Reversible Oxidation of Myometrial Voltage-Gated Potassium Channels with Hydrogen Peroxide

Abstract

The uteri, spontaneously active or Ca²⁺ (6 mM) induced, were allowed to equilibrate, and to inhibit voltage-gated potassium (𝐾_𝑉) channels 1 mM 4-amino pyridine (4-AP) was applied for 15 min before adding H₂O₂ .  H₂O₂ was added cumulatively: 2 μM, 20 μM, 200 μM, 400 μM, and 3 mM. Average time for H₂O₂ concentrations (2, 20, 200, and 400) μM to reach its full effect was 15 min. H₂O₂ 3 mM had a prolonged effect and therefore was left to act for 30 min. Two-way ANOVA showed significant differences in time dependency between spontaneous and Ca²⁺-induced rat uteri after applying 3 mM H₂O₂ (type of contraction, 𝑃=0.0280), but not 400 μM H₂O₂ (𝑃=0.9271). Our results indicate that H₂O₂ oxidises channel intracellular thiol groups and activates the channel, inducing relaxation. Cell antioxidative defence system quickly activates glutathione peroxidase (GSHPx) defence mechanism but not catalase (CAT) defence mechanism. Intracellular redox mechanisms repair the oxidised sites and again establish deactivation of 𝐾_𝑉 channels, recuperating contractility. In conclusion, our results demonstrate that 𝐾_𝑉 channels can be altered in a time-dependent manner by reversible redox-dependent intracellular alterations.

1. Introduction

Several studies have reported that hydrogen peroxide (H₂O₂) can mediate smooth muscle relaxation as an endothelium-derived hyperpolarising factor (EDHF) via activation of potassium (K⁺) channels [1–4]. Activation of K⁺ channels leads to hyperpolarisation and lowering of the calcium (Ca²⁺) concentration, resulting in a smooth muscle relaxation. To date, several subtypes of K⁺ channels have been identified in the rat uteri smooth muscle. The most abundant and most well studied include large conductance Ca²⁺- and voltage-sensitive K⁺ channels (BK_Ca), ATP-dependent K⁺ channels (𝐾_ATP), voltage-dependent K⁺ channels (𝐾_𝑉), and small-conductance Ca²⁺-sensitive K⁺ channels (SK). Our previous study showed that H₂O₂ induces relaxation in the smooth muscle of rat uteri [5]. In an attempt to identify the signaling pathways used by H₂O₂ in this tissue, we then performed a variety of experiments using a range of inhibitors: Nw-nitro-L-arginine methyl ester (L-NAME; NOS inhibitor), methylene blue (MB; cGMP signalling pathway inhibitor), propranolol (non-selective β-adrenoceptor antagonist), tetraethylammonium (TEA; nonselective K⁺ channel inhibitor), glibenclamide (selective ATP dependent K⁺ channel inhibitor), and 4-aminopyridine (4-AP; voltage-dependent K⁺ channel inhibitor). Our results indicated that H₂O₂-induced uterine relaxation is mediated predominantly through K⁺ channels as in the presence of K⁺ channel antagonists, higher doses of H₂O₂ were required to reduce uterine contractions compared with L-NAME, MB, and propranolol. The potency order of the K⁺ channels inhibitor effect was 4-AP > TEA > glibenclamide (the latter being far less effective), indicating that 𝐾_𝑉 channels play the most significant role of K⁺ channels in H₂O₂-induced smooth muscle relaxation of rat uteri [5]. These results were similar to those obtained by other investigators that employed arterial smooth muscles treated with H₂O₂ [6]. 𝐾_𝑉 channels are the biggest family of potassium channels. They include about 40 members divided in 12 subfamilies, 𝐾_𝑉1–𝐾_𝑉12, of which 𝐾_𝑉5, 𝐾_𝑉6, 𝐾_𝑉8, and 𝐾_𝑉9 are not independently functional, but are 𝐾_𝑉2 channels modulators. Smith and coworkers showed expression of many 𝐾_𝑉𝛼 subunits in nonpregnant and pregnant mouse myometrium [7]. Opening of 𝐾_𝑉 channels liberates positive charge leading to membrane repolarisation [8] and relaxation. Response to 𝐾_𝑉 channel inhibitor 4-AP disappeared in pregnant myometrium, what was correlated to loss of 𝐾_𝑉4.3 α expression [9], what is probably oestrogen dependent [7].

H₂O₂ is uncharged oxidant that can diffuse easily trough cell membranes being an eligible signal molecule in many physiological responses. Role of H₂O₂ in the regulation of myometrium smooth muscle contractile activity is not fully resolved and is still under investigation. In intact single fibers, there is evidence of complex multifactorial effects in response to H₂O₂. For instance, myofibrillar Ca²⁺ sensitivity increases early during exposure to high H₂O₂ concentrations and then declines. Moreover, H₂O₂ has little immediate effect on intracellular Ca²⁺, but prolonged exposure to H₂O₂ leads to decreased sarcoplasmic reticulum (SR) Ca²⁺ reuptake and increased resting (Ca²⁺)_i, suggestive of loss of Ca²⁺ homeostasis [10], also the opening probability of SR Ca²⁺ release channels increases after thiol oxidation [11]. (Ca²⁺)_i-increase is a constant feature of pathological states associated with oxidative stress [12]. Recent studies have underscored the notion that the Ca²⁺ and ROS signalling systems are intimately integrated such that Ca²⁺-dependent regulation of components of ROS homeostasis might influence intracellular redox balance and vice versa [13].

In this study, we have further examined the mechanism of 𝐾_𝑉 channels-dependant H₂O₂-relaxing effect on rat smooth muscle contractility and correlated these effects with changes in endogenous antioxidative defence, with respect to two types of activation: spontaneous and calcium-induced.

2. Material and Methods

2.1. Experimental System

Isolated uteri from virgin Wistar rats (200–250 g) in estrous, determined by examination of a daily vaginal lavage [14], were used. All protocols for handling rats were approved by the local ethics committee for animal experimentation that strictly follows international regulations.

2.2. Isolated Organ Bath Study of Uterine 𝐾_𝑉 Channels Time-Dependent Inhibition

All rats were killed by cervical dislocation. The uterine horns were rapidly excised and carefully cleaned of surrounding connective tissue and mounted vertically in a 10 ml volume organ bath containing De Jalon’s solution aerated with 95% oxygen and 5% carbon dioxide at 37°C.

The uteri, spontaneously active or Ca²⁺ (6 mM)-induced, were allowed to equilibrate at 1 g tension before addition of the experimental drugs. To inhibit 𝐾_𝑉 channels in uteri 1 mM 4-amino pyridine (4-AP) was applied for 15 min before adding H₂O₂. H₂O₂ was added cumulatively: 2 μM, 20 μM, 200 μM, 400 μM, and 3 mM. Myometrial tension was recorded isometrically with a TSZ-04-E isolated organ bath and transducer (Experimetria, Budapest, Hungary). Each H₂O₂ concentration was left to act for 15 min except 3 mM that was left for 30 min.

2.3. Determination of Uterine Antioxidative Enzyme Activities after High Impact of H₂O₂

To 7 h contracting uteri were applied 3.6 mM H₂O₂ (summed cumulative concentrations from previous experiment). Control group were uteri active equivalent time but untreated. After experiment, samples were immediately frozen, using liquid nitrogen, and then transferred to −80°C until enzyme analysis.

Thawed uteri were homogenised and sonicated in 0.25 M sucrose, 1 mM EDTA, and 0.05 M Tris-HCl buffer pH 7.4 before centrifugation for 90 min at 105000×g. The supernatant was used to determine enzyme activities (using a Shimadzu UV-160 spectrophotometer, Shimadzu Scientific Instruments, Shimadzu Corporation, Kyoto, Japan). Superoxide dismutase (SOD) activities were determined by the adrenaline method [15]. One unit of activity is defined as the amount of enzyme necessary to decrease by 50% the rate of adrenalin autooxidation at pH 10.2. Manganese SOD (MnSOD) activity was determined by incubating the samples with 8 mM KCN. Copper-zinc SOD (CuZnSOD) activity was calculated as the difference between total SOD and MnSOD activities. The activity of catalase (CAT) was determined by the rate of H₂O₂ disappearance measured at 240 nm, according to Claiborne [16]. One unit of CAT activity is defined as the amount of enzyme that decomposes 1 mmol H₂O₂ per minute at 25°C and pH 7.0. The activity of glutathione peroxidase (GSHPx) was determined by the GSH-dependent reduction of t-butyl hydroperoxide, using a modification of the assay described by Paglia and Valentine [17]. One unit of GSHPx activity is defined as the amount needed to oxidize 1 nmol NADPH per min at 25°C and pH 7.0. Glutathione reductase (GR) activity was determined using the method of Glatzle et al. [18]. This assay is based on NADPH oxidation concomitant with GSH reduction. One unit of GR activity is defined as the oxidation of 1 nmol NADPH per min at 25°C and pH 7.4. All enzyme activities were expressed as units·mg⁻¹ protein.

2.4. Statistical Analyses

Results were analysed with statistical GraphPad Prism (version 5.03), GraphPad Software, San Diego, CA, USA. Statistical significance was determined with t-test, one-way ANOVA, and post hoc tests: Dunett—comparison with a control group, Tukey—comparison among all groups and test for linearity trend; two-way ANOVA and post hoc Bonferroni test; regression analysis: Linear regression and nonlinear regression (dose-dependant inhibition model with variable Hill slope).

2.5. Reagents

The following reagents were used: H₂O₂ (ZORKA Pharma, Sabac, Serbia); 4-AP (Sigma Chemical Co, St Louis, MO, USA). All were dissolved in distilled water. De Jalon’s solution was comprised of (in gl-1): NaCl 9.0, KCl 0.42, NaHCO₃ 0.5, CaCl₂ 0.06, and glucose 0.5.

3. Results

3.1. 𝐾_𝑉 Channels Dependent H₂O₂ Effect on Contractile Activity

Inhibition of 𝐾_𝑉 channels with 1 mM 4-amino pyridine (4-AP) increased the basal tonus of uteri contractions (results not shown), confirming the presence of 𝐾_𝑉 channels in the uteri and their role in contractions. After rat uterine 𝐾_𝑉 channels were blocked with 1 mM 4-AP, H₂O₂ (400 μM and 3 mM) induced effect on contractile activity, both spontaneous and calcium induced. H₂O₂ first induced relaxation, after which contractions were gradually recovering. Additionally, H₂O₂ 3 mM had a prolonged effect and therefore was left to act for 30 min. Average time for other H₂O₂ concentrations (2, 20, 200, and 400) μM to reach its full effect was 15 min (Figure 1).

Figure 1 

A representative original trace of contractions in rat uteri treated with H2O2 (2, 20, 200, 400 μM, and 3 mM) in the presence of 4-amino pyridine (4-AP). It can be noted the time-dependent recovery of contractions after applying H2O2 400 μM and 3 mM, as well as prolonged effect of 3 mM H2O2.

3.2. Time-Dependent Changes in 𝐾_𝑉 Channel Inhibition

One-way ANOVA time-dependence analysis of H₂O₂ (400 μM and 3 mM) induced effect in spontaneous rat uteri showed time significance (time resp.: 𝑃=0.0246, 𝑃<0.0001). Post hoc test for linear trend showed significant trend of linear regression (400 μM H₂O₂: 𝑟²=0.3031, 𝑃=0.0102; 3 mM: 𝑟²=0.8142, 𝑃<0.0001). In Ca²⁺-induced rat, uteri were also shown significant time dependency and linear trend in the effect of H₂O₂ 3 mM (time: 𝑃<0.0001; 𝑟²=0.5800, 𝑃<0.0001) but not with 400 μM (time: 𝑃=0.6628; linear trend: 𝑟²=0.2673, 𝑃=0.3712) (Figure 2).

(a)

(b)

Figure 2 

Time-dependent changes in effect of H2O2 (400 μM and 3 mM) in spontaneous (a) and Ca2+-induced, (b) rat uteri contractile activity in the presence of 𝐾𝑉 channel inhibitor, 4-AP 1 mM. Contractile activity is expressed as change in contraction amplitude (%) after applying H2O2. Full line represents amplitude changes on every 15 min and dotted line on every 5 min after applying H2O2 (400 μM and 3 mM). 3 mM H2O2 had prolonged effect (time for contractions to equilibrate: 30 min). Values are expressed as mean ± error. 𝑥-axis represents the full experiment time starting from 30 min, which is the uteri equilibration interval.

Two-way ANOVA showed significant differences in time dependency between spontaneous and Ca²⁺-induced rat uteri after applying 3 mM H₂O₂ (type of contraction, 𝑃=0.0280), but not 400 μM H₂O₂ (𝑃=0.9271). Regression analysis of fitted lines also showed similar time dependency but different contraction intensity after applying 3 mM H₂O₂ (similar slope, but different intercepts) between spontaneous and Ca²⁺-induced rat uteri, as well as no significant differences after applying 400 μM H₂O₂ (no differences in slope and intercepts between spontaneous and Ca²⁺-induced).

3.3. Changes in Antioxidative Enzyme Activity in Rat Uteri after Impact of H₂O₂ High Concentration

t-test analysis for changes of antioxidative enzymes activity after applying 3.6 mM H₂O₂ (concentration that equals summed cumulative concentrations from previous experiment) comparing to Ca²⁺-induced uteri active equal time but without H₂O₂ treatment showed statistically significant increase of CuZnSOD (𝑃=0.0381) and GSHPx (𝑃=0.0344) activity after impact of 3.6 mM (Figure 3).

(a)

(b)

(c)

(d)

(e)

Figure 3 

Change of AOS enzyme activity (MnSOD (a), CuZnSOD (b), CAT (c), GR (d), and GSHPx (e)) in Ca2+-induced rat uteri after applying 3.6 mM H2O2. Control group were uteri active equivalent time interval without H2O2 treatment. Data are expressed as mean ± error. Groups were compared with t-test (𝑃<0.05, significant). Ns: non significant; *𝑃<0.05.

4. Discussion

Earlier studies have shown that H₂O₂ can act as contractile and relaxing agent, tissue dependent [19–21], and in some cases, it exhibits a biphasic effect [2, 22]. Role of H₂O₂ in the regulation of myometrial contractile activity is not fully resolved and is still under investigation. In some states as in the thrombosis postpartum [23] or during powerful myometrial contractions that restrict blood flow to the uterus, reperfusion/ischemia injury can occur [21, 24] possibly producing high impact of H₂O₂ on the uteri. In our study, we have observed that in 𝐾_𝑉 channel inhibitor (4-AP) pretreated uteri, high concentrations of H₂O₂ caused relaxation and recovery independent of the type of contractile activity (spontaneous or calcium induced), just with differences in intensity. Characteristic of this effect was its significant linear time dependency. After the first relaxation effect, there was a time dependent recovery of contractile activity. Additionally at 3 mM H₂O₂, effect lasted longer (30 min) then the equilibration time for other H₂O₂ concentrations (15 min). It is known that proteins are H₂O₂ targets and that K⁺ channels and proteins that regulate them are redox-sensitive elements [4]. Main protein modifications include direct oxidation, above all amino acids with thiol groups, as cysteine oxidative glication and carbonylation [25–28]. Several electrophysiological studies showed that H₂O₂ acts on cysteine of 𝐾_𝑉 modulatory subunits and found specific cysteins that determine the channel sensitivity. Mutation of these cysteins impeded H₂O₂-dependent channel activation [29]. After oxidation thiol groups may interact with nearby cysteins forming disulfide bonds [30]. Rogers and coworkers [4] showed that 𝐾_𝑉 channels are regulated in a redox-sensitive manner as H₂O₂ 1–10 mM-induced 𝐾_𝑉 currents in coronary artery smooth muscle cells were being antagonized by DTT, a thiol reductant, and blocked by NEM, a thiol-alkylating agent; but they did not observe rapid reversibility (i.e., the effect of H₂O₂ to increase 𝐾_𝑉 current was sustained). As a plausible explanation for their result, they suggest that H₂O₂ may oxidize an extracellular target, rather than a cytoplasmic one that could be repaired by endogenous intracellular reductants. They based this on the assumption that: (1) H₂O₂ crosses membrane easily and has access to extracellular and intracellular thiol groups; (2) thiol groups of extracellular components are always present outside the cell (e.g., extracellular loops of 𝐾_𝑉 channels); (3) intracellular targets include 𝐾_𝑉 channels or proteins that regulate them and always remain inside of the cell; (4) intracellular reductants do not cross the membrane but remain inside the cell having access only to intracellular components. Thus, if an extracellular target was oxidized, no “repair” (i.e., reduction) would be possible. Conversely, if an intracellular target was oxidized, it might be repaired by intracellular reducing mechanisms [4]. We believe that this oxidation could take place intracellular as well, since we did observe time dependent recovery of contractile activity after H₂O₂-induced relaxation in the presence of 4-AP. 4-AP blocks 𝐾_𝑉 channels intracellular [31, 32] and has small association and dissociation time (100 ms). 4-AP binding on the intracellular side of the 𝐾_𝑉 channel may be temporarily altered due to high concentration of highly diffusible H₂O₂. It is possible that H₂O₂ oxidises channel intracellular thiol groups and activates the channel, inducing relaxation. However, 4-AP gradually reassociates to intracellular channel sites as intracellular redox mechanisms repair the binding site and again establishes deactivation of 𝐾_𝑉 channels, recuperating contractility. That way H₂O₂ could directly interact with 𝐾_𝑉 channels on the intracellular side, but this bond could be overcome with time and 4-AP bond reestablished, making the H₂O₂ effect transient. Aikawa et al. also observed the transient response to high H₂O₂ concentrations in time. They showed that after 1 h of exposure to 6 different concentrations of H₂O₂ (0%, 0.0625%, 0.125%, 0.25%, 0.5%, and 1%), the contractile response of rat bladder smooth muscle decreased progressively to increase in H₂O₂ concentration [33]. In more recent study, Han at al. also found that increasing the duration of treatment with 3×10⁻⁴ g% H₂O₂ progressively decreased the contractile responses of the smooth muscle of bladder [34].

With an increase in H₂O_2, its toxic effect starts to appear. Therefore, timely elimination of messengers is important in cell signalisation. In our study after applying high concentration of H₂O₂ (3.6 mM) on a long-term activity, we observed increase in CuZnSOD and GSHPx activity. H₂O₂ is mainly scavenged by CAT and GSHPx. CuZnSOD, is a cytoplasmic O₂^•− scavenger, and as such implies O₂^•− increased production. Some researchers also showed that H₂O₂ causes increase in O₂^•− [35]. Others have observed decrease of reduced form of glutathione, a necessary factor in GSHPx activity [36]. As mentioned previously, we have observed increased activity of GSHPx, H₂O₂ scavenger. However, other important H₂O₂ scavenger, CAT, did not show any changes. It seems that cell antioxidative defence system quickly activates GSHPx defence mechanism but not CAT defence mechanism. CAT is mainly active in peroxisome though some of its activity is also present in mitochondria and endoplasmatic reticulum. CAT, comparing to GSHPx, has lesser affinity for H₂O₂ and as such is not effective in scavenging low concentrations of H₂O₂ [37, 38]. Therefore, GSHPx is considered as a H₂O₂ low concentration scavenger. However, it was shown that CAT is also active at H₂O₂ lower concentrations and that it loses its activity at H₂O₂ higher concentrations [39, 40], what is implying to a possible cause of the absence in its activity in our study.

In conclusion, our results demonstrate that 𝐾_𝑉 channels can be altered in a time-dependent manner by possible time and redox-dependent alterations of 𝐾_𝑉 channels intracellular binding sites or proteins that regulate them and that GSHPx mechanism is the primary scavenging mechanism in this H₂O₂ conditions. Further studies may help to find the possible solutions in protecting myometrium in pathological states including strong redox impact on the cell, as reperfusion ischemia or thrombosis postpartum.