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Am J Physiol Heart Circ Physiol 292: H1404-H1411, 2007. First published October 27, 2006; doi:10.1152/ajpheart.00696.2006
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H2O2 activates redox- and 4-aminopyridine-sensitive Kv channels in coronary vascular smooth muscle

Paul A. Rogers,1 William M. Chilian,1 Ian N. Bratz,2 Robert M. Bryan, Jr.,3 and Gregory M. Dick2

1Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, Louisiana; and 2Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana; and 3Department of Anesthesiology, Baylor College of Medicine, Houston, Texas

Submitted 30 June 2006 ; accepted in final form 25 October 2006


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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Previously, we demonstrated that coronary vasodilation in response to hydrogen peroxide (H2O2) is attenuated by 4-aminopyridine (4-AP), an inhibitor of voltage-gated K+ (KV) channels. Using whole cell patch-clamp techniques, we tested the hypothesis that H2O2 increases K+ current in coronary artery smooth muscle cells. H2O2 increased K+ current in a concentration-dependent manner (increases of 14 ± 3 and 43 ± 4% at 0 mV with 1 and 10 mM H2O2, respectively). H2O2 increased a conductance that was half-activated at –18 ± 1 mV and half-inactivated at –36 ± 2 mV. H2O2 increased current amplitude; however, the voltages of half activation and inactivation were not altered. Dithiothreitol, a thiol reductant, reversed the effect of H2O2 on K+ current and significantly shifted the voltage of half-activation to –10 ± 1 mV. N-ethylmaleimide, a thiol-alkylating agent, blocked the effect of H2O2 to increase K+ current. Neither tetraethylammonium (1 mM) nor iberiotoxin (100 nM), antagonists of Ca2+-activated K+ channels, blocked the effect of H2O2 to increase K+ current. In contrast, 3 mM 4-AP completely blocked the effect of H2O2 to increase K+ current. These findings lead us to conclude that H2O2 increases the activity of 4-AP-sensitive KV channels. Furthermore, our data support the idea that 4-AP-sensitive KV channels are redox sensitive and contribute to H2O2-induced coronary vasodilation.

reactive oxygen species; peroxides; sulfhydryl compounds; delayed-rectifier potassium channels; coronary circulation

H2O2 IS REPORTED TO ACT on both endothelial and vascular smooth muscle cells to produce vasodilation (30). Recently, H2O2 has been suggested as an endothelium-derived hyperpolarizing factor (14), a mediator of flow-induced dilation (17), and a factor influencing coronary autoregulation (35). The dismutated product of superoxide anion, H2O2 may come from mitochondria (13) or NAD(P)H oxidases (4). H2O2 may also be produced by endothelial nitric oxide synthase when supplies of tetrahydrobiopterin, a necessary cofactor, are limited (6). Importantly, however, the cellular and molecular mechanisms by which H2O2 elicits vasodilation remain to be determined, although smooth muscle hyperpolarization appears to be required (26).

The Na+-K+-ATPase and K+ channels are possible effectors of membrane hyperpolarization, and both have been implicated in H2O2-induced smooth muscle relaxation (10). Several studies have identified large-conductance Ca2+/voltage-sensitive K+ (BKCa) channels as putative targets in H2O2-induced vasodilation (1, 2, 30). BKCa channels definitely regulate vascular tone via electromechanical coupling (3); however, several patch-clamp studies indicate that H2O2 inhibits BKCa channel activity (7, 27–29), an effect that is shared by other oxidants (33) and is inconsistent with a role in vasodilation. Smooth muscle cells express a variety of voltage-gated K+ (KV) channels that also regulate arterial tone (11, 19, 21), but whether these channels serve as mediators of H2O2-induced coronary vasodilation is unknown. KV channels in pulmonary vascular smooth muscle function in a redox-sensitive manner, and H2O2 may be the regulatory factor (15, 16). H2O2 increases KV current in pulmonary neuroepithelial bodies (8) and activates cloned KV1.5 (5). Whether KV channels in the coronary vasculature are regulated in a redox-sensitive manner by H2O2 remains to be determined. However, because we previously demonstrated that 4-aminopyridine (4-AP) blocks H2O2-induced coronary vasodilation (23), the goal of the present study was to determine whether H2O2 increased 4-AP-sensitive K+ current in coronary vascular smooth muscle cells.


   METHODS
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 METHODS
RESULTS
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Protocols involving animals were approved by the Institutional Animal Care and Use Committee at Louisiana State University Health Sciences Center. Procedures complied fully with guidelines set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). The method of death conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia.

After dogs were anesthetized with pentobarbital sodium (30 mg/kg iv; Abbott Laboratories), the heart was fibrillated and excised in cold lactated Ringer solution. Third- and fourth-order branches of the left circumflex coronary artery (outer diameter ~0.5–1.0 mm) were dissected en bloc and transferred to a Sylgard-lined dish filled with cold nominally Ca2+-free salt solution containing (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 2 MnCl2, 10 glucose, 10 HEPES, and 5 Tris (pH 7.4). Mn2+ was used as an equimolar substitute for Ca2+, and this solution was used as the standard bath for patch-clamp experiments.

Arteries were gently cleaned of adipose and loose connective tissue and then digested with enzymes to isolate smooth muscle cells. Artery segments were cut into small rings and transferred to a glass tube containing a 1:1 mix of Ca2+-free Dulbecco's PBS and Hanks' balanced salt solution to give a Ca2+ concentration of 0.5 mM, which is near the optimal concentration for enzyme activity. The enzyme solution contained (in mg/ml): 2 type 2 collagenase (Worthington Biochemical, Lakewood, NJ), 1 trypsin inhibitor, 1 elastase, and 2 fatty acid-free BSA. Tissue was incubated for 30 min at 37°C before the enzyme solution was replaced with fresh buffer. The solution was gently agitated with a fire-polished Pasteur pipette to disperse single smooth muscle cells, which were kept at 4°C. Patch-clamp recordings were performed within 8 h after cell dispersion.

Whole cell K+ currents were measured at room temperature using the conventional dialyzed configuration of the patch-clamp technique. Drops of cell suspension were added to a recording chamber mounted on an inverted microscope. Cells were continuously superfused with nominally Ca2+-free salt solution (composition given above). Pipettes were fabricated from borosilicate glass, heat-polished, and had tip resistances of 2–4 M{Omega} when filled with solution containing (in mM) 140 KCl, 1 MgCl2, 3 ATP, 1 EGTA, 10 HEPES, and 5 Tris (pH 7.1). After whole cell access was established, series resistance and membrane capacitance were compensated as completely as possible. Currents were low-pass filtered at 1 kHz and digitized at 5 kHz. Intracellular and extracellular Cl– concentrations were 142 and 146 mM, respectively, and no adjustments were made for the small liquid junction potential that existed (calculated to be –4 mV). To rule out interactions of H2O2 with the Ag/AgCl reference electrode, a series of experiments was performed with the ground connected to the bath via an agar bridge containing 3 M KCl. There was no difference between H2O2-activated current with or without the agar bridge.

Unless otherwise indicated, chemicals were purchased from Sigma Chemical (St. Louis, MO). 4-AP was made as 1 M stock solution in H2O. Because of its basic pH, the solution was acidified with HCl to facilitate solubility and achieve a neutral pH. H2O2 (Calbiochem, San Diego, CA) was purchased as a 30% solution and diluted in bath solution to final concentrations of 1 and 10 mM.

Data are expressed as means ± SE of n experiments (where n represents the no. of smooth muscle cells). For all statistical tests, the criterion for differences was set as P < 0.05. Treatment differences in paired current-voltage relationships were determined by two-way repeated-measures ANOVA. Tukey's post hoc analysis was used where appropriate to identify differences at specific voltages.


   RESULTS
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 ABSTRACT
 METHODS
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DISCUSSION
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We used low-Ca2+ solutions to isolate KV current from BKCa current, since both types of channels are prominently expressed in vascular smooth muscle. Our previous work indicated that 4-AP, but not iberiotoxin (IbTx), blocked H2O2-induced vasodilation (23), and the goal of the present study was to determine whether H2O2 activated 4-AP-sensitive KV current. To demonstrate that IbTx-sensitive BKCa currents were expressed in our preparation, currents were recorded from smooth muscle cells with a physiological concentration of extracellular Ca2+ (2 mM) and 0.1 mM EGTA in the pipette (Fig. 1A). IbTx (100 nM) inhibited current at positive potentials (greater than or equal to +40 mV), typical of BKCa channels. In the continued presence of IbTx, 4-AP (3 mM) inhibited current in the physiological range of membrane potentials (Fig. 1A). To demonstrate that KV current can be separated from BKCa current without using IbTx, a very costly reagent, currents were recorded from smooth muscle cells dialyzed with a solution containing 1 mM EGTA and bathed in a nominally Ca2+-free solution (Fig. 1B). IbTx had very little effect on K+ current in cells under these low-Ca2+ conditions, even at very depolarized potentials. In contrast, 4-AP (3 mM) blocked a significant amount of current in the physiological range of membrane potentials (Fig. 1B). Low-Ca2+ solutions virtually eliminate the contribution of BKCa channels to whole cell current in these cells and thus are used to isolate KV current.


Figure 1
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  		Fig. 1. Isolation of voltage-gated K+ (KV) current from large-conductance Ca2+/voltage-sensitive K+ (BKCa) current using low-Ca2+ solutions. A: currents recorded from a smooth muscle cell with physiological Ca2+ concentrations. Iberiotoxin (IbTx; 100 nM) inhibited current at positive potentials. 4-Aminopyridine (4-AP, 3 mM) inhibited current in the physiological range of membrane potentials. IbTx- and 4-AP-sensitive currents are shown in the inset. B: currents recorded from a smooth muscle cell dialyzed with a solution containing 1 mM EGTA and bathed in a nominally Ca2+-free solution. IbTx (100 nM) had very little effect on K+ current, whereas 4-AP (3 mM) blocked a significant amount of current in the physiological range of membrane potentials. IbTx- and 4-AP-sensitive currents are shown in the inset. Low-Ca2+ solutions virtually eliminate the contribution of BKCa channels to whole cell current in these cells and thus are used to isolate KV current.

 
Importantly, however, it should be noted that H2O2 activates at least two components of K+ current in smooth muscle cells with physiological intracellular and extracellular Ca2+ concentrations (Fig. 2). When cells were bathed in a solution containing 2 mM Ca2+ and dialyzed with a solution containing 0.1 mM EGTA, H2O2 increased at least two components of whole cell current. Analysis of the H2O2-activated current (Fig. 2B) revealed the distinctive "hump" of delayed-rectifier current in the physiological range of membrane potentials and a more outwardly rectifying component that increased sharply at potentials positive to +40 mV. Thus the H2O2-activated current under physiological Ca2+ conditions is mediated by both KV and BKCa channels and has the same general shape as the 4-AP- and IbTx-sensitive current in these cells. However, it is 4-AP, not IbTx, that antagonizes H2O2-induced vasodilation in the canine coronary circulation (23).


Figure 2
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  		Fig. 2. H2O2 activates at least two components of K+ current in smooth muscle cells under physiological Ca2+ conditions. A: group data (n = 7) from cells bathed in a solution containing 2 mM Ca2+ and dialyzed with a solution containing 0.1 mM EGTA. H2O2 increased whole cell current. B: H2O2-activated current (b – a from A) is shown. A distinctive "hump" of delayed-rectifier current is observable in the physiological range of membrane potentials. A more outwardly rectifying component increases sharply at potentials positive to +40 mV. Inset shows the IbTx- and 4-AP-sensitive current from Fig. 1A, which has the same general shape as the H2O2-activated current.

 
H2O2 increased whole cell K+ current in canine coronary smooth muscle cells under the low-Ca2+ conditions used to isolate KV current (Fig. 3). Cells were held at –80 mV to remove any voltage-dependent channel inhibition, and voltage steps were applied in 20-mV increments from the holding potential. Subtracting the control current trace from that in the presence of 10 mM H2O2 revealed that H2O2 activated a time- and voltage-dependent current that did not inactivate during a 300-ms test pulse (Fig. 3C). H2O2-activated current deactivated slowly, as prolonged tail currents were evident at –40 mV (Fig. 3C). The effect of H2O2 to increase current was rapid and sustained, since washing H2O2-treated cells with H2O2-free buffer did not return current to the baseline level (Fig. 3D). Addition of 3 mM 4-AP to the bath reduced current magnitude below the baseline level, indicating KV channels are responsible for some portion of the outward current (Fig. 3D). Analysis of the steady-state current-voltage relationship indicated the effect of 10 mM H2O2 on current becomes significant at –20 mV (Fig. 4A). The effect of H2O2 to increase whole cell K+ current was concentration dependent, since 1 mM H2O2 also increased current (Fig. 4B). Furthermore, the data in Figs. 3 and 4 (i.e., kinetics, voltage dependence, low-Ca2+ concentration in the pipette and bath solutions, and 4-AP sensitivity) suggest H2O2 may act on KV channels.


Figure 3
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  		Fig. 3. H2O2 increases K+ current in canine coronary smooth muscle cells. A–C: representative current traces illustrating the effect of H2O2 (10 mM) to increase whole cell current. The cell was held at –80 mV, and membrane potential was stepped from –100 to 0 mV in 20-mV increments. Deactivating tail currents were observed at –40 mV following the various test potentials, indicative of KV channel activation. D: current at +20 mV in a single cell is plotted vs. time to demonstrate the rapid and sustained effect of H2O2. Current remained elevated after H2O2 was washed out; application of 4-AP (3 mM) reduced current below the baseline level.

 

Figure 4
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  		Fig. 4. H2O2 increases whole cell K+ current in canine coronary smooth muscle cells in a concentration-dependent manner. A: group data of the steady-state current-voltage (I-V) relationship with 10 mM H2O2 treatment [n = 40 cells; *P < 0.05 by 2-way repeated-measures (RM) ANOVA with Tukey's post hoc analysis]. B: group data demonstrate the increase in whole cell current with H2O2 is concentration dependent (1 mM, n = 17 cells and 10 mM, n = 40 cells). The difference current (i.e., current in the presence of H2O2 – control) is shown.

 
Thus we performed a series of experiments to characterize the voltage dependence of this putative whole cell KV current and determine whether H2O2 influenced the voltages of half-activation and/or -inactivation (Fig. 5). Tail currents were measured at –40 mV to determine the voltage dependence of activation, since these deactivating tails are a reflection of channel activation (–40 mV is not a strong enough depolarization to elicit current but is far enough removed from the Nernst equilibrium potential for K+ to provide a driving force through any channels opened in the preceding voltage step). Tail current amplitude before and after H2O2 stimulation is plotted vs. the conditioning voltage and fit by a single Boltzmann exponential function (Fig. 5A). H2O2 significantly increased tail current magnitude at conditioning pulses of –30 mV and above. The effects of H2O2 on KV current inactivation were determined by stepping to 0 mV after holding membrane potential at various levels for 10 s. The amplitude of this current reflects channel availability (i.e., channels that were not inactivated) at various potentials. H2O2 significantly increased available current (Fig. 5B). Currents in Fig. 5, A and B, were normalized to their respective maximum and minimum (assumed to be 0 pA for Fig. 5B) values to create conductance-voltage curves (Fig. 5, insets). The voltage of half-activation under control conditions was –18 ± 1 mV and was not significantly altered by H2O2 (–19 ± 1 mV). Likewise, the voltage of half-inactivation was not influenced by H2O2 (–36 ± 2 vs. –34 ± 2 mV). Together, these data indicate H2O2 increased KV current but does not change the intrinsic voltage sensitivity of KV channels.


Figure 5
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  		Fig. 5. Effects of H2O2 on the activation and inactivation of the voltage-gated current. A: tail currents measured at –40 mV after various depolarizing prepulses (n = 40). Tail current magnitude, which was increased by H2O2 (10 mM), is indicative of channel activation. Inset shows currents normalized to their respective maximum values [conduction-voltage (G-V) curves]. B: current at 0 mV after cells were conditioned for 10 s at the various potentials (n = 10). Current magnitude, which was increased by 10 mM H2O2, is a reflection of channel availability. Inset shows currents normalized to their respective maximum values (G-V curves).

 
In our previous study, we demonstrated that thiol oxidation mediates H2O2-induced dilation of coronary arteries and arterioles, since smooth muscle relaxation was reversed by dithiothreitol (DTT; a thiol-specific reducing agent; see Ref. 23). The identity of redox-sensitive elements was not determined; therefore, we performed experiments to determine whether the effect of H2O2 on KV current could be reversed by DTT. H2O2 increased outward KV current (Fig. 6A) and the tail current at –40 mV (Fig. 6B). DTT (1 mM) reduced KV current in H2O2-treated cells (Fig. 6, A and B). Interestingly, normalizing the tail currents revealed that DTT shifted the voltage of half-activation to more positive potentials (–10 ± 1 mV; Fig. 6C). The mechanism(s) by which DTT reduces the voltage sensitivity of activation remains to be determined; however, to confirm the potential role of reactive thiol groups, we examined them with another approach. Specifically, we treated cells with 5 mM N-ethylmaleimide (NEM), a thiol-alkylating agent. NEM reacts with and alkylates reduced thiol groups, thus preventing interactions with H2O2. NEM slightly, but significantly, inhibited outward KV current (Fig. 7A). NEM did not affect tail current (Fig. 7B). Importantly, however, NEM prevented the effect of H2O2 to increase KV current (Fig. 7).


Figure 6
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  		Fig. 6. Dithiothreitol (DTT) inhibits the voltage-gated H2O2-activated K+ current. A: steady-state I-V relationship (n = 15) of the effect of DTT (1 mM) on current activated by H2O2 (10 mM). B: H2O2 increased tail current at –40 mV, whereas DTT (added after H2O2) reduced tail current amplitude. C: tail currents were normalized to the maximum and plotted vs. the depolarizing prepulse. The voltage of half-activation was shifted to more positive potentials by DTT. P < 0.05 vs. control (* and #) and vs. H2O2 ($) by 2-way RM ANOVA with Tukey's post hoc analysis.

 

Figure 7
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  		Fig. 7. N-ethylmaleimide (NEM) prevents the effect of H2O2 to increase KV current. A: steady-state I-V relationship (n = 10) showing that NEM prevents the H2O2-induced increase in current. NEM (5 mM) was added after control current measurements, and the cell was treated for 5 min before current was measured in the presence of NEM. Cells were then treated with the combination of NEM and H2O2 (10 mM) for another 5 min before current was recorded. B: tail currents measured at –40 mV demonstrate that NEM inhibited the effect of H2O2 to increase KV current. P < 0.05 vs. control by 2-way RM ANOVA with Tukey's post hoc analysis (* and #).

 
Our final experiments were designed to characterize the pharmacological nature of the H2O2-activated current. To do so, we pretreated cells with various K+ channel inhibitors and then determined the ability of H2O2 to increase current. BKCa channels have been identified as possible mediators of H2O2-induced vasodilation in other species and tissues (2, 9, 30). Thus we first performed experiments with tetraethylammonium (TEA; 1 mM) and IbTx (100 nM), inhibitors of BKCa (Fig. 8). TEA slightly, but significantly, inhibited K+ current; however, TEA failed to block the effect of H2O2 to increase KV current. Experiments with IbTx further confirmed that BKCa channels are not activated by H2O2 under these conditions (i.e., with a nominally Ca2+-free bath, 1 mM intracellular EGTA, and depolarizations limited to +40 mV). Single cells in the recording chamber (volume ~1 ml) were treated with 10 ml of buffer (flow rate ~3 ml/min) containing 100 nM IbTx. The perfusion system was then turned off for 10 min while voltage-step protocols were performed to confirm steady-state block of BKCa channels by IbTx. The toxin blocked current at potentials positive to +80 mV. Flow in the recording chamber was reestablished, and the cells were treated with H2O2 in the continued presence of 100 nM IbTx. IbTx failed to inhibit the effect of H2O2 to increase KV current (Fig. 9). Last, we examined the effect of 4-AP (3 mM). The data in Fig. 3D demonstrate that current in the presence of H2O2 can be reduced by 4-AP. However, that experiment does not allow one to determine whether 4-AP is blocking the channels activated by H2O2. Therefore, we performed experiments where 4-AP was added before H2O2. 4-AP inhibited current at 0 mV by 76 ± 3% (Fig. 10A) and shifted the voltage of half-activation to +2 ± 1 mV (Fig. 10C). Most importantly, however, H2O2 failed to increase whole cell K+ current in the presence of 4-AP. These data suggest that H2O2 increases current through 4-AP-sensitive KV channels.


Figure 8
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  		Fig. 8. Tetraethylammonium (TEA, 1 mM) does not inhibit the ability of H2O2 to increase K+ current. The steady-state I-V relationship (n = 8) elicited by depolarizing pulses (A) and tail currents measured at –40 mV after depolarizing pulses (B) reveal that TEA (1 mM) does not block the ability of H2O2 (10 mM) to increase K+ current. P < 0.05 by 2-way RM ANOVA with Tukey's post hoc analysis (* and #).

 

Figure 9
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  		Fig. 9. IbTx fails to prevent the effect of H2O2 to increase K+ current. A: steady-state I-V relationship (n = 6) of the ability of H2O2 (10 mM) to increase whole cell, steady-state K+ current in the presence of IbTx (100 nM). B: tail currents measured at –40 mV depicting the ability of H2O2 to increase KV current in the presence of IbTx. *P < 0.05 vs. control and IbTx by 2-way RM ANOVA with Tukey's post hoc analysis.

 

Figure 10
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  		Fig. 10. 4-AP inhibits H2O2-induced increase in K+ current. Treating cells (n = 9) with 4-AP (3 mM) inhibited the ability of H2O2 (10 mM) to increase whole cell K+ current (A) and tail current (B) from KV channels. C: tail currents were normalized to the maximum and revealed 4-AP shifted the voltage of half-activation to +2 ± 0.6 mV. *P < 0.05 vs. 4-AP and 4-AP/H2O2 by 2-way RM ANOVA with Tukey's post hoc analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our purpose was to test the hypothesis that H2O2 increases current through 4-AP-sensitive KV channels of coronary vascular smooth muscle cells. Rationale for the study stemmed from observations that H2O2-induced coronary vasodilation was attenuated by 4-AP, an inhibitor of KV channels (23). Because no existing evidence directly linked H2O2 to the activation KV channels in coronary vascular smooth muscle, we focused our efforts on whole cell patch-clamp experiments to measure K+ current. Furthermore, we determined whether thiol oxidation represents a mechanism by which H2O2 acts. Major findings include 1) H2O2 increased a voltage- and time-dependent K+ current; 2) current elicited by H2O2 was antagonized by DTT, a thiol reductant, and blocked by NEM, a thiol-alkylating agent; and 3) effect of H2O2 to increase K+ current increase was blocked by 4-AP but not by IbTx or low concentrations of TEA. Thus our data support the hypothesis and indicate that 4-AP-sensitive KV channels, or proteins that regulate their activity, are redox sensitive and contribute to H2O2-induced coronary vasodilation. These findings, combined with our previous observations (23), indicate novel functions for KV channels in the regulation of coronary vascular tone.

Previous studies have focused on the contribution of BKCa channels to H2O2-induced coronary vasodilation (2, 30). The single channel studies of Barlow and White (2) indicated that H2O2 activated BKCa channels via a lipoxygenase-dependent pathway in porcine coronary smooth muscle cells; however, effects H2O2 on KV channels were not determined. Similarly, Thengchaisri and Kuo (30) found that IbTx partially inhibited H2O2-mediated dilation of porcine coronary arterioles, indicating the involvement of BKCa channels; however, the potential involvement of 4-AP-sensitive KV channels was not assessed. There may be species-specific differences between pig and dog, since our current and previous studies demonstrated that 4-AP-sensitive KV channels, not IbTx-sensitive BKCa channels, mediate H2O2-induced vasodilation of canine coronary arterioles (23). Although there may be species-specific differences between pigs and dogs, there seem to be none between dogs and rats in this matter. Specifically, Gao et al. (9) implicated 4-AP-sensitive KV channels, and not IbTx-sensitive BKCa channels, in H2O2-mediated vasodilation of rat mesenteric arteries. Furthermore, these investigators used perforated-patch techniques to demonstrate that 1 mM H2O2 activated 4-AP-sensitive KV channels in rat smooth muscle cells. Our data agree closely with those of Gao et al., and we draw the conclusion that coronary vascular smooth muscle KV channels are regulated in a redox-sensitive manner by H2O2.

We have not determined whether longer exposure to H2O2 will activate BKCa channels in canine coronary smooth muscle cells, an experiment that might more closely match the study of Barlow and White (2), who treated porcine coronary smooth muscle cells with 300 µM H2O2 for 30 min before measuring channel activity. In contrast, our data indicate that effects of 1–10 mM H2O2 on KV channels are mediated quickly (on the order of 2–3 min, depending on flow rate, concentration, and chamber size). Our findings are more similar to those of Gao et al. (9), who observed a very rapid effect of 1 mM H2O2 on whole cell K+ current. However, our findings differ from theirs in that we did not observe rapid reversibility (i.e., the effect of H2O2 to increase KV current was sustained). Such a difference might be depend on whether ruptured or perforated-patch techniques are used, since rupturing the cell membrane likely dialyzes out intracellular reductants, such as glutathione. This may promote a more permanent effect of H2O2. Another plausible explanation for our result is that H2O2 may oxidize an extracellular target, rather than a cytoplasmic one that could be repaired by endogenous intracellular reductants. This argument is based on assumptions that 1) H2O2 freely crosses the membrane and has access to both extracellular and intracellular thiol groups; 2) thiol groups on extracellular targets always remain outside of the cell (e.g., extracellular loops of KV channels); 3) intracellular targets include the KV channel or proteins (e.g., kinases) that regulate them and always remain inside the cell; and 4) intracellular reductants are membrane impermeable and remain inside the cell with access limited to intracellular targets. Thus, if an extracellular target were oxidized, no "repair" (i.e., reduction) would be possible. Conversely, if an intracellular target were oxidized, it might be repaired by intracellular reducing mechanisms. Future experiments will be geared toward determining whether KV channels and/or proteins that regulate KV channels are modified by H2O2. Once viable molecular candidates for H2O2-sensitive targets are identified, it will be important to determine the exact site and nature of the modifications.

The present study differs slightly from our previous study (23) in regard to the concentration dependence of H2O2 effects; however, the relative concentrations used for vasodilation and patch-clamp studies are similar to what has been observed by others. Specifically, our patch studies were performed with 1–10 mM H2O2, which is a log order higher than the 0.3 mM concentration used by Barlow and White (2) for their cell-attached and inside-out patch studies but similar to the 1 mM concentration used by Gao et al. (9) for their perforated-patch recordings. Other investigators, studying cloned KV channels, have used concentrations of H2O2 from 1 to 30 mM to demonstrate effects (20, 34). With respect to vasodilation, all of the studies mentioned above, including that of Thengchaisri and Kuo, indicate H2O2 EC50 concentrations of ~10–300 µM. Local concentrations of H2O2 and other reactive oxygen species in vivo remain unclear; however, 100 µM H2O2 is a value reported for normal human urine (32), and we measured H2O2 concentrations in excess of 200 µM in the interstitial fluid of the working myocardium (25). One factor that may underlie concentration-dependent differences between vasodilation and electrophysiology studies could be temperature. Currents were measured at room temperature (25°C), whereas vasodilation was measured at body temperature (≥37°C), and it is known that KV channels display marked differences in activity at room and body temperature (31). Another factor that might explain the concentration difference we observed could be related to methodology. We use the ruptured-patch technique, in which the intracellular milieu is likely disturbed by dialysis of some cellular constituents that might be necessary for the effect of H2O2.

Our experiments with DTT and NEM suggest that H2O2 acts on some thiol groups of proteins that are not dialyzed from the cell. Whether those thiol groups are in the KV channels themselves or present in proteins that regulate KV channels remains to be determined. DTT, a thiol reductant, reversed the effect of H2O2 to increase KV current, whereas NEM, a thiol-alkylating agent, prevented the effect of H2O2 to increase KV current. DTT affects gating of cloned rat KV1.4 channels (24) and decreases KV current in rabbit pulmonary arterial smooth muscle cells (18). NEM increases current from KV7 family channels (12, 22); however, we found that NEM inhibited native KV current and prevented the effect of H2O2 to increase it. Neither TEA nor IbTx prevented the effect of H2O2 to increase whole cell current, suggesting that H2O2 does not activate BKCa channels. TEA slightly, but significantly, inhibited whole cell current in a voltage range consistent with KV channels, suggesting 1 mM TEA is not ideal for selectively inhibiting BKCa channels. Thus experiments with IbTx were performed. IbTx inhibited current at much more positive potentials (consistent with inhibition of BKCa channels). Importantly, however, IbTx had no effect on current in the voltage range consistent with KV channels, nor did IbTx prevent the effect of H2O2 to increase current. In contrast, 4-AP significantly inhibited KV current and completely prevented the effect of H2O2 to increase it.

In conclusion, our data show that H2O2 activates 4-AP-sensitive KV channels in canine coronary smooth muscle cells. This finding is entirely consistent with our previous study that demonstrated H2O2-induced vasodilation was blocked by 4-AP. We propose that KV channels are redox-sensitive elements regulating coronary vascular tone.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
P. A. Rogers was supported by a Predoctoral Fellowship from the American Heart Association, Southeast Affiliate. This work was supported by National Institutes of Health Grants RR-018766 (G. M. Dick), HL-32788, HL-65203, and HL-73755 (W. M. Chilian), and NS-46666 (R. M. Bryan, Jr.).


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Johnathan D. Tune (Indiana University School of Medicine) and Drs. Dirar Khoury and Judy Ober (Baylor College of Medicine and Texas Heart Institute) for kindly providing canine coronary arteries. We appreciate the constructive criticism of Dr. James L. Kenyon (University of Nevada School of Medicine) regarding this work.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. Dick, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., MS 385, Indianapolis, IN 46202-5120 (e-mail: gdick{at}iupui.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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