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Am J Physiol Heart Circ Physiol 291: H2473-H2482, 2006. First published June 2, 2006; doi:10.1152/ajpheart.00172.2006
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H2O2-induced redox-sensitive coronary vasodilation is mediated by 4-aminopyridine-sensitive K+ channels

Paul A. Rogers,1 Gregory M. Dick,2 Jarrod D. Knudson,1 Marta Focardi,1 Ian N. Bratz,1 Albert N. Swafford, Jr.,1 Shu-ichi Saitoh,1 Johnathan D. Tune,2 and William M. Chilian1

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

Submitted 16 February 2006 ; accepted in final form 30 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hydrogen peroxide (H2O2) is a proposed endothelium-derived hyperpolarizing factor and metabolic vasodilator of the coronary circulation, but its mechanisms of action on vascular smooth muscle remain unclear. Voltage-dependent K+ (KV) channels sensitive to 4-aminopyridine (4-AP) contain redox-sensitive thiol groups and may mediate coronary vasodilation to H2O2. This hypothesis was tested by studying the effect of H2O2 on coronary blood flow, isometric tension of arteries, and arteriolar diameter in the presence of K+ channel antagonists. Infusing H2O2 into the left anterior descending artery of anesthetized dogs increased coronary blood flow in a dose-dependent manner. H2O2 relaxed left circumflex rings contracted with 1 µM U46619 [GenBank] , a thromboxane A2 mimetic, and dilated coronary arterioles pressurized to 60 cmH2O. Denuding the endothelium of coronary arteries and arterioles did not affect the ability of H2O2 to cause vasodilation, suggesting a direct smooth muscle mechanism. Arterial and arteriolar relaxation by H2O2 was reversed by 1 mM dithiothreitol, a thiol reductant. H2O2-induced relaxation was abolished in rings contracted with 60 mM K+ and by 10 mM tetraethylammonium, a nonselective inhibitor of K+ channels, and 3 mM 4-AP. Dilation of arterioles by H2O2 was antagonized by 0.3 mM 4-AP but not 100 nM iberiotoxin, an inhibitor of Ca2+-activated K+ channels. H2O2-induced increases in coronary blood flow were abolished by 3 mM 4-AP. Our data indicate H2O2 increases coronary blood flow by acting directly on vascular smooth muscle. Furthermore, we suggest 4-AP-sensitive K+ channels, or regulating proteins, serve as redox-sensitive elements controlling coronary blood flow.

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

UNCERTAINTIES SURROUND THE vasoactive properties of H2O2. Studies variably report H2O2 as a vasoconstrictor (17, 46), vasodilator (14, 48), or both (8, 31). Furthermore, different vascular responses have been described for endogenous and exogenous H2O2 (45). Some differences may depend on whether 1) studies are performed in vivo or in vitro, 2) conduit or resistance vessels are examined, and 3) tension or tone is spontaneous or pharmacologically induced. In addition, the key factor may be the specific vascular bed or vessel under investigation. Isolated mouse tail arterioles constrict (41), whereas mesenteric arterioles dilate (35), in response to H2O2. Thus, although no general consensus has been reached regarding the vascular effects of H2O2 or the mechanisms that mediate them, it is apparent that H2O2 is vasoactive and that much remains to be determined.

With regard to the coronary circulation, most reports indicate that H2O2 dilates and relaxes arteriolar and arterial vascular smooth muscle (37, 4). However, there remains controversy over whether the endothelium mediates H2O2-induced coronary vasodilation (47). H2O2 was suggested as an endothelium-derived hyperpolarizing factor (34, 35) and a mediator of flow-induced dilation (38); therefore, the endothelium is involved at least to such extent that it produces H2O2 to relax vascular smooth muscle. However, it is metabolism and oxygen consumption, and not endothelial/paracrine influences, that represent the major physiological determinants of coronary blood flow (50). Accordingly, H2O2 was suggested as a mediator of reactive hyperemia (26, 54) and a factor influencing coronary autoregulation in concert with nitric oxide and adenosine (55). Importantly, however, the cellular and molecular mechanisms by which H2O2 elicits vasodilation remain to be determined. Our goal in the present study was to test the hypothesis that K+ channels sensitive to 4-aminopyridine (4-AP) mediate H2O2-induced coronary vasodilation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animal protocols 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), and the method of euthanization conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia.

Mongrel dogs (n = 16) of either sex were sedated with morphine (3 mg/kg sc), anesthetized with {alpha}-chloralose (100 mg/kg iv), and ventilated with room air supplemented with oxygen. The femoral arteries and a femoral vein were catheterized for aortic pressure measurement, coronary perfusion, and administration of supplemental fluids and anesthetics. Sodium bicarbonate was administered intravenously to counteract the acidosis normally associated with {alpha}-chloralose, and ventilatory rate was adjusted to maintain normal blood gas parameters. The left anterior descending coronary artery (LAD) was isolated proximal to the first diagonal branch, and the artery was catheterized with a stainless steel cannula. The LAD was perfused with blood from the left femoral artery at constant pressure (100 mmHg) with a servo-controlled roller pump. After a 20-min recovery period, various intracoronary dose-response experiments were performed as described below. Coronary blood flow was measured with an in-line Transonics flow probe. Coronary plasma concentrations of drugs were calculated as [infusion rate/coronary blood flow (1 – hematocrit)] x infusion concentration.

H2O2 (Calbiochem, San Diego, CA) was infused into the coronary perfusion line. Flow at each H2O2 dose (20 nmol/min to 0.2 mmol/min) was recorded for 3 min at a stable rate. To determine the contribution of K+ channels to H2O2-induced coronary vasodilation, we used three different experimental protocols with the K+ channel antagonist tetraethylammonium (TEA) or 4-AP, or the combination of TEA and 4-AP. The calculated coronary plasma concentrations of TEA and 4-AP were 5 and 3 mM, respectively. Preliminary studies were performed to ascertain the feasibility of using 10 mM TEA; however, at this concentration, the heart developed ventricular tachycardia, thus precluding its use. Infusions of channel blockers started 10 min before H2O2 infusion and continued throughout the remainder of the experiment.

At the end of the in vivo experiments, the heart was excised and immersed in cold lactated Ringer solution. Left circumflex coronary arteries were dissected and cleaned of periadventitial fat. Arteries were cut into 3-mm rings and mounted in organ baths containing warmed Krebs buffer (in mM: 132 NaCl, 25 NaHCO3, 5 KCl, 2.5 CaCl2, 1.2 NaH2PO4, 1.2 MgCl2, and 10 glucose) for isometric tension studies. Optimal length was found by increasing passive tension in 1-g increments until there was <10% change in active tension in response to K+. Rings were contracted using U46619 [GenBank] (1 µM; Biomol, Plymouth Meeting, PA) or 60 mM K+, and H2O2 (1, 10, and 100 µM) was added in a cumulative manner. K+ channel blockers (10 mM TEA and/or 3 mM 4-AP) and indomethacin (20 µM; cyclooxygenase inhibitor) were added 10 min before U46619 [GenBank] contraction. In some experiments, rings were denuded of endothelium by rubbing the intimal layer with fine forceps. Effective denudation was determined by the lack of relaxation to 10 µM acetylcholine. To assess the role of thiol oxidation in H2O2-induced relaxation, we added dithiothreitol (1 mM DTT, a thiol reductant; Bio-Rad, Hercules, CA).

Coronary arterioles were dissected from left ventricular free wall tissue. A section of myocardium was placed in ice-cold PSS containing (in mM) 145 NaCl, 4.7 KCl, 2 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5 glucose, 2 pyruvate, 0.02 EDTA, and 3 MOPS. This solution contained 1% fatty acid-free bovine serum albumin and was buffered to pH 7.4 at 36.5°C and filtered through a 0.22-µm filter. Dissections were performed in a temperature-controlled chamber (4°C). Arterioles were cannulated with micropipettes and connected to reservoirs filled with PSS. Reservoir height was set to obtain an intraluminal pressure of 60 cmH2O. Arterioles that failed to maintain pressure were excluded from analysis. Internal diameter was measured with a charge-coupled device camera and video caliper system. Arterioles were slowly warmed to 37°C and assessed for the development of spontaneous tone. Vessels that did not develop tone spontaneously were constricted with 1–3 nM endothelin-1 and washed with PSS intermittently for 1.5 h before an experiment was begun. H2O2 responses in vessels were not significantly different between groups treated or not treated with endothelin. In initial preliminary studies, time control experiments were conducted to assess whether multiple H2O2 concentration-response curves could be performed on the same arteriole (e.g., dilation before and after K+ channel blockers). Two H2O2 concentration-response curves were conducted 20 min apart, interposed with multiple washes. Marked tachyphylaxis was observed. To determine whether decreased responsiveness was due to oxidized thiol residues, we washed arterioles for 5 min with PSS containing 1 µM DTT and performed a third H2O2 concentration-response curve. To determine the role of the endothelium in H2O2-induced dilation, we denuded arterioles of endothelium by perfusing them with PSS containing 0.4% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS; Bio-Rad) and then flushing them for 5 min with PSS alone. Effective endothelial removal was determined by responses to 1 µM acetylcholine before and after perfusion with CHAPS. To determine the role of K+ channels in H2O2-induced dilation, we treated arterioles with 0.3 mM 4-AP or 100 nM iberiotoxin. Effective channel block was confirmed by constriction to a new stable baseline diameter. Maximal dilation in vessels not treated with K+ channel antagonists was determined by administering a supramaximal dose of sodium nitroprusside (SNP; 100 µM). Because of NO-mediated signaling to K+ channels, maximal dilation in vessels treated with K+ channel antagonists was determined by administering SNP and 100 µM verapamil to remove all voltage-dependent tone.

Male Sprague-Dawley rats (125–150 g) were anesthetized with pentobarbital sodium (65 mg/kg ip), and their hearts were excised and placed in ice-cold PSS. Septal arteries and arterioles were dissected free from ventricular septal wall tissue. Arterioles were cannulated, pressurized, and examined in a manner identical to that described above for arterioles obtained from dog hearts. Septal arteries were mounted on a four-well Multi Myograph System model 610M (Danish Myo Technology) using 45-µm wire. Viability was tested using 60 mM KCl, and optimal passive tension was determined to be 0.5 g. Proximal segments were contracted with U46619 [GenBank] (1 µM) and did not respond to endothelin-1 (100 nM). More distal segments did not respond to U46619 [GenBank] but responded to endothelin-1. There was no difference in H2O2-induced responses in the presence of either agonist used to induce contraction. After agonist-induced contractions stabilized, H2O2 (0.001, 0.01, 0.1, and 1 mM) was applied to each well in a cumulative manner. After each application of H2O2, tension was allowed to plateau before the subsequent dose of H2O2 was applied. 4-AP was applied to vessel baths 10 min before agonist-induced contraction. Unless specifically indicated above, chemicals were purchased from Sigma Chemical (St. Louis, MO).

Data are expressed as means ± SE of n experiments, where n is the number of dogs, arteries, or arterioles as indicated. The criterion for significance in all statistical tests was P < 0.05. One-way repeated-measures ANOVA was used to test the effects of H2O2 on coronary blood flow, heart rate, blood pressure, artery relaxation, and arteriole diameter. Two-way ANOVA was used to test the effects of H2O2 on blood flow between control dogs and dogs receiving K+ channel blockers. Two-way ANOVA also was used to test the effects of H2O2 on artery relaxation and arteriole diameter between control and treatment groups. Two-way repeated-measures ANOVA was used to test the effect of multiple concentration responses of H2O2 on arteriole diameter. When statistical significance was found with ANOVA, Tukey's post hoc multiple comparison test was used to determine differences between treatment levels. A paired (same subject before and after) t-test was used to compare baseline blood flow before and after addition of K+ channel blockers. A paired t-test also was used to determine the effect of K+ channel blockers on arteriole diameter and on the effect of DTT on arterial tension and arteriole diameter.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Intracoronary infusion of H2O2 increased blood flow in a dose-dependent manner (Fig. 1A). Heart rate and mean arterial blood pressure were monitored throughout the experiments and did not change significantly (105 ± 22 beats/min and 60 ± 7 mmHg), suggesting that with intracoronary infusion, the cardiovascular effects of H2O2 were limited to the coronary circulation. Because perfusion pressure was held constant, increases in blood flow represent reductions in coronary vascular resistance, i.e., smooth muscle relaxation. Indeed, H2O2 relaxed coronary artery rings contracted with 1 µM U46619 [GenBank] (Fig. 1B) and dilated coronary arterioles pressurized to 60 cmH2O (Fig. 1C) in a concentration-dependent manner.


Figure 1
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  		Fig. 1. H2O2 increases coronary blood flow, relaxes epicardial arteries, and dilates isolated arterioles. A: intracoronary infusion of H2O2 increased blood flow in a dose-dependent manner (n = 4). B and C: increasing concentrations of H2O2 relaxed left circumflex artery rings (B; n = 21) and dilated coronary arterioles (C; n = 6). Inset in B shows a representative isometric tension recording. The ring was contracted with 1 µM U46619 and relaxed in response to 3 concentrations of H2O2. Scale bar represents 2.5 g and 2 min.

 
H2O2-induced relaxation was studied in rings with and without intact endothelium and in the presence or absence of 20 µM indomethacin (Fig. 2A). Coronary artery rings denuded of endothelium did not relax in response to acetylcholine (0 ± 1%; n = 4). Neither endothelial denudation nor inhibition of cyclooxygenase altered H2O2-induced relaxation of artery rings (Fig. 2B). Similarly, the endothelium had no influence on H2O2-induced dilation of coronary arterioles (Fig. 2C), given that responses to H2O2 were not different in arterioles perfused briefly with 0.4% CHAPS (which reduced acetylcholine-induced dilation from 85 ± 8 to 6 ± 4%; n = 5). Intraluminal CHAPS treatment did not negatively affect smooth muscle viability, because active tone was similar in intact and denuded arterioles (Table 1).


Figure 2
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  		Fig. 2. H2O2 relaxes coronary vessels independently of the endothelium or cyclooxygenase products. A: representative isometric tension traces demonstrate H2O2-induced relaxation in left circumflex coronary artery rings. Scale bars represent 2.5 g and 2 min. B: group data (n = 6–8) for isometric tension experiments. Indo, indomethacin. There were no differences between the groups as assessed by two-way ANOVA. C: dilation of endothelium-denuded coronary arterioles (n = 5) was not significantly different from that of endothelium-intact arterioles (n = 6) as measured using 2-way ANOVA.

 

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  		Table 1. Characteristics of isolated coronary arterioles

 
These data suggest that H2O2 evokes coronary vasodilation by acting directly on vascular smooth muscle; however, the molecular effectors of H2O2 remain to be identified. Proteins are major targets of H2O2 signaling, especially reactive thiol groups (cysteine residues). Thus we determined whether DTT, a thiol-specific reducing agent, could reverse H2O2-induced smooth muscle relaxation. Left circumflex artery rings were contracted with 1 µM U46619 [GenBank] and relaxed with 100 µM H2O2, and then 1 mM DTT was added. DTT reversed H2O2-induced smooth muscle relaxation (Fig. 3A) but had no effect on papaverine-induced smooth muscle relaxation (Fig. 3B). The effect of the thiol reductant was to reverse H2O2-induced relaxation by approximately one-half (Fig. 3C). Increasing the DTT concentration to 10 mM completely reversed H2O2-induced relaxation (3 ± 7%; n = 6). Importantly, addition of DTT to vascular rings contracted with 1 µM U46619 [GenBank] did not alter the maximum active tension (6.5 ± 0.5 vs. 6.4 ± 0.4 g; n = 4). Furthermore, addition of DTT to vascular rings in the absence of contractile agonist did not effect passive tension (4.2 ± 0.1 vs. 4.0 ± 0.1 g before and after addition of 1 mM DTT, respectively; n = 6). DTT did not affect viability of the ring tissue, because 60 mM K+-induced contractions were identical before and after addition of the reductant (9.9 ± 1.1 vs. 9.9 ± 1.3 g tension, respectively; n = 6). These data indicate the effect of DTT is specific for H2O2-induced relaxation and suggest that thiol groups in smooth muscle are targets of H2O2 signaling.


Figure 3
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  		Fig. 3. Dithiothreitol (DTT) reverses H2O2-induced smooth muscle relaxation. A: representative isometric tension trace shows the effect of 1 mM DTT to reverse relaxation elicited by 100 µM H2O2. B: representative isometric tension trace demonstrates inability of 1 mM DTT to reverse relaxation in response to 50 µM papaverine. Scale bars in A and B represent 1 g and 3 min. C: group data (n = 10 and 15 in H2O2 and papaverine groups, respectively) for isometric tension experiments. *P < 0.05 as measured using paired t-test.

 
A series of experiments was performed to determine whether H2O2 concentration-response curves could be repeated in the same arteriole. H2O2 dilated coronary arterioles in a concentration-dependent manner (Fig. 4). Arterioles were washed for 20 min to regain tone (active diameter: 88 ± 9 vs. 88 ± 8 µm; n = 6), and then a second H2O2 concentration-response experiment was performed. Again, H2O2 elicited concentration-dependent dilation; however, the second response to H2O2 was significantly attenuated (Fig. 4). To determine whether thiol oxidation was responsible for the diminished responsiveness, we conducted a series of experiments in which arterioles were treated with a reducing agent before a third H2O2 concentration-response experiment was performed. A subset of the arterioles were washed 5 min with buffer containing 1 µM DTT (active diameter: 98 ± 7 vs. 99 ± 9 µm; n = 3), and a third H2O2 concentration-response experiment was performed. DTT restored the H2O2 concentration-response relationship, because the curve shifted back to the left and was not significantly different from the first H2O2 administration (Fig. 4).


Figure 4
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  		Fig. 4. Tachyphylaxis in H2O2-induced coronary arteriole dilation: reversal with DTT. Dilation in arterioles to a second H2O2 administration was significantly decreased. *P < 0.05 compared with control (first administration) as measured using 2-way repeated-measures ANOVA and Tukey's post hoc analysis. After a 5-min wash with 1 µM DTT, dilation to a third administration of H2O2 was significantly recovered to control levels. #P < 0.05 compared with second administration. There was no significant difference between the first and third concentration-response curves.

 
H2O2 reportedly dilates coronary arteries and arterioles by activating large-conductance, Ca2+-activated K+ (BKCa) channels (4, 47). Using high extracellular K+ concentration and K+ channel antagonists, we determined whether hyperpolarization and K+ channels mediate H2O2-induced relaxation of left circumflex coronary artery rings. When a high extracellular K+ concentration (60 mM) was used to contract artery rings and limit smooth muscle hyperpolarization, 100 µM H2O2 could not elicit relaxation (Fig. 5; n = 4). In contrast, 50 µM SNP, a nitric oxide donor, could relax rings contracted with 60 mM K+ (Fig. 5). TEA was used to assess the contribution of the K+ channels to H2O2-induced smooth muscle relaxation. At a concentration of 1 mM, TEA is reported to be relatively specific for BKCa channels and has little effect on voltage-dependent K+ channels (44); however, 1 mM TEA (Fig. 6A; n = 11) did not significantly inhibit relaxation to H2O2 (compared with control; n = 15). Higher concentrations of TEA (5 and 10 mM), which inhibit a variety of K+ channels including voltage-dependent K+ (KV) channels, reduced relaxation in response to H2O2 in a concentration-dependent manner (Fig. 6A, n = 20 and 10, respectively). We attempted similar experiments with 3 mM 4-AP, a known KV channel antagonist; however, addition of U46619 [GenBank] caused phasic oscillations that prevented analysis (21). Addition of TEA stopped the oscillations; therefore, we performed experiments in the presence of TEA and 4-AP and inferred the effect of 4-AP by comparing with TEA alone. This combination of K+ channel antagonists abolished H2O2-induced relaxation (Fig. 6A, n = 10). Active tension in the groups receiving 1, 5, and 10 mM TEA (4.2 ± 0.9, 9.3 ± 0.8, and 6.9 ± 0.9 g, respectively) were not significantly different from the control group (7.0 ± 0.7 g), with the exception of the group receiving TEA and 4-AP (11.4 ± 0.5 g). These data indicate that H2O2-induced smooth muscle relaxation depends on K+ channel activation and membrane hyperpolarization; however, the data with high concentrations of K+, TEA, and 4-AP do not indicate a specific type of K+ channel. Thus we determined the effect of 100 nM iberiotoxin and 0.3 mM 4-AP, more specific antagonists of K+ channels (BKCa and KV, respectively), to inhibit H2O2-induced dilation of coronary arterioles. Intense vasoconstriction was observed with the addition of 3 mM 4-AP, thereby precluding the ability to perform experiments using a higher concentration of 4-AP. Addition of iberiotoxin or 4-AP significantly decreased arteriole diameter (Table 1) as observed previously (5, 25), indicating effective channel block. H2O2-induced dilation of coronary arterioles was significantly inhibited by 4-AP but not iberiotoxin (Fig. 6B; n = 7 and 5, respectively). These data suggest that 4-AP-sensitive KV channels, not BKCa channels, mediate coronary vasodilation to H2O2.


Figure 5
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  		Fig. 5. High-K+ depolarization abolishes H2O2-induced relaxation. A: representative isometric tension tracing. The coronary artery ring was contracted with 60 mM K+. H2O2 (100 µM) failed to elicit relaxation, whereas addition of sodium nitroprusside (SNP; 50 µM) caused substantial relaxation. B: group data (n = 4) summarize the effects of H2O2 and SNP on K+-induced contractions. *P < 0.05 as measured using paired t-test for relaxation between H2O2 and SNP.

 

Figure 6
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  		Fig. 6. Effect of K+ channel antagonists to inhibit H2O2-induced smooth muscle relaxation. A: group data (n = 10–20) for concentration-response relationship and effects of tetraethylammonium (TEA) with or without 4-aminopyridine (4-AP). *P < 0.05 vs. control; #P < 0.05 vs. 1 mM TEA; $P < 0.05 vs. 5 mM TEA; &P < 0.05 vs. 10 mM TEA, as measured using 2-way ANOVA and Tukey's post hoc analysis. B: H2O2 elicited concentration-dependent dilation under control conditions (n = 6). Dilation was unaffected by 100 nM iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ channels (n = 5). H2O2-induced vasodilation was attenuated by 0.3 mM 4-AP (n = 6). *P < 0.05 as measured using 2-way ANOVA and Tukey's post hoc analysis.

 
Experiments were designed to determine whether 4-AP-sensitive channels mediate H2O2-induced coronary blood flow responses in vivo. We infused K+ channel blockers into the LAD perfusion cannula simultaneously with H2O2. Infusion of TEA plus 4-AP (calculated plasma concentrations of 5 and 3 mM, respectively) significantly decreased baseline coronary blood flow (46 ± 14%; n = 4) and abolished H2O2-induced coronary vasodilation (Fig. 7A). TEA alone significantly attenuated, but did not abolish, H2O2-induced coronary vasodilation (Fig. 7B; n = 4). At a calculated plasma concentration of 5 mM, TEA would be expected to inhibit BKCa and KV channels. Infusing 4-AP alone (calculated plasma concentration of 3 mM) significantly decreased baseline coronary blood flow (54 ± 10%; n = 4) and abolished H2O2-induced coronary vasodilation (Fig. 7C). These data suggest that K+ channels sensitive to 4-AP, not BKCa channels, mediate coronary vasodilation to H2O2.


Figure 7
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  		Fig. 7. K+ channel antagonists inhibit H2O2-induced coronary blood flow responses. A: the combination of TEA (calculated coronary plasma concentration = 5 mM) and 4-AP (calculated coronary plasma concentration = 3 mM) blocked H2O2-induced increases in coronary blood flow. B: TEA alone attenuated H2O2-induced increases in coronary blood flow. C: 4-AP alone blocked H2O2-induced increases in coronary blood flow. Each group contains data from 4 dogs. *P < 0.05 at specific H2O2 dose rates as determined using 2-way ANOVA and Tukey's post hoc analysis.

 
Last, we performed experiments using rat septal arteries under isometric tension conditions and cannulated, pressurized (60 cmH2O) rat coronary arterioles to determine whether the involvement of 4-AP-sensitive K+ channels in mediating H2O2-induced coronary vasorelaxation is specific to the dog coronary vasculature (Fig. 8). Segments of the main rat septal coronary artery, constricted with endothelin-1 (100 nM) or U46619 [GenBank] (1 µM; see METHODS) relaxed in a concentration-dependent manner in response to H2O2 (Fig. 8A; n = 15). Addition of increasing concentrations of 4-AP did not cause obvious oscillations in tension as occurred in dog coronary arteries. Relaxation to H2O2 was not inhibited in the presence of 0.3 mM 4-AP (Fig. 8A; n = 7) but was inhibited in a concentration-dependent manner to 3 and 10 mM 4-AP (Fig. 8A; n = 7 and 10, respectively). Active tensions in groups receiving 0.3, 3, and 10 mM 4-AP (0.46 ± 0.06, 0.54 ± 0.05, and 0.59 ± 0.06 g, respectively) were not significantly different from control responses (0.43 ± 0.07 g). H2O2 also dilated rat coronary arterioles in a concentration-dependent manner (Fig. 8B; n = 14). Dilation to H2O2 was inhibited by 4-AP (0.3 and 3 mM 4-AP, n = 4 and 4, respectively) in a concentration-dependent manner and also by addition of catalase (1,000 units; H2O2 scavenger) to the tissue bath (Fig. 8B; n = 10). Addition of 4-AP significantly constricted rat coronary arterioles; however, there was no significant difference in passive diameter, active diameter, and tone among groups (Table 1). Finally, addition of increasing concentrations of DTT (0.001–1 mM) following H2O2 (1 mM)-induced dilation of arterioles resulted in a significant reversal of vasodilation (Fig. 8C; n = 5).


Figure 8
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  		Fig. 8. H2O2-induced vasorelaxation of rat coronary arterioles is sensitive to 4-AP and DTT. A: 4-AP inhibited vasorelaxation of rat septal coronary artery segments (n = 7–15) in response to H2O2 in a concentration-dependent manner. *P < 0.05 vs. control; #P < 0.05 vs. 3 mM 4-AP as measured using 2-way ANOVA and Tukey's post hoc analysis. B: 4-AP inhibited vasodilation of cannulated, pressurized (60 cmH2O) rat septal coronary arterioles in response to H2O2 in a concentration-dependent manner. Addition of catalase (1,000 units) to the vessel bath also inhibited vasodilation. *P < 0.05 vs. control; #P < 0.05 vs. 0.3 mM 4-AP as measured using 2-way ANOVA and Tukey's post hoc analysis. C: addition of DTT to the vessel bath significantly reversed H2O2 (1 mM)-mediated vasodilation of rat coronary arterioles in a concentration-dependent manner. *P < 0.05 vs. H2O2 as measured using 1-way repeated-measures ANOVA and Tukey's post hoc analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our goal was to test the hypothesis that 4-AP-sensitive K+ channels mediate H2O2-induced coronary vasodilation. One major finding, which extends and validates previous in vitro studies with isolated arteries (4) and arterioles (45, 47), is that H2O2 increases coronary blood flow in vivo. A second novel finding, which differentiates our study from others that focused on iberiotoxin-sensitive BKCa channels (3, 4, 47), is that 4-AP-sensitive K+ channels mediate H2O2-induced coronary vasodilation. The third major finding of our study is that 4-AP-sensitive K+ channels or accessory regulatory proteins in coronary smooth muscle are modified in a redox-sensitive manner, similar to what has been observed for KV channels in pulmonary vascular smooth muscle (36). Together, these data suggest that H2O2 activates 4-AP-sensitive K+ channels in coronary smooth muscle to cause vasodilation and increase blood flow.

In contrast to previous studies suggesting that H2O2-induced vasodilation is mediated by the endothelium, cyclooxygenase, or nitric oxide (47, 56), we found no role for such mechanisms in H2O2-induced vasodilation. Rather, our data suggest the effect of H2O2 is mediated directly on the smooth muscle and does not involve cyclooxygenase. When coronary rings were denuded of endothelium or treated with indomethacin, H2O2-induced smooth muscle relaxation was unchanged. Similar to what was observed in epicardial coronary arteries, denuding coronary arterioles had no effect on H2O2-induced vasodilation. Our results with endothelium-denuded canine coronary arterioles agree with those of Miura et al. (38) in human coronary arterioles; however, our findings contradict those of Thengchaisri and Kuo (47). These latter investigators demonstrated that H2O2 dilates porcine coronary arterioles, in part, through the production of endothelial-derived factors, including cyclooxygenase metabolites. Our data argue for a direct effect of H2O2 on vascular smooth muscle (e.g., hyperpolarization through K+ channel activation). We also want to mention that coronary arterioles appear to be more sensitive to H2O2 than large arteries. This is not surprising in view of the myriad differences in arterial versus arteriolar reactivity in the coronary circulation (23).

Previous studies implicated Ca2+-activated K+ (KCa) channels, specifically BKCa, as mediators of H2O2-induced smooth muscle relaxation (3, 38, 47); however, ATP-dependent K+ channels also may serve as targets for H2O2 in smooth muscle (15, 29). Less attention has been devoted to the role of 4-AP-sensitive KV channels in H2O2-induced smooth muscle relaxation (10). Regardless of the specific K+ channel type(s) involved, the resulting H2O2-induced smooth muscle hyperpolarization would lead to relaxation through indirect inhibition of L-type Ca2+ channels (39). L-type Ca2+ channels have been reported to be inhibited by oxidants (19); however, such a mechanism is unlikely to explain our results with H2O2. The data with high-K+ contractions (Fig. 5) show that H2O2 does not directly inhibit L-type Ca2+ channels, because K+-induced contractions are readily reversed by L-type Ca2+ channel blockers whereas H2O2 has no effect. Furthermore, H2O2 does not poison or nonspecifically damage arteries to cause relaxation, because K+-induced contraction is sustained in the presence of 100 µM H2O2. Thus the data suggest that H2O2-induced smooth muscle relaxation is mediated by membrane hyperpolarization, which could be produced by K+ channel activation [or other electrogenic mechanisms such as the Na+/K+-ATPase (22)].

To determine whether K+ channels are involved in H2O2-induced smooth muscle relaxation, we performed a variety of experiments using TEA, 4-AP, and iberiotoxin. TEA is a relatively specific blocker of BKCa at a concentration of 1 mM, a concentration that is five times the half-block constant of BKCa (5, 30). This concentration had no effect on H2O2-mediated relaxation of coronary rings. Similarly, 100 nM iberiotoxin, a specific inhibitor of BKCa channels, had no effect on H2O2-induced dilation of coronary arterioles. Effective block of BKCa channels by iberiotoxin was evidenced by significant vasoconstriction (5). Increasing the concentration of TEA to 5 and 10 mM, concentrations that block additional K+ channel types [e.g., 10 mM inhibits KV current by ~50% (44)], significantly attenuated H2O2-induced coronary artery relaxation. Experiments with 3 mM 4-AP alone in coronary artery rings were not possible, because phasic oscillations in tension were observed, as previously reported (12, 21, 42). Addition of 10 mM TEA stabilized the 4-AP-induced oscillations, and coronary artery rings treated with the combination of 4-AP and TEA did not relax to H2O2. Similarly, in arterioles, 3 mM 4-AP caused dramatic oscillations in diameter as well as very intense vasoconstriction; therefore, to complete the study, we had to use 0.3 mM 4-AP. This concentration of 4-AP elicited significant vasoconstriction and attenuated H2O2-induced vasodilation. We also performed experiments using rat coronary vessels to determine whether this observation applied to other species besides dogs. Indeed, addition of 4-AP inhibited vasorelaxation and vasodilation of rat coronary arteries and arterioles, respectively, in response to H2O2 in a concentration-dependent manner, indicating this property of targeting 4-AP-sensitive K+ channels by H2O2 may be shared across species.

Our findings contrast with recent investigations of Miura et al. (38), Barlow and White (3, 4), and Thengchaisri and Kuo (47). These investigators concluded that H2O2-induced smooth muscle relaxation involves Ca2+-activated K+ channels. Miura et al. (38) used a combination of the peptide channel blockers charybdotoxin and apamin. Charybdotoxin has been reported to inhibit a variety of K+ channels, including small-conductance KCa (SKCa) (18), BKCa (37), and various homomultimeric KV channels (11, 32). It is important to note that vascular smooth muscle cells express heteromultimeric channels (24, 42, 49). Differences in vascular bed studied and species also could explain the observed difference, because their arterioles were acquired from human right atrial tissue. The other investigators mentioned concluded that BKCa channels were involved in porcine coronary artery and arteriolar smooth muscle relaxation in response to H2O2 because iberiotoxin attenuated H2O2-induced responses (3, 4, 47). However, our results strongly suggest that H2O2-mediated vasodilation in the canine coronary vasculature is mediated via activation of K+ channels sensitive to 4-AP but not iberiotoxin (or low concentrations of TEA). Our results also suggest that this property of targeting channels sensitive to 4-AP is also observed in the rat coronary vasculature.

Because the most sensitive cellular protein target of H2O2 is thiol residues, we questioned whether thiol oxidation of a cellular target could regulate H2O2 vasodilation. Relaxation to H2O2 in left circumflex rings was significantly reversed by DTT, a thiol-reducing agent. DTT failed to reverse papaverine-induced relaxation, suggesting that thiol oxidation is specific to H2O2 and not a prerequisite for all vasodilators. Similarly, DTT reversed H2O2-induced vasodilation in a concentration-dependent manner in rat coronary arterioles. In arterioles studied with repeated administration of H2O2 dose-response curves, a diminished response was noted during the second dose response. We hypothesized that perhaps this represented a state in the vessel where sufficient reduction of oxidized thiol groups that led to the first vasodilatory response had not yet occurred, i.e., reduction via glutathione and glutathione reductase. Briefly bathing the vessel with DTT restored vessel response to H2O2, supporting this hypothesis. Although the generation of a diminished vessel response to H2O2 upon repeated exposure could argue against a significant physiological role for H2O2 as an important vasodilator, we speculate that this could be due to increased cellular oxidative stress as a by-product of the in vitro preparation (e.g., dissection of the microvessel, heating/cooling). Nevertheless, this indicates that H2O2 oxidizes a vascular thiol target activating 4-AP-sensitive K+ channels, leading to subsequent relaxation. Importantly, this also shows that H2O2 oxidizes its target to a reversible state. Reversal of an activated signaling pathway is an important quality found in physiological signaling schemes. Several electrophysiological studies suggest that H2O2 targets a cysteine residue on the modulatory beta-subunit of 4-AP-sensitive KV channels. Wang et al. (51) found that homomultimeric KV1.2 became much more sensitive to H2O2 when it was coexpressed with KVbeta1.3. Rettig et al. (43) found a specific cysteine residue in KVbeta1 that conferred redox-sensitivity to the channel. Mutation of this cysteine residue in the NH2 terminus eliminated channel activation by H2O2.

Finally, we performed an important series of experiments to determine whether our findings in left circumflex rings and isolated arterioles were relevant to the in vivo response to H2O2 administration. Simultaneous infusion of both channel blockers (TEA and 4-AP) abolished H2O2-induced increases in coronary blood flow, supporting the observations made in the left circumflex rings. Infusion of TEA alone significantly attenuated H2O2 effects, but infusion of 4-AP alone abolished increases in coronary blood flow. These data indicate that H2O2 activates 4-AP-sensitive K+ channels in vivo to decrease coronary vascular resistance. We interpret the partial block of H2O2 induced effects by TEA likely as partial inhibition of 4-AP-sensitive channels. The finding that 4-AP abolished relaxation in response to H2O2 in large canine coronary arteries but only attenuated responses in coronary arterioles could indicate the presence of an additional dilator mechanism in coronary arterioles. However, another likely explanation for the residual dilation in isolated coronary arterioles could be due to the required lower concentration of 4-AP (0.3 mM) needed to perform the experiments. Reported 4-AP concentrations needed to half block vascular smooth muscle KV channels, depending on tissue bed and species, are 0.2–1.1 mM (39). This explanation is supported by the in vivo data, because infusion of 4-AP (3 mM) alone effectively eliminated H2O2-induced blood flow responses.

The role of H2O2 as an important physiological signaling molecule is recently gaining more acceptance. The problem lies in regard to a lack of overwhelming evidence of target specificity that can only be overcome by further experimentation. It is clear, though, that H2O2 can interact with thiol moieties, particularly the thiolate anion (RS–), of protein cysteine residues. H2O2 also can interact with iron centers, such as the heme groups of enzymes like cyclooxygenase (28) and catalase (6, 53). It also should be noted that in some heme centers, the fifth coordination site for iron is a thiolate anion of a cysteine residue (7, 13). H2O2 has been reported to react with thiolate anions at rates of 10–105 M–1·s–1 (9). This reaction typically forms sulfinic acid (SOH) or its ionized form, the sulfenate anion (SO–). The oxidized thiol group also can interact with nearby cysteine residues to form a disulfide bridge. Oxidized thiol intermediates can be restored to the thiolate anion by either glutathione- or thioredoxin-mediated reactions. Redox signaling via H2O2 has been evaluated in various systems, including activation of mitogen-activated protein kinases (1) and protein tyrosine kinases (27) and inhibition of protein tyrosine phosphatases (16). With direct importance, H2O2 has been shown to activate soluble guanylate cyclase in lung extracts (52) and, through its catabolism by catalase, in bovine pulmonary artery smooth muscle (53). H2O2 may indeed interact directly with ion channels; however, we cannot preclude possible modification of associated signaling regulators. 4-AP-sensitive KV channels have been shown to be regulated via cGMP and cAMP pathways (2, 20), protein tyrosine kinases (40), and protein tyrosine phosphatases (33).

In conclusion, our data suggest that 4-AP-sensitive K+ channels serve as effective targets for H2O2. Importantly, H2O2 does not require endothelial elements but acts directly on vascular smooth muscle. Furthermore, protein thiol groups are targeted, thus suggesting that smooth muscle K+ channels or proteins that regulate them are redox-sensitive elements that are activated via oxidation to dilate the coronary vasculature and increase blood flow. A limitation of this study is that it is a purely pharmacological investigation; therefore, we cannot conclude with certainty the exact K+ channel type being activated. Further studies are needed to delineate the electrophysiological effects of H2O2 on coronary vascular smooth muscle cells. Importantly, it remains to be determined whether specifically 4-AP-sensitive K+ channels or associated regulatory/signaling proteins are oxidized by H2O2.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Health (NIH) Grants HL-32788, HL-65203, and HL-73755 (to W. Chilian), the Louisiana State University Center of Biomedical Research Excellence, and NIH Grants P20 RR018766 (to G. M. Dick) and HL-67804 (to J. D. Tune). P. Rogers was supported by a Predoctoral Fellowship from the American Heart Association-Southeast Affiliate.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Rogers, Dept. of Physiology, Louisiana State Univ. Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112 (e-mail: proger{at}lsuhsc.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.


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