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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1774.)
© 2008 American Heart Association, Inc.

Integrative Physiology/Experimental Medicine

Hydrogen Peroxide Potentiates the EDHF Phenomenon by Promoting Endothelial Ca²⁺ Mobilization

David H. Edwards; Yiwen Li; Tudor M. Griffith

From the Wales Heart Research Institute, School of Medicine, Cardiff University, UK.

Correspondence to Tudor Griffith, Department of Diagnostic Radiology, Wales Heart Research Institute, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. E-mail griffith{at}cardiff.ac.uk

	Abstract

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Objective— The purpose of this study was to test the hypothesis that H₂O₂ contributes to the EDHF phenomenon by mobilizing endothelial Ca²⁺ stores.

Methods and Results— Myograph studies with rabbit iliac arteries demonstrated that EDHF-type relaxations evoked by the SERCA inhibitor cyclopiazonic acid (CPA) required activation of K_Ca channels and were potentiated by exogenous H₂O₂ and the thiol oxidant thimerosal. Preincubation with a submaximal concentration of CPA unmasked an ability of exogenous H₂O₂ to stimulate an EDHF-type response that was sensitive to K_Ca channel blockade. Imaging of cytosolic and endoplasmic reticulum [Ca²⁺] in rabbit aortic valve endothelial cells with Fura-2 and Mag-fluo-4 demonstrated that H₂O₂ and thimerosal, which sensitizes the InsP₃ receptor, both enhanced CPA-evoked Ca²⁺ release from stores, and that the potentiating effect of H₂O₂ was suppressed by the cell-permeant thiol reductant glutathione monoethylester. CPA-evoked relaxations were attenuated by exogenous catalase and potentiated by the catalase inhibitor 3-aminotriazole, and were abolished by the connexin-mimetic peptide ⁴³Gap26, which interrupts intercellular communication via gap junctions constructed from connexin 43.

Conclusions— H₂O₂ can enhance EDHF-type relaxations by potentiating Ca²⁺ release from endothelial stores, probably via redox modification of the InsP₃ receptor, leading to the opening of hyperpolarizing endothelial K_Ca channels and an electrotonically-mediated relaxant response.

Pharmacological analysis of relaxation and imaging of intracellular Ca²⁺ mobilization has shown that H₂O₂ can contribute to electrotonically-mediated EDHF-type relaxations by promoting Ca²⁺ release from stores and secondary opening of K_Ca channels. The thiol oxidant thimerosal mimics this previously unrecognized endothelial action of H₂O₂, consistent with sensitization of the InsP₃ receptor.

Key Words: hydrogen peroxide • thimerosal • SERCA pump • EDHF

	Introduction

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The endothelium regulates arterial tone through the release of nitric oxide (NO) and vasodilator prostanoids and an NO/prostanoid-independent mechanism that involves smooth muscle hyperpolarization. Some workers have attributed this electrical response to a freely diffusible endothelium-derived hyperpolarizing factor (EDHF), whose identity has variously been proposed as H₂O₂, K⁺ ions or epoxyeicosatrienoic acid metabolites of arachidonic acid.^1,2 Definitive classification of underlying mechanisms has nevertheless been obscured by the existence of species- and vessel-specific differences in the contribution of such factors to relaxation. Indeed, it is now widely recognized that increases in endothelial [Ca²⁺]_i underpin the EDHF phenomenon by promoting the opening of Ca²⁺-activated K⁺ channels (K_Ca),² and there is evidence that the resulting hyperpolarization can then spread electrotonically through the vascular wall via myoendothelial and homocellular smooth muscle gap junctions to promote relaxation.³ Agents that elevate endothelial [Ca²⁺]_i by stimulating the generation of InsP₃ or activate store-operated Ca²⁺ entry (SOCE) by blocking Ca²⁺ sequestration via the endoplasmic reticulum SERCA pump, may thus evoke hyperpolarization and "EDHF-type" relaxations that can be suppressed either by pharmacological blockade of K_Ca channels or gap junctional communication.^3,4

See accompanying article on page 1691

In some artery types, there is evidence that exogenous H₂O₂ can evoke smooth muscle relaxation by activating hyperpolarizing BK_Ca channels and that EDHF-type relaxations and hyperpolarizations are associated with catalase-inhibitable endothelial H₂O₂ production, leading to the hypothesis that endogenously-generated H₂O₂ can serve as a freely diffusible EDHF.¹ By contrast, in human mesenteric arterioles endothelium-dependent agonists promote H₂O₂-dependent relaxation, but exogenous H₂O₂ causes constriction when such vessels are denuded of their endothelium, leading to the proposal that H₂O₂ releases a chemically distinct EDHF.⁵ A further permutation, not previously described in the context of the EDHF phenomenon, is that H₂O₂ promotes Ca²⁺ release from intracellular stores^6,7 thereby elevating [Ca²⁺]_i directly and enhancing SOCE, and contributing to a conducted hyperpolarizing response through the activation of endothelial K_Ca channels. To examine this scenario, we have therefore investigated the role of H₂O₂ in EDHF-type relaxations of the rabbit iliac artery (RIA), a vessel in which H₂O₂ cannot be regarded as a transferable EDHF because smooth muscle hyperpolarizations evoked by exogenous H₂O₂ are much smaller than those associated with the authentic EDHF phenomenon.⁸ Because there is evidence that H₂O₂ depletes Ca²⁺ stores by sensitizing the InsP₃ receptor, thereby mimicking the effects of agents that oxidize thiol groups such as the organic mercurial thimerosal,^6,7,9–12 we compared the effects of H₂O₂ and thimerosal on EDHF-type relaxations and Ca²⁺ mobilization, using fura-2 and the low affinity Ca²⁺ probe Mag-fluo-4, which selectively loads the endoplasmic reticulum (ER), to monitor [Ca²⁺]_i and [Ca²⁺]_ER in the rabbit aortic valve (RAV) endothelium.^13,14 This preparation was selected for Ca²⁺ imaging studies because H₂O₂ and thimerosal can both elevate vascular smooth muscle [Ca²⁺]_i, and in intact vessels Ca²⁺ signals can be transmitted from the media to the endothelium via myoendothelial gap junctions.^12,15,16 To avoid potentially confounding effects of H₂O₂ on receptor-coupled pathways mediated via phospholipase C (PLC),¹⁷ responses were evoked by the SERCA inhibitor cyclopiazonic acid (CPA). Although native arterial endothelial cells are widely recognized to express small- and intermediate-conductance SK_Ca and IK_Ca channels, also classified as SK1–3 and SK4 on the basis of structural and gating characteristics,^2,18–21 in some artery types the endothelium also expresses functional large-conductance BK_Ca channels.^22–26 The contributions of the 3 channel subtypes to EDHF-type relaxations were therefore investigated with selective SK_Ca, IK_Ca, and BK_Ca inhibitors (apamin, TRAM-34 and iberiotoxin, respectively), and the dominant role of electrotonic signaling confirmed with a synthetic peptide (⁴³Gap26) that blocks intercellular communication via gap junctions constructed from connexin 43.^13,27,28

	Methods

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Full details are provided in the supplemental materials (available online at http://atvb.ahajournals.org). Mechanical responses were studied in myograph-mounted iliac artery rings obtained from male NZW rabbits and incubated in Holmans buffer. In some experiments the endothelium was removed by gentle abrasion. To study endothelial Ca²⁺ mobilization rabbit aortic valve leaflets were isolated and loaded either with Fura-2 AM to assess [Ca²⁺]_i or the low-affinity Ca²⁺ indicator mag-fluo-4 to assess [Ca²⁺] in endothelial ER stores. Leaflets were imaged either with an inverted microscope to obtain background-corrected F_340/380 ratios and calculate [Ca²⁺]_i, or confocal microscopy to track the effects of interventions on Mag-fluo-4 fluorescence with changes in [Ca²⁺]_ER being assessed as fluorescence normalized to its value at the beginning of each experiment (F/F₀). All experiments were performed in the presence of L-NAME (300 µmol/L) and indomethacin (10 µmol/L) to inhibit the production of NO and prostanoids. Maximal percentage reversal of phenylephrine-induced constriction (R_max) by CPA or H₂O₂ and concentrations giving 50% reversal of this constrictor response (IC₅₀; in the case of CPA) or 50% of maximal relaxation (EC₅₀; in the case of H₂O₂) were determined. The use of IC₅₀ was necessary to allow for a small initial constriction to CPA that was observed in many experiments. All data are presented as mean±SEM and were compared by the Student t test or ANOVA followed by an appropriate post test. P<0.05 was considered significant; n denotes the number of animals studied for each data point.

	Results

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Mechanical Responses to CPA
In endothelium-intact rings constricted by phenylephrine (1 µmol/L), CPA-evoked relaxations were maximally equivalent to 85.6±1.4% of the constrictor response to phenylephrine (R_max), although the threshold for relaxation was variable, being 10 or 30 µmol/L in different preparations, with an overall IC₅₀ of 16 µmol/L (pIC₅₀ 4.79±0.02, n=54; data pooled from supplemental Table I). Relaxation was often preceded by small increases in tension (up to 10% at 3 µmol/L CPA), which are likely to reflect reduced buffering of Ca²⁺ influx following blockade of the smooth muscle SERCA pump.²⁸

Effects of H₂O₂ and Thimerosal
Preincubation of endothelium-intact RIA rings with 100 µmol/L H₂O₂ markedly potentiated relaxations evoked by CPA at concentrations of 1 to 10 µmol/L, without alteration in overall R_max (Figure 1A; supplemental Table I). Similarly, 100 µmol/L H₂O₂ potentiated the increases in [Ca²⁺]_i evoked by 1 to 10 µmol/L CPA in the endothelium of the RAV, but did not affect Ca²⁺ mobilization in response to 30 and 100 µmol/L CPA and minimally elevated basal [Ca²⁺]_i (Figure 1B). In the presence of 100 µmol/L H₂O₂ the increase in [Ca²⁺]_i evoked by 10 µmol/L CPA attained the same level observed with 30 µmol/L CPA and was not affected by the order in which these agents were administered (Figure 1B). Preincubation with GSH-MEE (1 mmol/L) blocked the ability of H₂O₂ to potentiate Ca²⁺ mobilization by 10 µmol/L CPA, but did not affect the control response to CPA (Figure 1B).

View larger version (11K): [in this window] [in a new window]   Figure 1. Effects of 100 µmol/L H2O2 on CPA-evoked responses. A, Potentiation of RIA relaxation (n=6). B, Synergistic elevation of RAV [Ca2+]i by H2O2 and 10 µmol/L CPA, whereas H2O2 was ineffective after 30 µmol/L CPA. Bar graphs confirm potentiation of RAV endothelial Ca2+ mobilization and its prevention by GSH-MEE (n=4 to 6). *P<0.05, **P<0.01 compared with corresponding CPA control.

Preincubation with 10 µmol/L thimerosal caused a pronounced leftward shift in the concentration-relaxation curve for CPA in endothelium-intact rings with potentiation again being evident over the range 1 to 10 µmol/L but R_max unaltered (Figure 2A; supplemental Table I). Thimerosal did not itself evoke relaxation at concentrations 10 µmol/L, but at concentrations 30 µmol/L induced a triphasic response consisting of an endothelium-dependent relaxation superimposed on a biphasic direct smooth muscle response in which constriction preceded relaxation (Figure 2B). At the subthreshold concentration of 10 µmol/L, thimerosal potentiated increases in [Ca²⁺]_i evoked by CPA over the range 1 to 10 µmol/L in the RAV endothelium, but not at 30 or 100 µmol/L CPA (Figure 2C). High concentrations of thimerosal themselves increased RAV [Ca²⁺]_i with an EC₅₀ of 60 µmol/L (pEC₅₀ 4.25±0.12, n=4) with no elevation being evident at concentrations 10 µmol/L (Figure 2D).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of thimerosal. A, Potentiation of CPA-evoked RIA relaxation by 10 µmol/L thimerosal (n=8). B, Endothelium-dependent and -independent relaxant effects of thimerosal (n=9). C, Concentration-dependent elevation of RAV [Ca2+]i by CPA in the presence and absence of 10 µmol/L thimerosal (n=7). D, Direct mobilization of Ca2+ by thimerosal in the RAV (n=4). *P<0.05, **P<0.01, and ***P<0.001 compared with corresponding control.

Mag-fluo-4 fluorescence in the endoplasmic reticulum of the RAV was decreased by 10 and 30 µmol/L CPA in a concentration-dependent fashion (Figure 3A through 3C). In the presence of 100 µmol/L H₂O₂ or 10 µmol/L thimerosal, depletion of the ER Ca²⁺ store by 10 µmol/L CPA increased to a level that was statistically similar to that observed with 30 µmol/L CPA alone (Figure 3B and 3C). Neither 100 µmol/L H₂O₂ nor 10 µmol/L thimerosal affected ER fluorescence in the absence of CPA.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effects of 100 µmol/L H2O2 and 10 µmol/L thimerosal on CPA-evoked depletion of ER Ca2+ stores loaded with Mag-fluo-4. A, Confocal images showing that H2O2 itself did not affect [Ca2+]ER but facilitated store depletion in the presence of 10 µmol/L CPA. B, Traces illustrating that the effects of H2O2 and CPA were synergistic. Changes in [Ca2+]ER are expressed as relative fluorescence ratio, F/Fo. C, Bar graphs showing that store emptying by CPA was potentiated to an equivalent extent by H2O2 and thimerosal (n=4 in each case). *P<0.05, **P<0.01 compared with 10 µmol/L CPA alone.

Mechanisms Contributing to CPA-Evoked Relaxation
CPA-evoked relaxations were unaffected by apamin (1 µmol/L) or TRAM-34 (10 µmol/L) individually, whereas R_max was reduced to 60% by preincubation either with the double combination apamin+TRAM-34 or iberiotoxin (IbTX, 100 nmol/L), and decreased to 35% in the presence of the triple combination apamin+TRAM-34+IbTX (Figure 4A; supplemental Table I). This residual relaxation was further attenuated to 20% by catalase (2000 U/mL). Control relaxations were almost abolished by the connexin-mimetic peptide ⁴³Gap26 (100 µmol/L; Figure 4B; supplemental Table I).

View larger version (28K): [in this window] [in a new window]   Figure 4. Mechanisms contributing to CPA-evoked relaxation. A, Combinatorial effects of apamin, TRAM-34 and iberiotoxin (n=4 to 25). Residual responses observed in the presence of apamin+TRAM-34+IbTX were further attenuated by catalase (n=9). B, Responses were effectively abolished by 43Gap 26 (n=4). C and D, Differential effects of catalase and apamin+TRAM-34+IbTX according to whether the threshold for relaxation was 10 or 30 µmol/L CPA (n=4 to 10). E, Relaxation was potentiated by 3-aminotriazole (n=6). F, U-73122 did not impair the potentiating effects of H2O2 on relaxation (n=5). *P<0.05, **P<0.01, ***P<0.001 compared with control (see also supplemental Table I).

Subanalysis showed that the ability of catalase to depress relaxation in individual rings correlated with their sensitivity to CPA, with inhibition being observed where the threshold was 10 µmol/L, but not in rings where the threshold for relaxation was 30 µmol/L (Figure 4C and 4D; supplemental Table I). In rings exhibiting a threshold at 30 µmol/L CPA, apamin+TRAM-34 or iberiotoxin effectively abolished relaxation, whereas in those with the lower threshold of 10 µmol/L, combined K_Ca channel blockade reduced R_max to 60% and this was further reduced by catalase to 40%. The catalase inhibitor 3-aminotriazole (ATZ, 50 mmol/L) selectively potentiated relaxations to 10 µmol/L CPA without affecting R_max (Figure 4E; supplemental Table I). Control CPA-evoked relaxations and their potentiation by 100 µmol/L H₂O₂ were unaffected by the PLC inhibitor U-73122 (10 µmol/L) (Figure 4F; supplemental Table I).

Relaxation to Exogenous H₂O₂
Concentration-relaxation curves for H₂O₂ were similar in rings with and without endothelium and were unaffected by IbTX, apamin+TRAM-34 (data not shown) or apamin+TRAM-34+IbTX in combination (Figure 5A; supplemental Table II). By contrast, in endothelium-intact rings preincubated with 10 µmol/L CPA, concentration-relaxation curves for H₂O₂ were shifted to the left with a small increase in R_max (Figure 5B; supplemental Table II), whereas no potentiation was seen in endothelium-denuded rings (data not shown). The potentiating effects of CPA in rings with endothelium were attenuated by IbTX, the combination of apamin+TRAM-34, and the triple combination apamin+TRAM-34+IbTX (Figure 5B; supplemental Table II), thus confirming the participation of the three K_Ca channel subtypes.

View larger version (34K): [in this window] [in a new window]   Figure 5. Interactive effects of CPA and KCa inhibitors on H2O2-evoked relaxation. A, Relaxant effects of H2O2 were unaffected by apamin+TRAM-34+iberiotoxin or endothelial denudation (n=5 to 8). B, Potentiation of H2O2-evoked relaxation by 10 µmol/L CPA in endothelium-intact rings was attenuated by iberiotoxin, apamin+TRAM-34, or apamin+TRAM-34+iberiotoxin (n=5 to 20). *P<0.05, **P<0.01, and ***P<0.001 compared with control (see also supplemental Table II).

	Discussion

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The principal finding of the present study is that H₂O₂ can promote EDHF-type relaxations of rabbit arteries via an endothelium-dependent mechanism that is distinct from its more widely-recognized smooth muscle action. The endothelial component of the response to H₂O₂ was shown to involve enhanced Ca²⁺ release from stores with secondary activation of K_Ca channels. H₂O₂ may thus contribute to relaxations mediated by the spread of endothelial hyperpolarization via gap junctions, rather than acting as a freely transferable EDHF, because its smooth muscle relaxing effects were insensitive to K_Ca channel blockade.

Preincubation of RIA rings with 100 µmol/L H₂O₂ markedly potentiated EDHF-type relaxations evoked by CPA when this SERCA inhibitor, which activates SOCE by depleting ER Ca²⁺ stores, was administered at concentrations 10 µmol/L. Correspondingly, preincubation of RAV leaflets with 100 µmol/L H₂O₂ amplified ER emptying and elevations in [Ca²⁺]_i evoked by CPA at concentrations 10 µmol/L, thus matching the range over which H₂O₂ potentiated relaxation. Because 100 µmol/L H₂O₂ did not itself induce changes in [Ca²⁺]_ER, this synergism suggests that H₂O₂ sensitizes the InsP₃ receptor. Indeed, heparin, an established antagonist of this receptor, abolishes H₂O₂-evoked Ca²⁺ release from ER stores in permeabilized endothelial cells,⁶ whereas the endothelium-specific SERCA3 pump isoform, which plays a key role in endothelium-dependent relaxation, is insensitive to H₂O₂, in contrast to the SERCA2b isoform found in smooth muscle.^29,30 Whereas millimolar concentrations of H₂O₂ can promote Ca²⁺ release from mitochondria or depress extrusion of cytosolic Ca²⁺ by the plasma membrane Ca²⁺ ATPase,^6,31–33 the participation of these ER-independent mechanisms was excluded by observations that (1) 100 µmol/L H₂O₂ minimally affected [Ca²⁺]_i and (2) relaxation and Ca²⁺ mobilization were maximal at CPA concentrations of 30 to 100 µmol/L and not further increased by 100 µmol/L H₂O₂. The possibility that the Ca²⁺ mobilizing effects of H₂O₂ might involve activation of PLC and enhanced InsP₃ synthesis¹⁷ was excluded by the demonstration that the PLC inhibitor U-73122 did not affect the potentiation of CPA-evoked relaxation by H₂O₂. This is consistent with reports that H₂O₂ does not increase endothelial InsP₃ synthesis, even at millimolar concentrations, and that U-73122 does not modulate H₂O₂-evoked Ca²⁺ mobilization in other cell types.^6,7,31,32

The generality of these conclusions was substantiated by experiments with thimerosal, which is known to sensitize the InsP₃ receptor to InsP₃ and Ca²⁺ via the oxidation of critical thiol groups,^9–12,32 and whose ability to facilitate intracellular Ca²⁺ mobilization has been dissociated from alterations in InsP₃ synthesis,^9,10 SERCA activity,¹¹ mitochondrial Ca²⁺ release^11,32 and blockade of the membrane extrusion Ca²⁺ ATPase.³² At concentrations 30 µmol/L, thimerosal itself evoked EDHF-type relaxations in the RIA and elevated basal [Ca²⁺]_i in the RAV endothelium. However, a subthreshold concentration (10 µmol/L) that did not affect [Ca²⁺]_i or [Ca²⁺]_ER mimicked the effects of H₂O₂ by potentiating relaxations and Ca²⁺ mobilization evoked by CPA at concentrations 10 µmol/L, but not further elevating [Ca²⁺]_i when stores were depleted by 30 or 100 µmol/L CPA. Evidence that oxidation of thiol groups similarly underpins the response to H₂O₂ was obtained in experiments showing that the cell permeant thiol reductant GSH-MEE abolished the synergy between H₂O₂ and CPA in RAV leaflets without compromising the ability of CPA to elevate [Ca²⁺]_i. While the precise molecular effects of H₂O₂ on the InsP₃ receptor remain to be delineated, it possesses a number of accessible cysteine residues that are susceptible to oxidative modification.³⁴ More specifically, thimerosal induces a conformational change in the N terminus of the receptor with the interaction between amino acids 1 to 225 (suppressor domain) and amino acids 226 to 604 (InsP₃-binding core) being strengthened interactively by Ca²⁺ and thimerosal, leading to the formation of a highly InsP₃-sensitive Ca²⁺-release channel.¹²

Thimerosal also promotes the formation of epoxyeicosatrienoic acid (EET) metabolites of CYP₄₅₀ epoxygenases by inhibiting acyl-coenzyme A (CoA)/lysolecithin acyltransferase, and in some species EETs may function as transferable EDHFs that activate smooth muscle BK_Ca channels.^2,35 In rabbit arteries, however, exogenously-administered EETs fail to relax endothelium-denuded preparations directly, thus excluding a role as an EDHF, whereas in preparations with endothelium they evoke indirect EDHF-type relaxations that involve signaling via gap junctions.^21,23,36 Endogenous EET production is nevertheless unlikely to contribute to the ability of thimerosal or H₂O₂ to potentiate the CPA-evoked Ca²⁺ mobilization demonstrated in the present study because (1) rabbit endothelial cells normally do not synthesize EETs,³⁷ (2) EETs elevate endothelial [Ca²⁺]_i by stimulating Ca²⁺ influx via TRP channels³⁸ rather than promoting intracellular Ca²⁺ release, and (3) H₂O₂ impairs EET synthesis by directly inhibiting CYP₄₅₀ epoxygenases.³⁹

The connexin-mimetic peptide ⁴³Gap 26 effectively abolished CPA-evoked relaxations, thus confirming the primarily electrotonic nature of the EDHF phenomenon in the RIA.^13,27,28 This peptide possesses homology with the first extracellular loop of Cx43, the dominant connexin expressed in the media of the RIA, and interrupts the spread of endothelial hyperpolarization via homocellular smooth muscle gap junctions without impairing CPA-evoked endothelial hyperpolarization.^13,27 Experiments with selective inhibitors of SK_Ca, IK_Ca, and BK_Ca channels provided evidence that all three subtypes contribute to this initiating hyperpolarization. Thus, apamin and TRAM-34, which block SK_Ca and IK_Ca channels, were individually ineffective against relaxation, but combined SK_Ca/IK_Ca blockade with apamin+TRAM-34 or BK_Ca blockade with iberiotoxin significantly reduced relaxation, and further inhibition was evident with the triple combination apamin+TRAM-34+iberiotoxin, leaving a residual response equivalent to 35% of control. It should be noted that considerable heterogeneity in the endothelial expression of these K_Ca subtypes and their functional contribution to the EDHF phenomenon across species and vessels is evident in the literature.^2,3,18–26 In the rabbit mesenteric artery, for example, EDHF-type relaxations and hyperpolarizations are insensitive to iberiotoxin,^20,21 whereas in the rabbit renal artery relaxation is partially inhibited by iberiotoxin and abolished by the combination of apamin and iberiotoxin, even though apamin alone is without effect.²³ Iberiotoxin also attenuates relaxation in 1st order rat mesenteric arteries, but is inactive in 3rd order branch arteries.²⁶ Indeed, in 3rd order arteries endothelium-dependent smooth muscle hyperpolarizations are abolished by apamin under resting conditions and therefore entirely attributable to the opening of SK_Ca channels, but when depolarized by phenylephrine the combination of apamin+TRAM-34 is necessary to abolish relaxation, suggesting a specific role for IK_Ca channels during repolarization.^19,26

Evidence that endogenously-generated H₂O₂ may contribute to EDHF-type relaxations was provided by experiments with catalase, which attenuated relaxation in RIA rings responsive to 10 µmol/L CPA, but was without effect in preparations where the threshold for relaxation was 30 µmol/L CPA. Correspondingly, 3-aminotriazole, which inhibits H₂O₂ degradation by binding to the active site of catalase,⁴⁰ significantly amplified EDHF-type relaxations evoked by CPA at 10 µmol/L, but not at other concentrations. These results are consistent with the finding that exogenous H₂O₂ potentiated EDHF-type relaxations evoked by low concentrations of CPA, but not at concentrations causing near-maximal depletion of the ER Ca²⁺ store (ie, 30 or 100 µmol/L). They also imply that endothelial [H₂O₂]_i during application of CPA will be lower than that attained after application of 100 µmol/L H₂O₂, because the threshold for relaxation was then reduced to 1 µmol/L CPA, as compared to 10 or 30 µmol/L CPA under control conditions. We attempted to assess endogenous H₂O₂ formation in RAV preparations with dichlorofluorescein (DCF), a probe that has been widely used to image [H₂O₂]_i (see supplemental Figure I), but were unable to detect changes in DCF fluorescence after stimulation with 10 or 30 µmol/L CPA or application of 100 µmol/L H₂O₂, whereas increased fluorescence was consistently observed with 1 mmol/L H₂O₂, as reported in pancreatic cells.³³ Although the findings with exogenous H₂O₂ at 100 µmol/L could in theory reflect an ability of intrinsic antioxidant mechanisms to establish an cytosolic/extracellular [H₂O₂] gradient of 1/10,⁴¹ DCF is resistant to oxidization by H₂O₂ in the absence of intracellular catalysts such as transition metal ions or haem-containing peroxidases, but is readily oxidized by species such as peroxyl radicals and peroxynitrite, thus questioning both its sensitivity and specificity as a H₂O₂ probe.⁴²

In preparations responding to 10 µmol/L CPA, residual relaxations observed in the presence of apamin+TRAM-34+iberiotoxin were attenuated by catalase, whereas in preparations with the higher threshold of 30 µmol/L, relaxation was effectively abolished by this triple combination of K_Ca channel inhibitors. These observations suggest that endogenously-generated H₂O₂ can modulate K_Ca channel activity. Further insights into this interaction were obtained in experiments with endothelium-intact rings preincubated with a submaximal concentration of CPA (10 µmol/L), which unmasked an endothelium-dependent relaxant response to exogenous H₂O₂ that was superimposed on its direct smooth muscle activity. This response exhibited characteristics similar to the relaxation evoked by CPA as it could be attenuated by combined SK_Ca/IK_Ca blockade or by BK_Ca blockade, thus providing additional evidence that concerted opening of endothelial BK_Ca, IK_Ca, and SK_Ca channels secondary to elevations in [Ca²⁺]_i underpins the EDHF phenomenon in the RIA. Observations that control concentration-relaxation curves for H₂O₂ were identical in RIA rings with and without endothelium, and were unaffected by the combination of apamin+Tram-34+iberiotoxin, exclude the possibility that H₂O₂ activates endothelial K_Ca channels directly or functions as a freely transferable EDHF that activates smooth muscle K_Ca channels. Indeed, there is evidence that millimolar concentrations of H₂O₂ inhibit, rather than activate, BK_Ca and IK_Ca channels in porcine and bovine endothelial cells,^25,43 and that 100 µmol/L H₂O₂ does not evoke significant smooth muscle hyperpolarization in the RIA (<3 mV).⁸ Because concentration-relaxation curves for H₂O₂ in denuded RIA rings are also unaffected by blockade of K_ATP or K_v channels or by inhibition of guanylyl and adenylyl cyclases,⁸ the precise nature of H₂O₂-evoked smooth muscle relaxation in the RIA remains unclear. One possibility is that H₂O₂ modulates the Ca²⁺ sensitivity of the contractile machinery.¹⁶ Indeed, in the rabbit aorta H₂O₂ can mediate relaxation while "paradoxically" elevating smooth muscle [Ca²⁺]_i, rather than causing reductions in [Ca²⁺]_i secondary to the hyperpolarization that would expected if H₂O₂ functioned as an EDHF.¹⁶

In conclusion, we have provided evidence that H₂O₂ may participate in the EDHF phenomenon by enhancing ER Ca²⁺ release and promoting the activation of endothelial K_Ca channels. Further studies are required to define the subcellular mechanisms that generate H₂O₂ in rabbit endothelial cells in view of evidence that Ca²⁺ influx evoked by SERCA inhibitors promotes H₂O₂ production by mitochondria,⁴⁴ whereas agonist-stimulated H₂O₂ production may be secondary to the generation of superoxide by NADPH oxidase.⁷ It also remains to be determined whether the increased endothelial oxidant stress that characterizes many vascular disease states enhances the EDHF phenomenon by potentiating intracellular Ca²⁺ release, thereby offsetting an associated reduction in NO bioavailability.

	Acknowledgments

 
Sources of Funding

This work was supported by the British Heart Foundation.

Disclosures

None.

	Footnotes

 
Original received January 2, 2008; final version accepted July 23, 2008.

	References

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