Role of In Vivo Vascular Redox in Resistance ArteriesNovelty and Significance
Vascular thiol redox state has been shown to modulate vasodilator functions in large conductance Ca2+-activated K+ channels and other related channels. However, the role of vascular redox in small resistance arteries is unknown. To determine how in vivo modulation of thiol redox state affects small resistance arteries relaxation, we generated a transgenic mouse strain that overexpresses thioredoxin, a small redox protein (Trx-Tg), and another strain that is thioredoxin-deficient (dnTrx-Tg). The redox state of the mesenteric arteries (MAs) in Trx-Tg mice is found to be predominantly in reduced state; in contrast, MAs from dnTrx-Tg mice remain in oxidized state. Thus, we created an in vivo redox system of mice and isolated the second-order branches of the main superior MAs from wild-type, Trx-Tg, or dnTrx-Tg mice to assess endothelium-dependent relaxing responses in a wire myograph. In MAs isolated from Trx-Tg mice, we observed an enhanced intermediate-conductance Ca2+–activated potassium channel contribution resulting in a larger endothelium-dependent hyperpolarizing (EDH) relaxation in response to indirect (acetylcholine) and direct (NS309) opening of endothelial calcium-activated potassium channels. MAs derived from dnTrx-Tg mice showed both blunted nitric oxide–mediated and EDH-mediated relaxation compared with Trx-Tg mice. In a control study, diamide decreased EDH relaxations in MAs of wild-type mice, whereas dithiothreitol improved EDH relaxations and was able to restore the diamide-induced impairment in EDH response. Furthermore, the basal or angiotensin II–mediated systolic blood pressure remained significantly lower in Trx-Tg mice compared with wild-type or dnTrx-Tg mice, thus directly establishing redox-mediated EDH in blood pressure control.
The endothelium plays an important role in maintaining normal vascular hemostasis by releasing vasoactive mediators, such as nitric oxide (NO), prostacyclin, and endothelium-dependent hyperpolarization (EDH) factors in response to hemodynamic, metabolic, and humeral stimuli.1 EDH is generally described as the relaxation that is independent of NO or prostacyclin.2 The relaxation attributable to EDH is dependent on the vessel type and size, being more pronounced in resistance-sized arteries (lumen diameter of <300 μm) and arterioles, which are important regulators of vasomotor tone, local tissue perfusion, and blood pressure.3,4 Calcium-activated potassium (KCa) channels are key players in the initiation of the EDH response. In the mesenteric arterial (MA) circulation, the small-conductance KCa (SK3 or KCa2.3) and intermediate-conductance KCa (IK1 or KCa3.1) are generally thought to mediate endothelial and smooth muscle hyperpolarization because pharmacological blockade of both these channels abolishes the acetylcholine-induced EDH response.4–9
Studies with large-conductance Ca2+-activated K+ channels have shown that chemical reductant dithiothreitol (DTT) promotes channel activity; in contrast, thiol-oxidizing agent diamide inhibits it in vitro.10 There are limited numbers of studies demonstrating that the large-conductance Ca2+-activated K+ channels are modulated by oxidative stress.10–12 For example, NO activates this channel leading to channel opening, whereas oxidizing agent H2O2 inactivates this channel in isolated porcine renal arteries.13 Redox modification of sulfhydryl groups has been shown to alter large-conductance Ca2+-activated K+ channel gating, in a manner that oxidizing agents inhibited, but reducing agents such as DTT and β-mercaptoethanol stimulated its activity in smooth muscle cells.10,14 All these studies were conducted in vitro, treating vessels with chemical oxidizers or reducers ex vivo.
Small MAs isolated from mice are commonly used to study endothelium-dependent relaxations. We reasoned that endothelial KCa channels could be redox sensitive and thereby susceptible to redox regulation of their activity. To determine the effect of in vivo thiol-disulfide reductase on vascular function, we generated a transgenic mouse strain (Trx-Tg) that overexpresses human thioredoxin and another strain that is deficient (dnTrx-Tg) in endogenous active thioredoxin. Because thioredoxin knockout mice are embryonic lethal,15 we generated the dnTrx-Tg transgenic mouse line, which expresses thioredoxin that is redox inactive because the catalytic cysteines are mutated to serine (C32S, C35S). Thioredoxin is a thiol-disulfide reductase with antioxidant properties and is a major regulator of cellular redox state.16 Together with thioredoxin reductase (TrxR), it regenerates oxidatively inactivated proteins to restore their normal function and nicotinamide adenine dinucleotide phosphate provides reducing equivalents for regeneration of thioredoxin. Although potentially important, the role of thioredoxin in vessel redox homeostasis remains virtually unknown.
The major goal of this study was to determine whether in vivo modulation thioredoxin redox state increases EDH response in resistance arteries. We hypothesized that redox-active thioredoxin controls the endothelial KCa channel activity via its thiol reductase function leading to an enhanced EDH response in murine small MAs. We also determined whether increased levels of thioredoxin would enhance the IK1 activity, which may increase acetylcholine-induced EDH response. We used our newly created Trx-Tg mice and dnTrx-Tg mice to demonstrate in vivo effects of thioredoxin on SK3- and IK1-dependent EDH in mice MAs. We demonstrate that whereas MAs from Trx-Tg mice show increased EDH, the vessels from dnTrx-Tg mice had significantly lower EDH. As a result, systolic blood pressure is reduced in Trx-Tg mice compared with wild-type (WT) or dnTrx-Tg mice.
See the online-only Data Supplement for detailed methods.
To understand the effect of in vivo redox modulation on EDH response, we generated transgenic mice overexpressing cytosolic thioredoxin and another strain that contains decreased level of active thioredoxin in a dominant-negative manner attributable to mutation of active-site cysteines to serine (C32S, C35S) as shown in Figure S1 in the online-only Data Supplement.
Noninvasive Systolic Blood Pressure Measurements by Tail Cuff Method
To obtain reliable blood pressure measurements in conscious mice via the tail-cuff method (CODA system, Kent Scientific), mice were trained for 1 to 2 weeks using the tail-cuff system. After this training period, systolic blood pressures were recorded and the values obtained during 3 consecutive days were averaged.
Vascular Contractile Responses
After a 30-minute washout period, cumulative concentration–response curves were performed to the vasoconstrictor phenylephrine (0.01–30 μmol/L), which causes α1-adrenergic–mediated contractions. The role of NO in suppressing phenylephrine-induced contractions was assessed by incubating the same MAs with nonselective NO synthase blocker Nω-nitro-l-arginine methyl ester (l-NAME;100 μmol/L) for 30 minutes followed by a concentration–response curve to phenylephrine.
Vascular Relaxation Responses
During contraction with a single concentration of phenylephrine (10–30 μmol/L), relaxing responses to the endothelium-dependent muscarinic vasodilator acetylcholine (0.01–10 μmol/L) were recorded in the absence of any inhibitors (control).
EDH-Mediated Relaxing Responses
EDH-mediated relaxations were recorded in the presence of l-NAME and indomethacin. To study the contribution of each KCa channel subtype, EDH-mediated relaxations were always studied in the combined presence of l-NAME and indomethacin to rule out any potential interference of NO and prostaglandins with KCa channels.17
Direct Opening of SK3 and IK1 Channels by NS309
In a subset of MAs, the relaxing responses of the KCa channel activator 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309; 0.01–3 μmol/L)18 were recorded. The same segments were then incubated 30 minutes with indomethacin (10 μmol/L) and l-NAME (100 μmol/L), and relaxing responses to NS309 were repeated.
Role of Sulfhydryl-Modifying Agents on EDH Responses
In MAs derived from WT mice, the EDH-mediated relaxing responses were assessed with sulfhydryl-modifying agentdiamide as a reversible oxidizing agent and DTT as a reducing agent.
Redox Assay of Thioredoxin
MAs were excised from Trx-Tg and dnTrx-Tg mice, and carboxymethylation of MA homogenates was performed as described in our previous publications.19
Thioredoxin, Thioredoxin Reductase, and Peroxiredoxin Assays
Thioredoxin and TrxR activity assays were performed as described in our previous publications.19 Peroxiredoxin assay was performed with H2O2 as substrate using reduced thioredoxin Trx-SH as electron donor in presence of mammalian TrxR and nicotinamide adenine dinucleotide phosphate.20
Vessel Redox Environment Is Predominantly Oxidized in dnTrx-Tg Mice, but Maintained in Reduced State in Trx-Tg Mice
Because thioredoxin knockout mice are embryonic lethal, we generated the dnTrx-Tg transgenic mouse line, which expresses thioredoxin that is redox inactive because the catalytic cysteines are mutated to serine (C32S, C35S). This mutant protein competitively inhibits the reduction of endogenous thioredoxin by TrxR, with a Ki of 1.8 μmol/L.21 Thus, by blocking the regeneration of endogenous thioredoxin, dnTrx-Tg functions as dominant negative.21–23 For comparative studies, we generated a Trx-Tg transgenic mouse line that overexpresses human thioredoxin. To confirm the expression patterns, we conducted immunoblots with an anti-thioredoxin antibody that recognizes both WT and mutant thioredoxin and found increased transgenic protein levels in MA lysates of dnTrx-Tg as well as in Trx-Tg mice (Figure 1A). We could not detect endogenous mouse thioredoxin in the same blot although the antibody is reactive to mouse thioredoxin. Increased expression of thioredoxin by both dnTrx-Tg and Trx-Tg mice was detected in various organs (Figure S2).To establish that increased expression of thioredoxin (or dnTrx) correlates with increased (or decreased) thioredoxin function, respectively, we determined the activity of thioredoxin in the MAs isolated form WT, Trx-Tg, or dnTrx-Tg. As expected, Trx-Tg mice showed significantly higher levels of thioredoxin activity compared with WT or dnTrx-Tg mice (Figure 1B) in the MAs. However, the activity of TrxR that regenerates thioredoxin using nicotinamide adenine dinucleotide phosphate remained unchanged among MAs isolated from WT, Trx-Tg, or dnTrx-Tg mice (Figure 1C). To determine the effect of increased or decreased expression of thioredoxin on vessel redox state, we performed the redox state assay of thioredoxin in MAs isolated from Trx-Tg or dnTrx-Tg mice. As shown in Figure 1D, the redox state of MAs from dnTrx mice is predominantly oxidized as we did not detect any reduced thioredoxin in pooled samples of MAs. However, significantly higher level of reduced thioredoxin was detected in the MAs from Trx-Tg mice (Figure 1D, lane 1, lower band). These data show that the overall redox state of MAs from dnTrx-Tg mice is oxidized compared with Trx-Tg mice, which is reduced. Thus, our mice system represents an in vivo redox model, and we further studied MAs in this mice system.
Structure of WT, Trx-Tg, and dnTrx-Tg MA
The optimal diameters of segments of MAs measured in the wire myograph were comparable for all 3 mice groups: 203±6 μm in WT, 205±8 μm in Trx-Tg, and 194±9 μm in dnTrx-Tg mice.
Comparable Contractile Responses in MAs From All 3 Mice Groups
Addition of 60 mmol/L K+ Krebs-Ringer buffer resulted in tensions that did not differ between MAs derived from all 3 mice groups (Figure 2A), and phenylephrine contracted MAs in a concentration-dependent manner (Figure 2B). The sensitivity (pEC50, negative logarithm of the half maximal effective concentration) to phenylephrine did not differ significantly between arteries from WT, Trx-Tg, and dnTrx-Tg mice (5.61±0.07, 5.72±0.09, and 5.74±0.09, respectively; Figure 2B). The maximal tension (in mN/mm) in response to 30 μmol/L phenylephrine was significantly increased in Trx-Tg (2.89±0.12) compared with WT (2.52±0.12) and dnTrx-Tg mice (2.39±0.20; Figure 2B). We also studied phenylephrine-mediated contractions in the presence of l-NAME to determine the contribution of basal NO levels in suppressing these contractions. Figure S3 shows that l-NAME caused significantly larger tension levels in response to phenylephrine in MAs from WT and Trx-Tg mice, but not in dnTrx-Tg mice.
Thioredoxin Deficiency Resulted in Decreased NO-Mediated Relaxations in MAs From dnTrx-Tg Mice
Because generation of NO is a major contributor to vessel relaxation, we determined the effect of in vivo redox modulation on NO-mediated relaxations in MAs. As shown in Figure 2C, endothelium-dependent acetylcholine-mediated relaxations were significantly enhanced in MAs derived from Trx-Tg mice compared with WT mice. Sensitivity (EC50) to acetylcholine averaged 0.16±0.06 μmol/L in Trx-Tg compared with 0.42±0.20 μmol/L in WT mice. The maximal relaxation (Emax) in response to 10 μmol/L was higher in MAs from Trx-Tg mice (96±1%) compared with WT (92±2%) mice, but this did not reach statistical significance. In contrast, these acetylcholine-mediated responses were severely blunted in MAs from dnTrx-Tg mice, as reflected both by a significantly larger EC50 (1.41±0.65 μmol/L) and by a lower Emax (79±4%) compared with either WT or Trx-Tg mice. This impairment in the relaxing responses of MAs from dnTrx-Tg mice was specific to the endothelium, because relaxing responses to the endothelium-independent NO donor sodium nitroprusside were comparable in MAs from all 3 mice groups (Figure 2D). Next, we determined NO-mediated relaxing responses in MAs by treating them with a cocktail of indomethacin and the endothelial KCa channel blockers TRAM-34 (1 μmol/L) and UCL-1684 (1 μmol/L) to block vasorelaxing prostanoid release and EDH responses, respectively. As shown in Figure 2E, NO-mediated relaxing responses were significantly reduced in MAs from dnTrx-Tg mice compared with MAs from WT or Trx-Tg mice. Sensitivity (EC50) for acetylcholine averaged 29±14 μmol/L in dnTrx-Tg mice compared with 4.0±2.2 μmol/L and 1.2±0.3 μmol/L in WT or Trx-Tg mice, respectively (Figure 2E). Similarly, Emax values were 47±7% in dnTrx-Tg, compared with 73±4% and 77±3% in WT and Trx-Tg mice, respectively (Figure 2E).
EDH Relaxations Are Increased in MAs From TRX-Tg Mice
EDH-mediated relaxing responses were analyzed by incubating MAs in the combined presence of l-NAME and indomethacin. EDH responses were markedly enhanced in MAs from Trx-Tg mice, compared with WT or dnTrx-Tg mice (Figure 2F). EC50 values were significantly lower in Trx-Tg (2.7±1.3 μmol/L) compared with WT and dnTrx-Tg mice (9.8±4.7 and 16.7±6.7 μmol/L, respectively). Maximal relaxation averaged 74±6% in Trx-Tg mice, which was significantly larger compared with WT and dnTrx-Tg mice (56±8% and 51±7%, respectively).
Effect of Thioredoxin Redox on IK1 and SK3 Channels in Acetylcholine-Mediated EDH Relaxation
The selective IK1 channel blocker TRAM-34 inhibited the EDH response to comparable levels in MAs from all 3 mice groups (Figure 3A). Consequently, the percent inhibition by TRAM-34 was significantly higher in MAs derived from Trx-Tg mice compared with WT or dnTrx-Tg mice (see inserted graph, Figure 3A). The selective SK3 channel blocker UCL-1684 also resulted in similar residual relaxations in MAs of all 3 mice groups (Figure 3B). Combined inhibition of IK1 and SK3 channels by TRAM-34 and UCL-1684 almost completely blunted the acetylcholine-mediated relaxations (Figure 3C).
Direct Opening of Endothelial KCa Channels by NS309 Is More Sensitive in MAs From Trx-Tg Mice
Because acetylcholine indirectly activates KCa channels, we investigated the effect of direct opening of IK1 and SK3 channels by NS309 on the EDH-mediated relaxation in MAs. We studied NS309-induced responses in endothelium-intact as well as endothelium-denuded segments. NS309 (0.01–3 μmol/L) induced potent relaxations in endothelium-intact MAs (Figure 4A). However, these relaxations were decreased in dnTrx-Tg compared with WT or Trx-Tg mice. EC50 values averaged 0.5±0.2 μmol/L in dnTrx-Tg compared with 0.08±0.02 μmol/L and 0.07±0.02 μmol/L in WT or Trx-Tg mice, respectively (Figure 4A). Endothelial denudation inhibited NS309-induced relaxations in MAs from all 3 mice groups, indicating that the effects are endothelium dependent (Figure 4A). Inhibition with l-NAME and indomethacin reduced NS309-induced relaxations in MAs from all 3 mice groups (Figure 4B). However, sensitivity (EC50) to NS309 was significantly higher in MAs of Trx-Tg (0.3±0.1 μmol/L) compared with WT (0.9±0.2 μmol/L) or dnTrx-Tg (0.7±0.2 μmol/L) mice. The pharmacological endothelial KCa channel activator naphto [1,2-d] thiazol-2-ylamine (SKA-31) has a 10-fold higher potency for IK1 than SK3.24 Incubation of l-NAME– and indomethacin-treated MAs with SKA-31 (1 μmol/L) resulted in an enhanced acetylcholine-mediated EDH response in all mice groups, but to a greater extent in MAs from Trx-Tg mice (Figure 4C). Sensitivity was significantly larger in MAs from Trx-Tg mice (0.7±0.2 μmol/L), compared with WT (4.3±2.1 μmol/L) or dnTrx-Tg (4.7±1.6 μmol/L) mice, suggesting a greater contribution of IK1 channels in the EDH response in MAs from Trx-Tg mice.
Opening of Endothelial KCa Channels by NS309 Is Primarily Mediated by IK1
Because both IK1 and SK3 are activated by NS309, we determined the effect of thioredoxin redox on specific KCa channel opening by using NS309 in combination with a specific inhibitor of either IK1 or SK3. Inhibition of IK1 channels with TRAM-34 led to a greater rightward shift in the response to NS309 in MAs from Trx-Tg mice compared with WT and dnTrx-Tg mice (Figure 5A). This was evident by a significantly larger shift in sensitivity (pEC50) for NS309 caused by TRAM-34 in Trx-Tg mice compared with WT or dnTrx-Tg mice (Figure 5A, insert). Blockade of SK3 channels with UCL-1684 resulted in comparable NS309-induced relaxations (Figure 5B). However, a significant leftward shift in the response to NS309 occurred in WT compared with Trx-Tg or dnTrx-Tg mice (Figure 5B, insert). Furthermore, combined blockade by TRAM-34 and UCL-1684 led to similar relaxations as compared with TRAM-34 alone (Figure 5C).
Redox Control of EDH Response
Because thioredoxin is a protein disulfide reductase, we reasoned that the enhanced EDH response in Trx-Tg mice might be mediated by the thiol reductase effect of thioredoxin. To address this, we used diamide as a reversible sulfhydryl oxidant and DTT as a disulfide-reducing agent. In MAs isolated from WT mice and contracted with phenylephrine (3–10 μmol/L), diamide (0.01–100 μmol/L) caused a concentration-dependent inhibition of the active tension (Figure 6A). The IC50 of diamide was found to be ≈2.3 μmol/L. In contrast, DTT resulted in small relaxations at lower concentrations (<100 μmol/L) and resulted in recovery of phenylephrine-induced active tension at higher concentrations (0.1–1 mmol/L; Figure 6A). Based on these characteristics, we chose 2 concentrations for diamide and DTT, both in the micromolar range (0.1 and 1 μmol/L). To determine whether diamide-mediated relaxing effects were attributable to sulfhydryl oxidation, we examined whether DTT was able to reverse the diamide-mediated effect. As shown in Figure 6B, treatment of 1 mmol/L DTT completely reversed the diamide (20 μmol/L)-mediated reduction in phenylephrine-induced active tension. Because of this fast-acting effect of DTT, we also assessed combinations of diamide and DTT in modulating the EDH response. Figure 6C shows that 0.1 μmol/L diamide caused a significant reduction in the EDH response. Emax averaged 28±3% in the presence of 0.1 μmol/L diamide and 44±3% in the absence of diamide (Figure 6C). Incubation of 1 μmol/L diamide caused a similar impairment as 0.1 μmol/L diamide (28±6%; Figure 6C). Coincubation of diamide with TRAM-34 (1 μmol/L) resulted in comparable residual relaxations (Figure 6C, insert). In contrast to diamide, DTT dose-dependently enhanced the EDH response in MAs (Figure 6D). Emax averaged 53±11% in the presence of 0.1 μmol/L DTT and 68±11% in the presence of 1 μmol/L DTT (Figure 6D). Again, these effects seemed to be mediated via IK1 channel activation, because coincubation with TRAM-34 resulted in equal residual relaxations (Figure 6D, insert). The lowest concentration of DTT tested (0.1 μmol/L) was unable to reverse the diamide (0.3 μmol/L)-induced blunted EDH response, but 1 and 10 μmol/L DTT significantly restored the EDH response (Figure 6E).
DTT Improved Acetylcholine-Induced Relaxations in MAs From dnTrx-Tg Mice
The dominant-negative production of catalytically inactive human thioredoxin (C32S; C35S) in dnTrx-Tg mice competes for reduction by TrxR with oxidized thioredoxin. Therefore, significant amount of WT mouse thioredoxin and all of the mutant human thioredoxin appears to remain as oxidized thioredoxin in the redox assay. Although DTT could reduce the oxidized WT mouse thioredoxin, it cannot reduce the C32S and C35S mutant thioredoxin because cysteine is substituted with serine. Therefore, we reasoned that MAs from dnTrx-Tg mice would be minimally relaxed by DTT. Incubation of MAs from dnTrx-Tg mice with 10 μmol/L DTT for 30 minutes, prior contraction with phenylephrine (10 μmol/L) followed by cumulative concentrations to acetylcholine, improved endothelium-dependent relaxations (Figure 6F), albeit with less potency.
H2O2-Mediated Relaxations Are Less Sensitive in MAs From Trx-Tg Mice
H2O2 is a sulfhydryl-oxidizing agent that has been shown to act as an endothelium-derived hyperpolarizing factor (EDHF).25 Although H2O2 causes endothelium-independent relaxations in many arteries, its effect on a specific channel or its specific role in endothelial cells and smooth muscle cells remains far from clear.26 Nevertheless, H2O2 does induce relaxations in MAs in mice, albeit at concentrations that appear toxic to vascular cells (>3 μmol/L). We speculated that the relaxation demonstrated in response to H2O2 could actually be attributable to its effect as an oxidizing agent similar to thiol-disulfide exchanges that we observed in diamide and DTT (Figure 6). Our data in Figure 7 show that relaxing responses to H2O2 were significantly less sensitive in MAs from Trx-Tg mice compared with WT and dnTrx-Tg mice. These responses were significantly inhibited by catalase (Figure 7B). These results suggest that increased levels of thioredoxin could decrease the applied concentrations of H2O2 via 2-Cys peroxiredoxins that draw their reducing equivalents from thioredoxin. Peroxiredoxin expression is known to be induced by increased levels of thioredoxin.27 Therefore, we performed peroxiredoxin activity assay of MAs isolated from WT, Trx-Tg, and dnTrx-Tg mice. As shown in Figure 7C, peroxiredoxin activity is significantly higher in Trx-Tg mice compared with either WT or dnTrx-Tg mice. Therefore, H2O2-mediated decreased relaxation in Trx-Tg mice could be attributable to increased peroxiredoxin activity in Trx-Tg mice, but not in WT or dnTrx-Tg mice.
Increased Levels of Thioredoxin Is Correlated With Decreased Blood Pressure in Trx-Tg Mice, but Not in WT or dnTrx-Tg Mice
Our studies demonstrated that vascular relaxations are affected by redox state of the MAs. Because relaxation of MAs is directly related to blood pressure control, we determined whether modulation of vascular thioredoxin redox would affect the basal blood pressure levels in our transgenic mice system. As shown in Figure 8, Trx-Tg mice showed significantly lower level of basal blood pressure compared with either WT or dnTrx-Tg mice, indicating a direct effect of vascular redox homeostasis of MAs on blood pressure regulation. Further, we also determined whether higher levels of thioredoxin could provide protection against increased blood pressure in response to angiotensin II. As shown in Figure 8B, angiotensin II infusion (1000 ng/kg per minute) caused an increase in blood pressure level in WT and Trx-Tg mice; however, the increase in Trx-Tg mice was significantly lower compared with WT mice, demonstrating that angiotensin II–mediated hypertension is attenuated by increased levels of thioredoxin.
The major finding of this study is that EDH relaxations in small MAs are dependent on in vivo redox condition of vessels. This is the first report demonstrating that increased thioredoxin expression in Trx-Tg mice results in an increased EDH response in small MAs. In contrast, mice that are deficient in active thioredoxin show severely blunted endothelium-dependent relaxations attributable to both a reduced NO- and EDH-mediating relaxation. Functionally, the reduced NO and EDH response in dnTrx-Tg mice was reflected by a significantly higher systolic blood pressure; in contrast, increased NO and EDH response was correlated with decreased blood pressure in Trx-Tg mice, confirming the important role of endothelial KCa channels in modulating blood pressure. Additional in vitro studies show that the EDH relaxations could be modulated by exogenous addition of sulfhydryl-modifying agents such as diamide or DTT, where diamide blunted but DTT enhanced the EDH relaxation. Furthermore, we demonstrate a pivotal role for IK1 channel activation in mediating the EDH response via redox state modulation.
It is now well accepted that endothelial SK3 and IK1 channels initiate and conduct the EDH response.4–8,28 Because combined blockade of SK3 and IK1 channels blunted acetylcholine-mediated relaxations, it is likely that these channels mediate the EDH response in these small MAs and suggest that large-conductance Ca2+-activated K+ channels play a minor or no role as observed by others.5–7,9,29 Endothelial denudation drastically reduced the relaxations mediated by NS309 demonstrating that NS309 is an endothelium-dependent KCa channel opener as confirmed by others.30,31 In the presence of l-NAME and indomethacin, NS309-induced responses were similar in MAs from WT and dnTrx-Tg mice, but both were significantly lower compared with Trx-Tg mice, suggesting an enhanced EDH response in the latter mice. NS309 has been demonstrated to initiate NO release in addition to contributing to the main EDH component in rat small MAs.31 Our observation that NS309-induced relaxations were comparable in MAs from WT or dnTrx-Tg mice in the presence of l-NAME and indomethacin (whereas these responses were enhanced in the absence of these inhibitors for MA from WT compared with dnTrx-Tg mice) might be attributed to the fact that NO release was inhibited.
SKA-31 has a 10-fold higher affinity for IK1 compared with SK3 channels,24 suggesting that IK1 channels are predominantly activated. To assess this, we analyzed the role of IK1 channels in mediating NS309-induced relaxations. TRAM-34 blunted NS309-induced EDH responses to a greater extent in MAs derived from Trx-Tg mice, compared with WT or dnTrx-Tg mice (Figure 5A), suggesting a greater contribution of IK1 channels in mediating the EDH response in Trx-Tg mice. UCL-1684 did not alter NS309-induced relaxations in MAs from Trx-Tg and dnTrx-Tg mice, but caused a paradoxical increase in the sensitivity for NS309 in arteries from WT mice.
It is likely that in MAs from Trx-Tg mice an increased IK1 channel activity is present. We questioned whether higher levels of thioredoxin increase IK1 channel activity. Diamide is a thiol oxidant that rapidly crosses the membrane by diffusion.32 Thioredoxin has been shown to be inactivated because of oxidation by diamide.33 Diamide has been shown to cause relaxation in contracted rat pulmonary arteries and bovine coronary arteries, because of closure of L-type voltage-operated calcium channels and inhibition of Ca2+ influx, thereby inhibiting the contraction.34,35 The effects of diamide on MAs are unknown. Here, we showed that diamide causes concentration-dependent relaxations in phenylephrine-contracted MAs with an EC50 value of ≈2 to 3 μmol/L, being substantially lower than the reported EC50 value in rat pulmonary arteries (58 μmol/L).34 DTT was able to reverse the diamide-induced inhibition of contraction, demonstrating the fast-acting interchangeable nature of the cellular redox state. EDH-mediated relaxations were negatively modulated by diamide and positively modulated by DTT in MAs from WT mice. Strikingly, TRAM-34 blocked EDH relaxations in the presence of diamide or DTT to comparable levels, suggesting that IK1 modulation is involved in mediating the EDH relaxation. Interestingly, DTT was able to reverse the inhibitory actions of diamide on the EDH relaxation and to improve acetylcholine-mediated relaxations in isolated small MAs from dnTrx-Tg mice. Because in dnTrx-Tg mice human thioredoxin is in the oxidized and mutated form, only the endogenous oxidized form could be reduced by DTT, but the mutant human thioredoxin cannot be reduced by DTT. Hence, the beneficial effects of DTT are most likely attributable to reducing the endogenous murine thioredoxin.
H2O2 causes relaxation of small arteries, which has been shown to be endothelium-independent and has nonspecific effects on smooth muscle cells.36 However, some studies have suggested that H2O2 is an EDHF; but others have shown that catalase does not inhibit EDH-mediated relaxing responses in small MAs from WT mice.26 Furthermore, the specific effect of H2O2 on KCa channels remains largely unknown.26 We speculated that H2O2 might act as an oxidizing agent, because we observed that the relaxing responses only occur in ≥10 μmol/L concentrations of H2O2. At this concentration, H2O2 could act as a potent oxidizing agent. The decrease in relaxing responses in the MAs isolated from Trx-Tg mice could be attributable to the removal of H2O2 by peroxiredoxin because its activity increased in MAs of Trx-Tg mice, but not in dnTrx-Tg mice. A recent study has demonstrated that MAs express lower levels of peroxiredoxin compared with aorta,37 and therefore, H2O2 is a potent EDHF in MAs, but not in aorta. However, in the MAs of our Trx-Tg mice, the activity of peroxiredoxin is higher, and therefore, H2O2 could be effectively neutralized causing a dampening in relaxation and hence may not be an EDHF. These data further support that KCa could be effective as EDHF in MAs of Trx-Tg mice.
In conclusion, our study demonstrated that in vivo thioredoxin redox state plays an important role in regulating the endothelium-dependent arterial relaxations and arterial blood pressure. In addition, the difference in blood pressure among 3 strains of mice could have resulted in vascular remodeling resulting in differential vascular reactivity in vessels of these animals. The reduced form of thioredoxin is able to enhance EDH responses resulting in an increased IK1 channel activity and lower systolic blood pressure. In contrast, loss of active thioredoxin (-oxidation of thioredoxin) resulted in both a reduced NO- and EDH-mediated relaxation in response to acetylcholine reflected by a higher systolic blood pressure. Pathological modulation of vascular thioredoxin redox state (Figure 9) might directly result in loss of normal relaxing ability of vessels and set the stage for endothelial dysfunction and hypertension. Our observations provide new insights into the vasoprotective effects of thioredoxin in vivo and its beneficial role in the control of hypertension.
Hypertension is a major risk factor for several cardiovascular diseases including myocardial infarction and stroke. Therefore, a clear understanding of underlying mechanisms of hypertension is paramount to discovery of novel therapeutics. Our current study provides novel insight to control of vascular relaxation by modifying the redox state of vessels resulting in lower blood pressure. This new regulatory mechanism of vascular relaxation could provide important clues to develop therapeutic agent for control of hypertension.
Sources of Funding
Research reported in this publication is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award No. R01HL107885 and R01HL109397. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.114.04473/-/DC1.
- Received August 21, 2014.
- Revision received September 4, 2014.
- Accepted September 21, 2014.
- © 2014 American Heart Association, Inc.
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- Heinemann SH,
- Weissbach H,
- Brot N,
- Hoshi T
- Das KC,
- Guo XL,
- White CW
- Oblong JE,
- Berggren M,
- Gasdaska PY,
- Powis G
- Das KC
- Gallegos A,
- Gasdaska JR,
- Taylor CW,
- Paine-Murrieta GD,
- Goodman D,
- Gasdaska PY,
- Berggren M,
- Briehl MM,
- Powis G
- Sankaranarayanan A,
- Raman G,
- Busch C,
- Schultz T,
- Zimin PI,
- Hoyer J,
- Köhler R,
- Wulff H
- Félétou M,
- Vanhoutte PM
- Berggren MI,
- Husbeck B,
- Samulitis B,
- Baker AF,
- Gallegos A,
- Powis G
- Hilgers RH,
- Webb RC
- Hashemy SI,
- Holmgren A
- Schach C,
- Xu M,
- Platoshyn O,
- Keller SH,
- Yuan JX
- Iesaki T,
- Wolin MS
- Burgoyne JR,
- Prysyazhna O,
- Rudyk O,
- Eaton P
Novelty and Significance
What Is New?
Modulation of in vivo vessel redox state affected relaxations in resistance-sized arteries, demonstrating that endothelium-dependent hyperpolarizing and nitric oxide–mediated responses are regulated by vessel redox status, and the blood pressure is directly related to vessel redox perturbations.
What Is Relevant?
The redox state of vessels is critical for relaxation responses either because of nitric oxide or endothelium-dependent hyperpolarization and directly correlates with lower blood pressure.
A shift in vessel redox to oxidized state in pathological conditions or aging could result in hypertension attributable to decreased relaxation by both nitric oxide and endothelium-dependent hyperpolarization. Therapies that target to restore the vessel redox in reduced state could be important for control or reversal of hypertension.
The study provides a mechanistic understanding of in vivo vessel redox state that influences vascular relaxations that is important in cardiovascular diseases such as hypertension and endothelial dysfunction. We demonstrated that redox perturbations modulate blood pressure by regulating the activity of endothelial calcium-activated potassium channels that result in nitric oxide– and endothelium-dependent hyperpolarization–mediated relaxations. In addition, calcium-activated potassium channels, specifically the IK1 channel in mesenteric arteries is modulated by redox state perturbations.