposed to
HG.
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Contractile Response to 4-AP
Resting diameters of small CAs incubated in NG, LG, and
HG were not different (147±14, 159±19, and 140±16 µm,
respectively; n=8 for each group).
Figure 6A shows diameter changes in response to four
increasing concentrations of 4-AP. A similar concentration-dependent
contraction to 4-AP was seen in CAs incubated in NG or LG (maximum
constriction 30±7% and 34±4%, respectively). In contrast, the
maximal constriction to 4-AP in arteries exposed to HG was lower
(13±5%). Passive diameters induced by sodium nitroprusside were
175±14, 182±16, and 164±13 µm
(P>0.05) in CAs exposed to NG,
LG, and HG, respectively. Maximal constriction to 4-AP was similar in
vessels with and without endothelium (for NG, 28±7%
before and 27±13% after denudation; for HG, 12±4% before and 9±3%
after denudation; n=3 for denudation groups). Thus, elevations in
glucose impair Kv channel function in coronary
VSMCs.
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Figure 6B shows similar KCl-induced constriction (20 to
40 mmol/L) between NG and HG arteries.
Figure 6C indicates that arteries incubated in NG or HG
showed similar dilator responses to the L-type
Ca2+ channel antagonist
nifedipine (10, 100, and 1000 nmol/L). These results
indicate that 24-hour incubation in HG did not affect coronary
vascular reactivity nonspecifically but appeared to selectively impair
vasoactive responses dependent on functional Kv
channels.
Effect of Antioxidants on Contractile
Responses to 4-AP
Similar to
electrophysiological observations, SOD and
CAT restored constrictions of HG arteries to increasing concentrations
of 4-AP but had little effect on arteries incubated in NG or LG
(Figures 7A through 7C), further indicating that ROS
contribute to the impaired function of Kv channels exposed to
HG.
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Discussion
The present study demonstrates for the first time that short-term exposure of small CAs to HG suppresses Kv current in VSMCs. This impairment is manifested by a direct reduction of Kv current in coronary VSMCs and by reduced vasomotor responses to Kv channel activity. The novel findings of the present study are that exposure of small CAs to HG for 24 hours (1) increases O2·- production, (2) decreases 4-AP–sensitive K+ current in dissociated VSMCs, and (3) reduces 4-AP–induced contraction. Furthermore, the reduced Kv current and contractile response to 4-AP are linked to O2·- production, because treatment with radical scavengers partially restores these processes in arterial preparations exposed to HG. These findings may relate to coronary vasomotor regulation in diabetes, because NO-mediated dilation is impaired in DM and because compensatory dilator responses, including hyperpolarization factors, may rely on Kv channels.
Role of Kv Channels in the Circulation
Kv channels are highly expressed in
VSMCs23 24 and
contribute to membrane potential, particularly at lower levels of
intracellular Ca2+, at which the
BKCa channel is less
active.14 In the present
study, inhibition of Kv channels elicited a substantial
vasoconstriction. Because Kv channels are activated by
depolarization, they may serve to limit membrane depolarization during
vasoconstriction. Kv channels also mediate pharmacological
vasodilation. Forskolin, an activator of adenylate
cyclase, increases 4-AP–sensitive K+
currents in rabbit CAs.25
The ß-adrenoceptor agonist isoproterenol enhances 4-AP–sensitive
currents in rabbit portal
veins.26 In the
coronary circulation, activation of histamine
H1-receptors inhibits 4-AP–sensitive currents,
consistent with a role for Kv channels in coronary
vasospasm.27 The vasomotor
effects of angiotensin
II28 and
pH29 are also in part
mediated by Kv currents.
The predominant K+ current in cells from arteries either freshly isolated (no incubation) or incubated with NG or LG was sensitive to 4-AP. 4-AP (3 mmol/L) caused a 37% and 38% reduction of resting diameter of NG and LG arteries, respectively. Thus, Kv channels contribute to the resting membrane potential of rat small CAs and may be involved in coronary blood flow regulation.
Because the patch-clamp results show reduced Kv current in cells from HG arteries and because Kv is involved in resting tone, a reduction in resting diameter would be expected after incubation in HG media. However, resting diameters of arteries exposed to NG, LG, or HG were similar. This could be due to a compensatory increase in the contribution of other K+ channels, such as BKCa. Such compensation has been demonstrated for loss of NO during hypercholesterolemia.12
HG Increases Oxidative Stress
Elevated glucose induces ROS generation by several
mechanisms. Glucose auto-oxidation forms
O2·-, hydroxyl
radicals, and hydrogen
peroxide.7 Increased flux
through glycolytic and polyol pathways results in an excess
production of sorbitol. This is coupled with the NADPH-mediated
generation of ROS. Acute elevations in glucose also depress natural
antioxidant defenses. Incubation of purified bovine CuZn SOD with HG
(10 to 100 mmol/L) reduces enzyme activity by
60%.30 In the present
study, O2·-
(fluorescence microscopy) was increased in small CAs incubated
with high glucose. The increased fluorescence was reduced by
SOD, confirming the involvement of
O2·-.
The source of O2·- generation in the CA model is not known. In conduit arteries, the endothelium likely contributes,8 whereas other studies have implicated the adventitial layer.31 In hypercholesterolemia, elevated O2·- is seen throughout the vascular wall.32 Thus, multiple cell types may contribute to the generation of O2·- during exposure to HG. In the present study, auto-oxidation of glucose was not the source of O2·-, because virtually none was detected in the media alone. Future studies should examine the cellular source (endothelium, VSMCs, or adventitia) and the chemical reactions responsible, eg, XO and NAD(P)H oxidase, for O2·- generation in the coronary microcirculation in response to HG.
Effect of Superoxide Associated With HG on Kv
Channel Activity
Little is known about the effect of
O2·- on vascular
mechanisms of hyperpolarization. Gating of
large-conductance
BKCa33
and Kv34 channels may be
modified by the redox state of the cell. In the present study, the
4-AP–sensitive current was reduced in cells from arteries incubated
with HG. In parallel, a reduced 4-AP contractile response was observed
in the CA. Treatment with antioxidants partially restored Kv current
and contraction to 4-AP, suggesting that
O2·- generated by
HG reduces Kv current and function. The importance of this finding is
that Kv channels contribute largely to the total
K+ current in rat CAs and may be responsible
for physiological and pharmacological
dilation.
The mechanism by which O2·- inhibits Kv channels is unknown. It may involve an increase in intracellular Ca2+ concentration by O2·-.35 Several reports indicate that high cytosolic Ca2+ levels inhibit Kv channel activity.36 Oxidation of specific amino acid residues within the Kv channel could also alter its gating.37
Kv channels have not been a major therapeutic focus because they represent a superfamily of K+ channels for which specific gene family blockers are unavailable for distinct gene family subtypes. Because 4-AP effectively blocks most Kv channels, the molecular composition of the specific subtype(s) of Kv channels in CAs affected by HG remains unclear. Future identification of Kv channel structure may provide new insight into potential sites of interaction between Kv channel subunits and ROS.
Although hyperosmolarity can alter vasoactive responses in CAs,38 the reduced Kv current caused by elevated glucose was not due to hyperosmotic effects in the present study, because substituting an equimolar concentration of the nonmetabolizable isomer, L-glucose, had no effect on either Kv currents or 4-AP constriction of isolated vessels.
Potential Problems
It is possible that ROS other than
O2·- participate
in the vascular dysfunction associated with hyperglycemia. However,
most data implicate
O2·- as the
primary culprit in DM and
HG.39 The present study
did not distinguish between endogenous production
of O2·- or
hydrogen peroxide because SOD and CAT were always given together.
However, HE fluorescence and the effect of exogenous generation
of O2·- suggest
that this radical plays an important role in the oxidant stress
associated with HG.
Because SOD does not readily penetrate cells, the restorative effect of SOD observed in the present study may underestimate the true role of O2·- in vasomotor abnormalities. This problem may be minimized in the present study by the small size of the vessels and, consequently, short diffusion distances. Furthermore, the effectiveness of combined SOD and CAT during vasomotor and fluorescence techniques suggests some degree of intracellular penetrance or removal of excess O2·- from within or near the cell membrane where the Kv channels are located. In support of this idea, others have used SOD effectively to improve vasomotor responses in conditions associated with the elevated production of O2·-.8 9 In the present study, the improvement with SOD was less than complete. The use of polyethylene glycol–SOD or a cell-permeable mimetic, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), might further improve Kv channel function after exposure to HG.
The reduction in K+ current density at membrane potentials between 0 and 30 mV is greater in cells from arteries exposed to HG than in cells treated with XA+XO. Possible explanations include the following: (1) O2·- generated by HG likely occurs inside the cell, whereas O2·- generated by XA+XO was primarily extracellular with subsequent entry into the cell. If inhibition of Kv channels depends on intracellular O2·-, this could explain the difference. Unfortunately, because O2·- detection by HE is not quantitative, we cannot determine the amount of O2·- generated in our experiments. (2) HG may act through multiple mechanisms to reduce Kv current, including the generation of other free radicals, such as peroxynitrite, to inhibit K+ currents.40
Finally, the reduction in 4-AP–sensitive K+ currents was observed at membrane potentials positive to 0 mV, whereas vessel constriction to 4-AP presumably occurred at more negative potentials, at which little Kv current was observed in patch-clamped VSMCs. This is likely due to the fact that although Kv channels show low open-state probabilities at negative voltage, small changes in their activation levels can significantly alter membrane potential (and, therefore, vasomotor tone) because of the high membrane resistance of VSMCs.
Physiological
Implications
Our findings based on
electrophysiological and functional data
illustrate that the Kv channel importantly regulates the resting
membrane potential of small CAs. In DM, in which NO-mediated dilation
is reduced, the attendant hyperglycemia may also impair Kv
channel–mediated dilation and thereby reduce myocardial perfusion. We
speculate that ß-adrenergic dilation, an important response to
sympathetic activation, may also be impaired in DM, inasmuch as
cAMP-mediated dilation involves the activation of Kv
channels.25 Thus, a better
understanding of the mechanisms by which hyperglycemia reduces
K+ channel–mediated coronary
vasodilation may enable the design of new therapies to improve
myocardial perfusion in
DM.
Acknowledgments
This study was supported by the National Institutes of Health (grants R01 HL-59238 to Dr Rusch and RO1 HL-52869 to Dr Gutterman), by a Veterans Administration Merit Award to Dr Gutterman, and by the Gutterman Foundation. Dr Liu is the recipient of a Scientist Development Grant from the American Heart Association.
Footnotes
Original received January 22, 2001; revision received May 18, 2001; accepted May 18, 2001.
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