Hydrogen peroxide activates store‐operated Ca²⁺ entry in coronary arteries

Abstract

Background and Purpose

Abnormal Ca²⁺ metabolism has been involved in the pathogenesis of vascular dysfunction associated with oxidative stress. Here, we have investigated the actions of H₂O₂ on store‐operated Ca²⁺ (SOC) entry in coronary arteries and assessed whether it is impaired in arteries from a rat model of metabolic syndrome.

Experimental Approach

Simultaneous measurements of intracellular Ca²⁺ concentration and contractile responses were made in coronary arteries from Wistar and obese Zucker rats, mounted in microvascular myographs, and the effects of H₂O₂ were assessed.

Key Results

H₂O₂ raised intracellular Ca²⁺ concentrations, accompanied by simultaneous vasoconstriction that was markedly reduced in a Ca²⁺‐free medium. Upon Ca²⁺ re‐addition, a nifedipine‐resistant sustained Ca²⁺ entry, not coupled to contraction, was obtained in endothelium‐denuded coronary arteries. The effect of H₂O₂ on this voltage‐independent Ca²⁺ influx was concentration‐dependent, and high micromolar H₂O₂ concentrations were inhibitory and reduced SOC entry evoked by inhibition of the sarcoplasmic reticulum ATPase (SERCA). H₂O₂‐induced increases in Fura signals were mimicked by Ba²⁺ and reduced by heparin, Gd³⁺ ions and by Pyr6, a selective inhibitor of the Orai1‐mediated Ca²⁺ entry,. In coronary arteries from obese Zucker rats, intracellular Ca²⁺ mobilization and SOC entry activated by acute exposure to H₂O₂ were augmented and associated with local oxidative stress.

Conclusion and Implications

H₂O₂ exerted dual concentration‐dependent stimulatory/inhibitory effects on store‐operated, IP₃ receptor‐mediated and Orai1‐mediated Ca²⁺ entry, not coupled to vasoconstriction in coronary vascular smooth muscle. SOC entry activated by H₂O₂ was enhanced and associated with vascular oxidative stress in coronary arteries in metabolic syndrome.

Abbreviations

[Ca²⁺]_i

intracellular Ca²⁺ concentration

CPA

cyclopiazonic acid

CRAC

Ca²⁺ release Ca²⁺‐activated

EDH

endothelium‐derived hyperpolarization

ER

endoplasmic reticulum

IP₃

inositol trisphosphate

KPSS

125 mM K⁺ physiological saline solution

LZR

lean Zucker rat

OZR

obese Zucker rat

PSS

physiological saline solution

Pyr6

N‐(4‐(3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl)phenyl)‐3‐fluorisonicotinamide

SERCA

sarcoendoplasmic reticulum Ca²⁺‐ATPase

SOC

store‐operated Ca²⁺

SR

sarcoplasmic reticulum

STIM1

stromal interaction molecule 1

Tables of Links

TARGETS
Ligand‐gated ion channels a
IP3receptor
Ion channels b
L‐type Ca 2+ channels (Ca V 1.x)
TRPC1
TRPM2

LIGANDS
CPA, cyclopiazonic acid
IP3
Nifedipine
H2O2

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (^{a b}Alexander et al., 2013a, 2013b).

Introduction

Reactive oxygen species (ROS) derived from various sources in the vascular wall are involved in the regulation of redox‐sensitive physiological processes such as vasoconstriction, proliferation and migration. A role of ROS as intracellular signalling molecules in the regulation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of different cell types is now well established (Trebak et al., 2010; Bogeski et al., 2011). For instance, H₂O₂ and other ROS can activate Ca²⁺ mobilization from intracellular stores and stimulate Ca²⁺ entry through ion channels in the plasma membrane to increase the overall [Ca²⁺]_i. Conversely, the activity of ROS‐generating enzymes such as NADPH oxidase and NOS can be regulated by stimuli inducing changes in [Ca²⁺]_i (Touyz, 2005; Trebak et al., 2010).

ROS have been reported to regulate Ca²⁺ influx through both voltage‐dependent and non‐voltage‐dependent plasmalemmal Ca²⁺ channels (Trebak et al., 2010). Voltage‐gated l‐type Ca²⁺ channels play a key role in increasing the global [Ca²⁺]_i during vascular smooth muscle (VSM) contraction, and both superoxide anion (O₂ ⁻) and H₂O₂ exogenously applied or endogenously produced in response to vasoconstrictors and growth factors have been shown to activate L‐type Ca²⁺ entry in VSM (Chaplin and Amberg, 2012; Santiago et al., 2013), as well as in cardiac myocytes (Hool, 2000; Hool and Arthur, 2002; Viola et al., 2007). Ca²⁺ entry through non‐voltage‐dependent channels is produced following receptor activation and/or falling of Ca²⁺ levels of the intracellular stores in the endoplasmic reticulum (ER), the latter known as capacitative Ca²⁺ entry that takes place through store‐operated Ca²⁺ (SOC) channels (Parekh and Putney, 2005). Modulation of Ca²⁺ entry through non‐voltage–gated channels by ROS has been characterized mainly in mammalian non‐excitable cells including endothelial cells (Hu et al., 1998; Az‐ma et al., 1999; Redondo et al., 2004a,2004b) and to a lesser extent in excitable VSM cells, where information remains scarce (Lin et al., 2007; Pourmahram et al., 2008).

Accumulated evidence suggests that ROS are critically involved in the physiological regulation of coronary blood flow. H₂O₂ plays a role along with NO and adenosine in coronary autoregulation (Yada et al., 2003) and is released by shear stress in coronary arterioles (Miura et al., 2003) and involved in the pacing‐induced metabolic coronary vasodilatation to couple coronary blood flow to myocardial O₂ consumption (Saitoh et al., 2006; Yada et al., 2007). H₂O₂ is an endogenous mediator of the endothelium‐derived hyperpolarization (EDH) of coronary microvessels (Matoba et al., 2003; Shimokawa, 2010), through activation of Ca²⁺‐activated K⁺ channels (Barlow and White, 1998) and of the Na⁺/K⁺ pump (Wong et al., 2014). We have recently demonstrated that H₂O₂ can also induce endothelium‐dependent vasoconstriction tightly coupled to increased Ca²⁺ influx through voltage‐dependent l‐type Ca²⁺ channels in coronary arteries (Santiago et al., 2013), a mechanism shared by cardiac myocytes where endogenous peroxide activates l‐type Ca²⁺ currents and has been proposed to sensitize these Ca²⁺ channels and increase the responses to β‐adrenoceptor stimulation (Hool, 2000; Hool and Arthur, 2002). However, H₂O₂ also induced a nifedipine‐resistant [Ca²⁺]_i rise in coronary VSM (Santiago et al., 2013) consistent with the fact that H₂O₂ might mobilize intracellular Ca²⁺ and subsequent Ca²⁺ entry through non‐selective cation channels, as reported in pulmonary arterial myocytes (Lin et al., 2007; Pourmahram et al., 2008).

In the present study, we sought to determine whether H₂O₂ mobilized Ca²⁺ from intracellular stores and activated store depletion‐dependent Ca²⁺ entry in coronary arteries. Furthermore, the effect of H₂O₂ on SOC entry in coronary arteries was also assessed under pathological conditions of high vascular oxidative stress such as metabolic syndrome (Furukawa et al., 2004), a constellation of metabolic and cardiovascular abnormalities including obesity, insulin resistance, dyslipidemia and hypertension, which represent a risk condition for ischaemic heart disease (Arbel et al., 2015).

Methods

Animals and tissue preparation

All animal care and experimental protocols conformed to the European Union Guidelines for the Care and the Use of Laboratory Animals (European Union Directive 2010/63/EU) and were approved by the IACUC at Complutense University (Madrid, Spain). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). A total of N animals were used in the experiments described here.

Male Wistar rats 12–14 weeks old (n = 55) and Zucker rats 17–18 weeks old (n = 20, provided by Charles River Laboratories France, 69592 L'Arbresle Cedex France) were used. Animals were anesthetized with sodium pentobarbital (40 mg·kg⁻¹, i.p.) and killed by cervical dislocation and exsanguination. The heart was quickly removed and transferred into cold (4°C) physiological saline solution (PSS) of the following composition (mM): 119 NaCl, 4.7 KCl, 1.18 KH₂PO₄, 1.17 MgSO₄, 1.5 CaCl₂, 24.9 NaHCO₃, 0.027 EDTA, and 11 glucose, pH = 7.4. Second‐order branches of the left anterior descending coronary artery were dissected and mounted in microvascular myographs (Danish Myotechnology, Denmark) for isometric tension recording. The arteries were equilibrated for 30 min in PSS at 37°C while continuously gassed with a mixture of 95% O₂–5% CO_2. The internal circumference L₁₀₀ corresponding to a transmural pressure of 100 mmHg for a relaxed vessel in situ was calculated, and the arteries were set to L₁ equal to 0.9 times L₁₀₀ (L₁ = 0.9 × L₁₀₀) (Mulvany and Halpern, 1977).

The obese Zucker rat (OZR) was used as a well‐established model of insulin resistance and the metabolic syndrome, caused by a dysfunctional gene of the leptin receptor. Male OZR (fa/fa, n = 13)) and their control counterparts, lean Zucker rats (LZR) (fa/‐, n = 7)), were purchased from Charles River Laboratories (Barcelona, Spain) at 8–10 weeks of age, housed at the Pharmacy School animal care facility and maintained on standard chow and water ad libitum, until they were used for study, at 16–18 weeks of age (Villalba et al., 2009).

Simultaneous measurements of [Ca²⁺]_i and tension

Simultaneous measurements of [Ca²⁺]_i and tension were performed in intact arterial segments using Fura‐2‐acetoxymethylester (Fura‐2‐AM) fluorescence as described previously (Villalba et al., 2008; Santiago et al., 2013). Coronary arteries were incubated in the dark at 37°C in PSS containing the indicator 4 μM Fura‐2‐AM and 0.05% Cremophor EL for a 2 h period. They were washed three times in PSS to remove remaining Fura‐2‐AM, and the solution was changed to PSS with fresh Fura‐2‐AM after 1 h. After Fura‐2‐AM loading, arteries were washed for 45 min in PSS. Experiments were performed in PSS (37°C) continuously gassed with a mixture of 95% O₂–5% CO₂ to maintain pH at 7.4. The myograph was mounted on an inverted microscope (Zeiss Axiovert S100 TV) equipped for dual excitation wavelength microfluorimetry (Deltascan, Photon Technology International).The coronary artery was alternately illuminated at two different wavelengths, 340 and 380 nm, and the intensity of emitted fluorescence was collected through a 510 nm filter using a photomultiplier and monitored together with the tension. At the end of each experiment, Ca²⁺‐insensitive signals determined by quenching of Fura‐2‐AM with Mn²⁺ were subtracted from the measured emission levels at 340 (F₃₄₀) and 380 nm (F₃₈₀). The ratio (R) F₃₄₀/F₃₈₀ corrected for autofluorescence was taken as a measure of [Ca²⁺]_i.

Experimental procedures for the functional experiments

At the beginning of each experiment, the viability of each artery was tested by stimulating it twice with 124 mM K⁺ PSS (KPSS), similar to PSS except that NaCl was replaced by KCl on an equimolar basis. In some experiments, the endothelial cell layer was mechanically removed by guiding a human hair through the vessel lumen and gently moving it back and forward as described previously. Absence of functional endothelium was assessed by the lack of relaxation to ACh (10 μM). The role of intracellular Ca²⁺ in the H₂O₂‐induced vasoconstriction was assessed by stimulating the arteries with a single dose of H₂O₂ (100 μM) in the absence or presence of extracellular Ca²⁺. In order to evaluate whether store depletion by H₂O₂ induces ‘capacitative Ca²⁺ entry’, the bath solution was exchanged for Ca²⁺‐free PSS for 5 min and then for nominally Ca²⁺‐free PSS with nifedipine (1 μM), a selective blocker of l‐type voltage‐operated Ca²⁺ channels, for a period of 10 min. H₂O₂ (30, 100 μM and 300 μM) or cyclopiazonic acid (CPA; 20 μM), a selective inhibitor of the sarcoendoplasmic reticulum Ca²⁺‐ATPase (SERCA) was then added for a period of 8 min, after which 1.5 mM Ca²⁺ or Ba²⁺ was re‐introduced into the bath solution and kept for 20 min.

The effects of H₂O₂ on the SOC entry in coronary VSM were characterized in endothelium‐denuded coronary arteries. The inositol trisphosphate receptor (IP₃R) antagonist heparin (200 µg·mL⁻¹) (Saleem et al., 2014), Gd³⁺ ions (50 μM), as a non‐selective cation channel blocker and Pyr6 (3 μM; N‐(4‐(3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl)phenyl)‐3‐fluorisonicotinamide), a new selective blocker of the stromal interaction molecule 1 (STIM1) and Orai1‐coupled Ca²⁺ release Ca²⁺‐activated (CRAC)‐mediated SOC entry (Schleifer et al., 2012) were tested on the non‐l‐type Ca²⁺ entry induced by store depletion with either H₂O₂ or CPA. In order to assess thiol oxidation‐dependency of H₂O₂ actions on Ca²⁺ entry, the effect of H₂O₂ on the SOC entry in coronary arteries was assessed in arteries treated with the reducing agent DTT (100 μM).

Measurement of O₂  ⁻ production by chemiluminescence

Levels of production O₂ ⁻ by coronary arteries and myocardial tissue from control (LZR) and metabolic syndrome (OZR) rats under basal conditions were detected by lucigenin‐enhanced chemiluminescence as previously described in heart tissue (Santiago et al., 2013). Briefly, segments of coronary arteries and samples of myocardial tissue were dissected and equilibrated in PSS for 30 min at room temperature and then incubated in the presence or absence of tempol (30 μM) at 37°C. Samples were then transferred to microtiter plate wells containing 5 μM lucigenin in air‐equilibrated Krebs solution buffered with 10 mM HEPES‐NaOH, in the absence and presence of tempol, and H₂O₂ (100 μM) was then applied. Chemiluminescence was measured in a luminometer (BMG Fluostar Optima). Baseline values were subtracted from the observed values under the different experimental conditions, and O₂ ⁻ production was normalized to the weight of the tissue samples.

Data analysis

Mechanical responses of the arteries were measured as force and expressed as active wall tension, which is the increase in force, ΔF, divided by twice the segment length (Mulvany and Halpern, 1977). Changes in [Ca²⁺]_i were measured as the increase in the Fura‐2‐AM ratio taking as baseline the average value of the baseline in PSS and baseline in Ca²⁺‐free PSS (ΔF340/F380). The results are expressed as either absolute values of tension (as N m⁻¹) or in units of ratio of fluorescence (F340/F380), or as a percentage of the responses induced by KPSS. All the results are reported as means ± SEM; n represents the number of animals. One arterial segment per animal was used in the calcium experiments, and one to two arterial or myocardial samples per animal were used in the chemiluminescence experiments for O₂ ⁻ measurement. Statistical differences between means were analyzed by unpaired Student's t‐test for comparison between two groups when assessing the effect of various treatments on the changes in [Ca²⁺]_i and tension activated by H₂O₂ or CPA. Two‐factor repeated measures one‐way ANOVA followed by a Bonferroni post hoc test was applied to assess statistically significant differences in changes in either [Ca²⁺]_i or tension activated by H₂O₂ or CPA (factor 1) over time (factor 2). The time at which Ca²⁺ re‐addition was made was taken as time 0. P <0.05 was considered significant. All calculations were made using a standard software package (Prism 5.0; GraphPad Software).

Materials

The sources of the compounds used were as follows: barium chloride, gadolinium chloride, H₂O₂, indomethacin and lucigenin were obtained from Sigma Aldrich (Madrid, Spain), Fura‐2‐AM and ionomycin from Invitrogen (Life Technologies SA, Madrid, Spain), CPA, heparin and nifedipine from Tocris (Bristol, UK) and Pyr6 from Calbiochem (Merck‐Millipore, Madrid, Spain).

Results

H₂O₂ stimulates Ca²⁺ entry and mobilizes Ca²⁺ from intracellular stores in coronary arteries

To assess whether H₂O₂ might induce Ca²⁺ mobilization from intracellular stores thus activating SOC entry, coronary arteries were stimulated with a single submaximal vasoconstrictor concentration of H₂O₂ (100 μM) (Santiago et al., 2013), in the absence and presence of extracelullar Ca²⁺. In PSS containing 1.5 mM Ca²⁺, H₂O₂ induced an increase in [Ca²⁺]_i (∆F₃₄₀/F₃₈₀ = 0.33 ± 0.06, n = 8) that was accompanied by a simultaneous sustained vasoconstriction on baseline tension (0.67 ± 0.11 Nm⁻¹, n = 8) (Figure 1). Both the [Ca²⁺]_i rise and the increase in tension induced by H₂O₂ were markedly reduced after removal of Ca²⁺ from the extracellular medium, although a small fraction of the [Ca²⁺]_i rise (∆F₃₄₀/F₃₈₀ = 0.05 ± 0.01, n = 8) and contraction (0.11 ± 0.02 Nm⁻¹, n = 8) still persisted in Ca²⁺‐free medium (Figure 1). These data suggest that H₂O₂ induces Ca²⁺ mobilization from intracellular stores.

Figure 1.

Rise in [Ca²⁺]_i and vasoconstriction induced by H₂O₂ are in part to due to mobilization of Ca²⁺ from intracellular stores in coronary arteries. (A) Simultaneous recordings showing changes in [Ca²⁺]_i (top) and tension (bottom) induced by KPSS and H₂O₂ in the presence and absence of extracellular Ca²⁺ in coronary arteries with intact endothelium. (B) Summarized data showing the changes in [Ca²⁺]_i (top) and tension (bottom) in response to H₂O₂ (100 μM) in the presence and absence of extracellular Ca²⁺. Results are expressed as a percentage of the KPSS‐induced contraction and are means ± SEM of eight animals. ***P < 0.001; significantly different from control; Student's t‐test.

H₂O₂ stimulates SOC entry not coupled to contraction in coronary arteries

In Ca²⁺‐free PSS, addition of H₂O₂ produced a small Ca²⁺ mobilization and further re‐addition of Ca²⁺ (1.5 mM) to the medium resulted in a marked and sustained increase in [Ca²⁺]_i after 20 min (∆F₃₄₀/F₃₈₀ = 0.48 ± 0.16, n = 5) similar in magnitude to the increase in [Ca²⁺]_i produced by stimulation with KPSS in the same arteries (∆F₃₄₀/F₃₈₀ = 0.52 ± 0.16, n = 5) (Figure 2A, top). However, vasoconstriction induced by H₂O₂ under these conditions (0.29 ± 0.07 Nm⁻¹) was about one‐third of the KPSS‐elicited contraction (0.88 ± 0.22, n = 5) (Figure 2A, bottom). The changes in [Ca²⁺]_i induced by H₂O₂ were not affected by the presence of the voltage‐dependent l‐type Ca²⁺ channel blocker nifedipine (Figure 2B, top), suggesting that Ca²⁺ entry occurs through voltage‐independent Ca²⁺ channels activated by emptying of intracellular Ca²⁺ stores induced by H₂O₂. Nifedipine blunted the small contraction induced by H₂O₂ (Figure 2B, bottom).

Figure 2.

H₂O₂ induces l‐type voltage channel‐independent Ca²⁺ entry in coronary arteries. (A) Simultaneous recordings of [Ca²⁺]_i (A, top) and tension (A, bottom) showing the marked increase in [Ca²⁺]_i and moderate vasoconstriction produced by H₂O₂ (100 μM) in a Ca²⁺‐free medium after the re‐addition of 1.5 mM Ca²⁺ in endothelium‐intact coronary arteries. (B) Summarized data showing the changes in [Ca²⁺]_i (top) and tension (bottom) induced by H₂O₂ (100 μM) in response to the re‐addition of Ca²⁺ to the extracellular medium, in the absence and presence of the l‐type channel blocker nifedipine (1 μM). Results are expressed as a percentage of the KPSS‐induced contraction and are means ± SEM of five animals. ^a P < 0.05, ^b P < 0.01, ^c P < 0.001; significantly different from [Ca²⁺]_i or tension at time 0 (Ca²⁺ re‐addition); one‐way ANOVA. *P < 0.05; significantly different from control (absence of nifedipine); Student's t‐test.

Characterization of SOC entry activated by H₂O₂ in coronary VSM

Because vascular endothelium is involved in the Ca²⁺ mobilization induced by H₂O₂ in the coronary arterial wall (Santiago et al., 2013), experiments were performed in coronary arteries in which endothelium was mechanically removed in order to assess the SOC entry activated by H₂O₂ in coronary VSM. In endothelium‐denuded arteries kept in a Ca²⁺‐free medium under conditions of l‐type channel blockade, H₂O₂ (100 μM) mobilized intracellular Ca²⁺, and readmission of Ca²⁺ to the medium evoked a large and sustained increase in [Ca²⁺]_i (Figure 3A) similar to that obtained in endothelium‐intact arteries and not significantly different from the one induced by depolarization with KPSS in the same arteries. This [Ca²⁺]_i increase was not accompanied by significant increases in tension (Figure 3B). These data suggest that H₂O₂ activates SOC entry in VSM not coupled to contraction. Re‐introduction of Ca²⁺ in the absence of peroxide evoked a minor [Ca²⁺]_i increase in endothelium‐denuded coronary arteries (Figure 3A).

Figure 3.

H₂O₂ promotes store‐operated Ca²⁺ entry not coupled to contraction in coronary vascular smooth muscle. Average changes in (A) [Ca²⁺]_i and (B) contraction evoked by Ca²⁺ re‐addition to endothelium‐denuded coronary arteries kept in Ca²⁺‐free medium (PSS₀) and activated by H₂O₂ under conditions of l‐type channel blockade. In the presence of nifedipine (1 μM) and 100 μM H₂O₂, re‐admission of 1.5 mM Ca²⁺ to the extracellular medium caused a large rise in [Ca²⁺]_i (A, left) similar to the one induced by KPSS (A, right), which was not accompanied by significant changes in tension (B). In control experiments, Ca²⁺ re‐addition was conducted in arteries kept in Ca²⁺‐free PSS in the absence of activating H₂O₂. Results are expressed as a percentage of the KPSS‐induced responses (A, B right) or as absolute values of F340/380 (A, left) and of tension (as N m⁻¹: B, left) and are means ± SEM of four to nine animals. ^a P < 0.05, ^b P < 0.01, ^c P < 0.001; significantly different from [Ca²⁺]_i or tension at time 0 (Ca²⁺ re‐addition); one‐way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001; significantly different from control (absence of H₂O₂); Student's t‐test.

SOC entry activated by H₂O₂ in coronary VSM was compared with the Ca²⁺ entry stimulated by passive sarcoplasmic reticulum (SR) Ca²⁺ depletion by SERCA inhibition with CPA. In endothelium‐denuded coronary arteries kept in Ca²⁺‐free PSS, CPA (20 μM) induced a moderate increase in [Ca²⁺]_i significantly larger than that evoked by 100 μM H₂O₂ (Figure 4A–C). Re‐addition of Ca²⁺ to the medium resulted in a marked rise in [Ca²⁺]_i in the presence of CPA (Figure 4B) that was about 1.5‐fold larger than the one induced by H₂O₂ (Figure 4C and D).

Figure 4.

Store‐operated Ca²⁺ entry induced by SERCA inhibition and by H₂O₂ in coronary vascular smooth muscle. (A, B) Representative traces showing changes in [Ca²⁺]_i induced by H₂O₂ (100 μM) (A) and by the SERCA nhibitor CPA (20 μM) (B) in response to the re‐addition of Ca²⁺ to the extracellular Ca²⁺‐free medium in an endothelium‐denuded coronary artery. (C) Comparative average effects of CPA and 100 μM H₂O₂, on [Ca²⁺]_i in the absence of extracellular Ca²⁺ and after Ca²⁺ re‐addition. (D) Average time‐dependent changes in [Ca²⁺]_i in response to the re‐addition of Ca²⁺ to the extracellular medium in endothelium‐denuded arteries activated with CPA or H₂O₂. Results are expressed as absolute values of F340/380 (C) or as a percentage of the KPSS‐induced ΔF340/380 (D) and are means ± SEM of four to nine animals. *P < 0.05, **P < 0.01; significantly different from control (100 μM H₂O₂); Student's t‐test.

To assess whether the action of H₂O₂ on SOC entry was concentration dependent, the effect of three different concentrations of H₂O₂ on the Ca²⁺ entry upon Ca²⁺ re‐addition to the extracellular medium was compared. While 100 μM H₂O₂ increased [Ca²⁺]_i about 116% of the KPPS‐induced response, 30 μM H₂O₂ induced about half this response, while 300 μM H₂O₂ had an inhibitory effect compared with the effect at 100 μM (Figure 5A–D). Moreover, treatment of endothelium‐denuded coronary arteries with 300 μM H₂O₂ for 30 min reduced by half SOC entry induced by SERCA inhibition with CPA (Figure 5E). At 1 mM H₂O₂, SOC entry was nearly abolished (not shown). Subsequent experiments were performed at 100 μM H₂O₂.

Figure 5.

H₂O₂ has dual concentration‐dependent effects on non‐l‐type Ca²⁺ entry in coronary vascular smooth muscle. (A, B, C) Representative traces showing the increase in [Ca²⁺]_i produced by the re‐addition of Ca²⁺ (1.5 mM) to the extracellular medium after stimulation with 30 (A), 100 (B) or 300 μM H₂O₂ (C) in endothelium‐denuded coronary arteries. (D) Mean values of the changes in [Ca²⁺]_i in response to the re‐addition of Ca²⁺ (1.5 mM) into the extracellular medium after stimulation 30, 100 and 300 μM H₂O₂. (E) Mean values of the changes in [Ca²⁺]_i in response to the re‐addition of Ca²⁺ (1.5 mM) into the extracellular medium in the presence of CPA (20 μM) in control conditions and after 30 min treatment with 300 μM H₂O₂. Results are expressed as a percentage of the KPSS‐induced ΔF340/380 and are means ± SEM of three to nine animals. *P < 0.05, **P < 0.01, significantly different from control (D, 100 µm H₂O₂) (E, 20 μM CPA); Student's t‐test.

Activation of IP₃Rs and Orai1 is involved in the H₂O₂‐induced Ca²⁺ entry of coronary VSM

To investigate whether stimulation of IP₃Rs and the subsequent intracellular Ca²⁺ release underlie H₂O₂‐induced Ca²⁺ entry, coronary arteries were treated with the IP₃R antagonist heparin (200 µg·mL⁻¹). Acute exposure to heparin reduced the nifedipine‐resistant Ca²⁺ influx activated by H₂O₂ (Figure 6A) and caused a large inhibition of SOC entry obtained by SERCA inhibition with CPA (Figure 6B). Heparin also reduced the rises in intracellular Ca²⁺ induced by CPA but not by H₂O₂ in the absence of extracellular Ca²⁺ (Figure 6C).

Figure 6.

Non‐l‐type Ca²⁺ entry induced by H₂O₂ is inhibited by the IP₃R antagonist heparin. (A, B) Average values of the changes in [Ca²⁺]_i induced by H₂O₂ (100 μM) (A) and CPA (20 μM) (B) in response to the re‐addition of Ca²⁺ to the extracellular medium in the absence and after 10 min exposure to heparin (200 µg•mL⁻¹) in coronary arteries. (C) Comparative average effects of heparin on the CPA‐induced and H₂O₂‐induced rises [Ca²⁺]_i in the absence of extracellular Ca²⁺. Results are expressed as a percentage of the KPSS‐induced ΔF340/380 and are means ± SEM of four animals. *P < 0.05, **P < 0.01, ***P < 0.001, significant effects of heparin; Student's t‐test.

The contribution of non‐selective cation channels to the Ca²⁺ entry stimulated by H₂O₂ in coronary VSM was evaluated by using Ba²⁺ fluorimetry instead of Ca²⁺ to investigate the influx of divalent cations activated by peroxide treatment without the intervention of Ca²⁺ extrusion and of Ca²⁺ storage mechanisms (Villalba et al., 2008). In endothelium‐denuded coronary arteries kept in Ca²⁺‐free PSS under conditions of l‐type channel blockade and stimulated with H₂O₂ (100 μM), addition of Ba²⁺ (1.5 mM) to the Ca²⁺‐free solution increased the Fura‐2‐AM ratio to a similar extent to the rise elicited by Ca²⁺ re‐addition (Figure 7A, B and D). Moreover, treatment of coronary arteries with Gd³⁺ (50 μM), a non‐selective blocker of cation channels, significantly reduced the increase in [Ca²⁺]_i activated by H₂O₂ upon extracellular Ca²⁺ re‐addition (Figure 7C and E). These results suggest the involvement of non‐selective cation channels in the capacitative Ca²⁺ influx activated by H₂O₂ in coronary VSM.

Figure 7.

Non‐l‐type Ca²⁺ entry induced by H₂O₂ is mediated by non‐selective cation channels. (A, B, C) Representative traces showing (A) the increase in [Ca²⁺]_i induced by 100 μM H₂O₂ in a Ca²⁺‐free medium upon Ca²⁺ re‐addition, (B) the increase in Fura‐2‐AM fluorescence in arteries stimulated with 100 μM H₂O₂ in a Ca²⁺‐free medium after re‐addition of 1.5 mM Ba²⁺ and (C) the increase in [Ca²⁺]_i induced by 100 μM H₂O₂ in a Ca²⁺‐free medium upon Ca²⁺ re‐addition after treatment with 50 μM Gd³⁺ in endothelium‐denuded coronary arteries. (D) Average comparative effects on the increase in F340/380 upon Ca²⁺ and Ba²⁺ re‐addition in coronary arteries activated with 100 μM H₂O₂ in a Ca²⁺‐free medium. (E) Mean values of changes in [Ca²⁺]_i in response to H₂O₂ in the presence and absence of the non‐selective cation channel inhibitor Gd³⁺ (50 μM). Results are expressed as a percentage of the KPSS‐induced ΔF340/380 and are means ± SEM of three to four animals. *P < 0.05, **P < 0.01, significant effects of Gd³⁺; Student's t‐test).

The role of Orai1 channels in the nifedipine‐resistant Ca²⁺ entry induced by H₂O₂ was further assessed by treatment of coronary arteries with the new selective inhibitor of the Orai1‐mediated SOC entry Pyr6. In the presence of Pyr6 (3 μM), Ca²⁺ influx activated by the re‐addition of Ca²⁺ in coronary arteries activated with either H₂O₂ (Figure 8A, C) or CPA (Figure 8 B,D) was abolished and reduced to levels similar to those obtained by Ca²⁺ re‐addition in the absence of either H₂O₂ or CPA in control experiments (Figure 3A).

Figure 8.

Non‐l‐type Ca²⁺ entry induced by H₂O₂ is inhibited by Pyr6, a selective blocker of Orai1‐mediated Ca²⁺ entry. (A, B) Representative traces showing the increase in [Ca²⁺]_i induced by H₂O₂ (A) and by CPA (B) in the presence of Pyr6 (3 μM) in response to re‐addition of Ca²⁺ to the extracellular medium. (C, D) Mean values of the changes in [Ca²⁺]_i induced by H₂O₂ (100 μM) (C) and CPA (20 μM) (D) in response to the re‐addition of Ca²⁺ to the extracellular medium in the absence and presence of Pyr6 (3 μM) in coronary arteries. Results are expressed as a percentage of the KPSS‐induced ΔF340/380 and are means ± SEM of four animals. *P < 0.05, **P < 0.01, ***P < 0.001, significant effects of Pyr6; Student's t‐test).

Thiol oxidation‐dependent effects of H₂O₂ on SOC entry in coronary VSM

Treatment of endothelium‐denuded coronary arteries with the thiol‐specific reducing agent DTT (100 μM) induced a marked inhibition of the voltage‐independent Ca²⁺ entry stimulated by H₂O₂ (Figure 9A) and to a lesser but not significant extent that induced by SERCA inhibition with CPA (Figure 9B), which suggests that the action of H₂O₂ on SOC entry in VSM is due to oxidation of thiol groups.

Figure 9.

H₂O₂‐induced SOC entry is due to thiol oxidation. Average changes in [Ca²⁺]_i induced by H₂O₂ (100 μM) (A) and CPA (B) in response to the re‐addition of Ca²⁺ to the extracellular medium in the absence and after treatment with the thiol specific reducing agent DTT (100 μM) in endothelium‐denuded coronary arteries. Results are expressed as a percentage of the KPSS‐induced ΔF340/380 and are means ± SEM of four to eight animals. *P < 0.05, **P < 0.01, significant effects of DTT; Student's t‐test.

Effect of H₂O₂ on SOC entry in coronary VSM in metabolic syndrome

Figure 10 shows that basal O₂ ⁻ production was markedly enhanced in coronary arteries (Figure 10A) but not myocardium (Figure 10B) from insulin resistant OZR compared with their lean counterparts and was quenched by the O₂ ⁻ scavenger tempol. These data suggest higher levels of vascular oxidative stress in animals with metabolic syndrome.

Figure 10.

ROS generation and effects of H₂O₂ on [Ca²⁺]_i and vasoconstriction in coronary arteries from LZR and OZR. (A, B) Basal O₂ ⁻ production is increased in coronary arteries (A) but not myocardium (B) from OZR. Effect of the free radical scavenger tempol (30 μM) on basal superoxide production in coronary arteries (A) and myocardium (B) from LZR and OZR. (C, D) H₂O₂ induces rises in [Ca²⁺]_i and vasoconstriction of similar magnitude in LZR and OZR. Changes induced by H₂O₂ (100 μM) in resting [Ca²⁺]_i (C) and basal tension (D) in the presence of Ca²⁺ in the extracellular medium in endothelium‐denuded coronary arteries from LZR and OZR. (E) Intracellular Ca²⁺ mobilization induced by H₂O₂ in a Ca²⁺‐free medium was larger in coronary arteries from OZR. (F) Increases in [Ca²⁺]_i induced by H₂O₂ (100 μM) in response to the re‐addition of Ca²⁺ (1,5 mM) to the extracellular medium in the presence of nifedipine (1 μM) was enhanced in coronary arteries from OZR compared with LZR. (A, B) Results are expressed as cpm (mg tissue)^‐1 and bars represent mean ± SEM of 4–13 animals. (C–F) Results are expressed either as absolute values of ratio F340/380 (C, E) or as a percentage of the KPSS‐induced responses (D, F) and are means ± SEM of three to four animals. *P < 0.05, significantly different from basal; †P < 0.05, ††P < 0.01, significantly different from LZR; one‐way ANOVA.

In order to determine whether the increase in [Ca²⁺]_i and vasoconstriction elicited by H₂O₂ in coronary VSM was affected by the conditions of metabolic syndrome, endothelium‐denuded coronary arteries from LZR and OZR were stimulated with a single concentration of H₂O₂ (100 μM). H₂O₂ induced a sustained increase in [Ca²⁺]_i (Figure 10C) that was accompanied by a simultaneous and sustained vasoconstriction (Figure 10D) of similar magnitude in coronary arteries from LZR and OZR. Ca²⁺ responses to H₂O₂ were largely inhibited upon removal of extracellular Ca²⁺, and mobilization of intracellular Ca²⁺ by H₂O₂ was higher in arteries from OZR compared with LZR (Figure 10E). Furthermore, SOC entry activated by H₂O₂ in coronary VSM from OZR was impaired. Thus, in a Ca²⁺‐free medium in the presence of nifedipine, Ca²⁺ entry stimulated by H₂O₂ upon Ca²⁺ re‐addition after 20 min was significantly larger in coronary VSM from OZR than in that from LZR (Figure 10F). However, increases in [Ca²⁺]_i induced by depolarization with KPSS were unchanged in OZR (∆F₃₄₀/F₃₈₀ 0.20 ± 0.02, n = 5) compared with LZR (∆F₃₄₀/F₃₈₀ = 0.21 ± 0.05, n = 6).

Discussion

Interactions between ROS and the molecular machinery involved in non‐l‐type Ca²⁺ entry in VSM are relatively little explored. The present study provides evidence for a redox regulation of SOC entry in coronary VSM, as well as oxidative stress‐associated changes in this store depletion‐dependent Ca²⁺ influx under conditions of vascular dysfunction in the metabolic syndrome.

ROS regulation of voltage‐independent Ca²⁺ entry has previously been reported mainly in non‐excitable cells including blood and endothelial cells, where SOC entry is a major route for Ca²⁺ entry (Hu et al., 1998, 2000; Az‐ma et al., 1999; Redondo et al., 2004a,2004b; Florea and Blatter, 2008; Sun et al., 2011). In the present study, we show that H₂O₂ mobilizes Ca²⁺ from intracellular stores and activates a significant non‐l‐type Ca²⁺ entry in intact coronary arteries. Because H₂O₂‐induced [Ca²⁺]_i rises in coronary arteries are partially dependent on the endothelium (Santiago et al., 2013), SOC entry was further characterized in endothelium‐denuded coronary arteries. Our data confirmed a nifedipine‐resistant Ca²⁺ entry activated by H₂O₂ in coronary VSM of similar magnitude to the one obtained by maximum high‐K⁺ depolarization leading to Ca²⁺ entry through l‐type channels and about two‐thirds the SOC entry obtained by inhibition of SERCA and passive depletion of the SR with CPA.

Although H₂O₂ is known to produce vasoconstriction via activation of Ca²⁺ entry through l‐type Ca²⁺ channels in coronary arteries (Santiago et al., 2013), the current results reveal an uncoupling between the nifedipine‐resistant Ca²⁺ entry and contraction activated by H₂O₂ in coronary VSM. H₂O₂ is a well‐recognized endogenous mediator of the EDH response in coronary arteries (Matoba et al., 2003; Miura et al., 2003; Yada et al., 2003; Shimokawa, 2010), and therefore, the possibility that H₂O₂ activates vasodilator mechanisms involving K_Ca channels (Barlow and White, 1998) or the Na⁺/K⁺ pump (Wong et al., 2014) cannot be ruled out. However, uncoupling of the nifedipine‐resistant Ca²⁺ entry stimulated by H₂O₂ and contraction in coronary arteries, as shown in the present study, is consistent with the uncoupling between the capacitative Ca²⁺ entry induced by SERCA inhibition with CPA and vasoconstriction in the same arteries. This dissociation between [Ca²⁺]_i and contraction is in agreement with that earlier observed in other small arteries and arterioles and suggests that Ca²⁺ entering through SOC channels is confined to a subcellular compartment that has limited access to the contractile apparatus (Flemming et al., 2002; Snetkov et al., 2003; Villalba et al., 2007). In fact, SOC entry, CRAC currents and channel proteins involved in SOC entry have been reported to be up‐regulated in synthetic/proliferative VSM cells compared with quiescent/contractile vascular myocytes and associated to gene expression, proliferation and migration (Berra‐Romani et al., 2008; Potier et al., 2009; Li et al, 2011).

Studies on the role of ROS in SOC entry in non‐excitable cells have often provided controversial data, and both enhanced and attenuated ROS‐induced capacitative Ca²⁺ entry have been reported, these reciprocal effects being ascribed to the concentration, the time of exposure and the type of oxidant species used in acute experimental protocols. In human platelets (Redondo et al., 2004a), endothelial cells (Elliott et al., 1989; Hu et al., 1998; Florea and Blatter, 2008) and pulmonary artery myocytes (Lin et al., 2007), concentrations of H₂O₂ in the micromolar range up to 500 μM, dose‐dependently stimulated intracellular Ca²⁺ release and store depletion‐dependent Ca²⁺ entry, while higher millimolar concentrations had an inhibitory effect on SOC entry that was also time‐dependent (Florea and Blatter, 2008). In agreement with these observations, the present results indicate that redox regulation of SOC entry in coronary VSM is also dependent on the local levels of ROS and, whereas a maximum nifedipine‐resistant Ca²⁺ entry was obtained upon exposure to 100 μM H₂O₂ for 30 min, this Ca²⁺ response and also SOC entry elicited by SERCA inhibition with CPA were decreased at higher concentrations and practically abolished at millimolar concentrations of H₂O₂ in coronary arteries, which suggests an inhibition of store‐dependent Ca²⁺ entry under conditions of elevated vascular oxidative stress. Concentrations of H₂O₂ up to 100 μM have been reported in inflammatory microenvironments surrounding macrophages (Dröge, 2003). The inhibitory effects of high levels of H₂O₂ are currently explained on the basis of irreversible oxidations of thiol groups of proteins at higher oxidant conditions, considered as oxidative damage (Nunes and Demaurex, 2014). Consistent with this view, concentrations of H₂O₂ up to 100 μM have been shown to activate the Na⁺/K⁺ ATPase and the EDH‐type relaxant responses in porcine coronary arteries, while exposure to millimolar concentrations of H₂O₂ inhibited the pump and caused non‐specific relaxations by impairing VSM contraction (Ellis et al., 2003; Wong et al., 2014). The present findings further suggest that high oxidant stress might induce coronary relaxation by reducing Ca²⁺ entry in VSM.

ROS can influence SOC entry via either direct effects on the molecular machinery of SOC including Orai, the STIM1 and transient receptor potential canonic (TRPC) channels or indirectly by influencing the content of the intracellular SR Ca²⁺ stores (Trebak et al., 2010; Nunes and Demaurex, 2014). H₂O₂ has been reported to induce intracellular Ca²⁺ release via IP₃R activation in blood cells (Parekh and Penner, 1995; Redondo et al., 2004b), and NADPH‐derived endothelial H₂O₂ has been shown to increase the sensitivity of intracellular Ca²⁺ stores to IP₃ in human endothelial cells (Hu et al., 2000; Zheng and Shen, 2005). In the present study, the inhibitory effect of the IP₃R antagonist heparin suggests that H₂O₂‐induced SOC Ca²⁺ entry in coronary VSM is partially mediated by store depletion through activation of IP₃R. These findings are consistent with data obtained in DT40 B‐lymphocytes where both pharmacological inhibition of IP₃R and molecular knock‐out of all three IP₃Rs strongly reduced H₂O₂‐induced CRAC currents (Grupe et al., 2010). Heparin also significantly reduced intracellular Ca²⁺ mobilization and SOC entry activated by CPA in coronary arteries, which suggests that passive depletion of the SR by SERCA inhibition is blunted upon blockade of the IP₃R.

The molecular identity of the non‐voltage‐gated Ca²⁺ channels mediating CRAC currents and SOC entry has been clarified during the last decade, and the ER Ca²⁺ sensor STIM1 (Roos et al., 2005), the plasma membrane Ca²⁺ selective pore‐forming subunit Orai1 (Vig et al., 2006) and also the channel proteins of the TRPC subfamily, including TRPC1 (Ong et al., 2007; Jardin et al., 2008), are known to interact to allow SOC entry. In the present study, the nifedipine‐resistant Ca²⁺ entry activated by H₂O₂ in coronary VSM exhibited classical pharmacological features of SOC entry in other cells, namely, inhibition by low concentrations of lanthanides and reproduction by Ba²⁺ fluorescence, indicative of Ba²⁺ influx through non‐selective cation channels (Parekh, 2006). Furthermore, the selective inhibitor of Orai1‐mediated Ca²⁺ entry, Pyr6 (Schleifer et al., 2012), abolished Ca²⁺ entry obtained by SERCA inhibition and also the non‐l‐type‐dependent Ca²⁺ entry activated in the presence of H₂O₂, thus indicating that H₂O₂ activates SOC entry in a Orai1‐dependent manner in coronary VSM. These data are consistent with recent observations in several native cell lines demonstrating that at micromolar concentrations, H₂O₂ activates Orai1‐mediated Ca²⁺ influx in a STIM1‐dependent manner, also dependent on activation of IP₃R activity rather than modification of Orai1/STIM1 itself (Grupe et al., 2010). On the other hand, the present data showing that H₂O₂ mobilizes Ca²⁺ from intracellular stores and activates a non‐l‐type Ca²⁺ entry in coronary VSM agreed in part with that earlier reported in vascular myocytes from rat intralobar pulmonary arteries, where H₂O₂ releases Ca²⁺ from IP₃‐sensitive and ryanodine‐sensitive intracellular stores and further activates a nifedipine‐resistant Ca²⁺ entry and a Ni²⁺‐sensitive cation current (Lin et al., 2007). However, this voltage‐independent Ca²⁺ entry was not inhibited by either lanthanides or TRPC blockers, and hence, TRPM2 channels were proposed to be involved in Ca²⁺ influx activated by H₂O₂ in pulmonary arterial myocytes.

It is well established that H₂O₂ regulates intracellular signalling pathways through reactions with the highly reactive cysteine residues in target proteins, these oxidative modifications including the reversible formation of disulfide bridges or oxidation products such as sulphenic acid, or the irreversible production of sulphinic or sulphonic acids (Trebak et al., 2010; Bogeski et al., 2011). The data provided here suggest that thiol oxidation produced store depletion‐dependent Ca²⁺ entry in coronary VSM, because the nifedipine‐resistant [Ca²⁺]_i rise induced by H₂O₂, but not by SERCA inhibition with CPA, was abolished by the oxidant thiol reductant DTT. These findings are in line with functional studies demonstrating redox‐dependent regulation of coronary blood flow (Saitoh et al., 2006, 2007) and H₂O₂‐mediated EDH‐dependent relaxation (Wong et al., 2014) through oxidation of thiol groups in the redox sensitive p38 MAPK and the Na⁺/K⁺ ATPase respectively.

The fact that H₂O₂ mobilizes Ca²⁺ from intracellular stores through IP₃R activation along with the inhibitory effect of DTT on H₂O₂‐induced SOC entry in coronary arteries further suggests that H₂O₂ might regulate IP₃R activity through thiol oxidation to induce store depletion, as earlier shown in blood (Parekh and Penner, 1995; Redondo et al., 2004a) and endothelial cells where Ca²⁺‐induced Ca²⁺ release via the IP₃R is enhanced by oxidant‐induced glutathionylation (Lock et al., 2012). However, involvement of other Ca²⁺ transport protein oxidation in the activation of SOC entry induced by H₂O₂ in coronary VSM, such as ryanodine receptors (Du et al., 2005), SERCA (Lancel et al., 2010), the Na⁺–Ca²⁺ exchanger (Kuster et al., 2010), Orai1 (Bogeski et al., 2010) and/or STIM1 Hawkins et al., 2010), cannot be ruled out.

Oxidative stress underlies the pathogenesis of vascular disease in diabetes, obesity and other insulin resistant states (Furukawa et al., 2004; Muñoz et al., 2015). On the other hand, accumulated evidence suggests a critical involvement of abnormal VSM [Ca²⁺]_i homeostasis in the augmented vasoconstriction, remodelling and vascular dysfunction in insulin‐resistant states (Touyz, 2005; Villalba et al., 2008; Linde et al., 2012; Contreras et al., 2013). The present findings confirm high levels of oxidative stress in coronary arteries from OZR, an experimental model of the metabolic syndrome and insulin resistance, consistent with the high levels of ROS reported in the myocardium of these rats (Serpillon et al., 2009), despite the compensatory increase in antioxidant defences including the H₂O₂‐generating enzymes superoxide dismutases (SOD) (Conti et al., 2004). In our study, we found that mobilization of Ca²⁺ from intracellular stores and SOC entry obtained upon acute exposure to H₂O₂ were enhanced in arteries from obese compared with lean rats, as shown by the higher [Ca²⁺]_i rise in a Ca²⁺‐free medium and by the larger nifedipine‐resistant Ca²⁺ entry induced by H₂O₂, and in contrast to the preserved l‐type‐mediated Ca²⁺ entry elicited by KPSS depolarization. These observations suggest that SR Ca²⁺ release and SOC entry activated by exogenous H₂O₂ are sensitized under conditions of increased vascular levels of ROS and would be consistent with the increased sensitivity of the intracellular Ca²⁺ stores to IP₃R activation by H₂O₂ reported in human endothelial cells (Hu et al., 2000). Likewise, enhanced intracellular Ca²⁺ mobilization and Ca²⁺ entry have been found along with increased IP₃R and SERCA2 expression in mesenteric artery myocytes from genetic hypertensive rats (Linde et al., 2012), where high levels of vascular oxidative stress have been reported (Touyz, 2005). Augmented Ca²⁺ signalling was associated with increased vasoconstriction in hypertensive rats, in contrast to the augmented H₂O₂‐induced SR Ca²⁺ release and entry not coupled to contraction in coronary arteries from obese rats. The latter would be consistent with the increased SOC entry and CRAC currents found in proliferative VSM myocytes (Berra‐Romani et al., 2008; Potier et al., 2009; Li et al, 2011).

In conclusion, we provide evidence that Orai1‐mediated SOC entry in coronary VSM is redox sensitive and can be activated by micromolar concentrations of H₂O₂ in part through the release of Ca²⁺ from IP₃‐sensitive intracellular stores, whereas higher H₂O₂ concentrations are inhibitory and blunt SOC entry. Under conditions of vascular oxidative stress in the metabolic syndrome, SR Ca²⁺ release and store‐dependent Ca²⁺ entry responses to acute exposure to H₂O₂ are sensitized, which suggests abnormal Ca²⁺ handling, not related to augmented coronary vasoconstriction.

Author contributions

E. S. and D. P. conceived and designed the experiments. E. S., B. C. and M. M. performed the experiments. E. S., B. C., M. M. and D. P. analysed the data. B. C., A. G. S., L. R. and D. P. contributed reagents/materials/analysis tools. E. S. and D. P. wrote the manuscript. E. S., B. C., A. G. S., L. R. and D. P. revised the article critically for important intellectual content.

Conflict of interest

None declared.

Santiago, E. , Climent, B. , Muñoz, M. , García‐Sacristán, A. , Rivera, L. , and Prieto, D. (2015) Hydrogen peroxide activates store‐operated Ca²⁺ entry in coronary arteries. British Journal of Pharmacology, 172: 5318–5332. doi: 10.1111/bph.13322.