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(Circulation Research. 2001;89:146.)
© 2001 American Heart Association, Inc.

Cellular Biology

High Glucose Impairs Voltage-Gated K⁺ Channel Current in Rat Small Coronary Arteries

Yanping Liu, Ken Terata, Nancy J. Rusch, David D. Gutterman

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

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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

	Introduction

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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⁺ (BK_Ca) 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

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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

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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.

View larger version (21K): [in this window] [in a new window]   Figure 1. A, Ethidium bromide fluorescence of rat small CAs incubated for 10 minutes in HE immediately after dissection (control) or after 24 hours in NG, LG, or HG. All image gain settings were identical. Relative fluorescence intensity (red) corresponds to the rate of O2·- formation in the vessel wall. Exposure to HG increased the production of O2·- within the vessel wall. Dotted lines demonstrate edge of vessel wall. B, Relative intensity ratio of arteries exposed to NG, LG, and HG normalized to that of control vessels. The fluorescence intensity is significantly increased only in HG arteries (*P<0.05 vs NG and LG).

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).

View larger version (20K): [in this window] [in a new window]   Figure 2. A, Sample images comparing fluorescence intensity in freshly isolated arteries (control) and in arteries treated with HG with or without SOD and CAT. Fluorescence is markedly increased in HG arteries compared with control vessels. SOD+CAT abolished ethidium bromide fluorescence in HG. Dotted lines indicate edge of vessel wall. B, Summary of fluorescence intensity (normalized to control) in 6 arteries from each group. Fluorescence intensity was significantly reduced in HG arteries treated with SOD and CAT (*P<0.05 vs HG).

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.

View larger version (41K): [in this window] [in a new window]   Figure 3. A, Whole-cell K+ current (IK) in coronary VSMCs from arteries incubated with NG, LG, and HG. Currents were elicited by incremental 10-mV depolarizing steps from -60 to 60 mV. Cell capacitance was similar between cells: NG was 10 pF; LG, 11 pF; and HG, 10 pF. Compared with outward currents in cells from arteries incubated in NG or LG, outward currents in cells from arteries exposed to HG were reduced. 4-AP (3 mmol/L) blocked a large component of the outward current in cells from NG and LG arteries but caused less inhibition in cells from HG arteries. B, Current-voltage relationships of IK densities in cells from arteries exposed to NG, LG, or HG. Density was significantly reduced in cells from HG arteries compared with cells from NG and LG arteries (*P<0.05 vs NG and LG). C, Current-voltage relationships showing the effect of 4-AP on macroscopic IK in NG, LG, and HG cells. VSMCs from arteries incubated with HG show less 4-AP–sensitive IK compared with cells from arteries exposed to NG and LG (*P<0.05 vs NG and LG). D, Comparison of voltage dependence of IK between cells from NG and HG arteries. Inset shows sample traces of tail currents recorded at -60 mV after a prepulse step to 60 mV to maximally activated IK. The plot of peak tail current amplitudes vs prepulse potentials indicates that tail currents were reduced in cells from HG arteries, whereas voltage dependence of IK was not altered.

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, I_act=I_max/[1+exp(V_m-V_0.5)/k], where I_max is the maximal tail current amplitude at 60 prepulse, I_act is the tail current observed at a given prepulse voltage (V_m), V_0.5 is the voltage for half of I_max, and k is the steepness factor (slope) indicative of voltage sensitivity. Figure 3D shows that HG reduced I_max but had little effect on the voltage dependence of K⁺ channels. V_0.5 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 I_max but did not change the voltage dependence of K⁺ channel activation. V_0.5 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.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of O2·- generated by XA and XO on IK. A, IKs generated at a membrane potential of +60 mV when the cell was superfused with XA, XA+XO, or XA+XO+4-AP. Current traces were not leak-subtracted. B, Averaged IK density as a function of membrane potential. XA+XO reduced IK density with no further reduction in response to 4-AP (*P<0.05 vs XA). C, Voltage dependence of IK in response to O2·- generation. XA+XO decreased peak tail current amplitudes (*P<0.05 vs XA) but had no significant effect on voltage dependence. Respective V0.5 and k values given by the Boltzmann equation are as follows: XA, -10 mV and 17; XA+XO, -9 mV and 18. Voltage protocol was the same as in Figure 3D.

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.

View larger version (25K): [in this window] [in a new window]   Figure 5. Current-voltage relationships comparing IK densities of patch-clamped NG, LG, or HG cells in response to SOD (150 U/mL) and CAT (500 U/mL). A and B, SOD and CAT did not alter IK density in cells from arteries incubated in NG or LG. C, However, SOD and CAT partially restored IK in cells from arteries incubated in HG (*P<0.05 vs control). Insets show original currents recorded at 60 mV before and after cells were superfused with SOD and CAT. Current traces were not subtracted.

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.

View larger version (28K): [in this window] [in a new window]   Figure 6. A, Effect of 4-AP on the resting diameter of rat CAs incubated with NG, LG, and HG is shown (*P<0.05 vs NG and LG). Contractions induced by 4-AP were reduced in arteries exposed to HG compared with those exposed to NG or LG (n=7 each). B, Similar contractile responses to graded doses of KCl were observed in arteries incubated with either HG or NG (n=6, P=NS). C, Nifedipine induced similar dilator responses (percent maximal [Max.] dilation) in arteries incubated with NG and HG (n=6, P=NS).

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.

View larger version (25K): [in this window] [in a new window]   Figure 7. Diameter changes of small CAs in response to 4-AP before and after SOD and CAT. A and B, SOD and CAT did not affect the amplitude of 4-AP–induced contractions in vessels incubated in either NG or LG, respectively (n=7, P=NS each). C, SOD and CAT improved the contractile response to 4-AP in arteries incubated with HG (n=7) (*P<0.05 vs control).

	Discussion

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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 VSMCs^23,24 and contribute to membrane potential, particularly at lower levels of intracellular Ca²⁺, at which the BK_Ca 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 BK_Ca. 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 BK_Ca³³ 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.

Received January 22, 2001; accepted May 18, 2001.

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