Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogen-deficient female rats

Vascular Pharmacology xxx (xxxx) xxx–xxx

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Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogendeficient female rats Rogério Faustino Ribeiro Juniora,⁎, Jonaina Fiorima, Vinicius Bermond Marquesa, Karoline de Sousa Ronconia, Tatiani Botelhoa, Marcella D. Grandob, Lusiane M. Bendhackb, Dalton Valentim Vassalloa, Ivanita Stefanona a b

Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES, Brazil Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, São Paulo, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords: Estrogen Na+-K+ ATPase activity K+ channels Nitric oxide Oxidative stress

The goal of the present study was to evaluate vascular potassium channels and Na+-K+-ATPase activity in estrogen deficient female rats. Female rats that underwent ovariectomy were assigned to receive daily treatment with placebo (OVX) or estrogen replacement (OVX + E2, 1 mg/kg, once a week, i.m.). Aortic rings were used to examine the involvement of K+ channels and Na+-K+-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 μM) and K+ channels blockers: tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 mM), charybdotoxin (ChTX, 0.1 mM) and iberiotoxin plus apamin. When aortic rings were pre-contracted with KCl (60 mM) or pre-incubated with TEA (5 mM), 4-aminopyridine (4-AP, 5 mM) and iberiotoxin (IbTX, 30 nM) plus apamin (0.5 μM), the ACh-induced relaxation was less effective in the ovariectomized group. Additionally, 4-AP and IbTX decreased the relaxation by sodium nitroprusside in all groups but this reduction was greater in the ovariectomized group. Estrogen deficiency also increased aortic functional Na+-K+ ATPase activity evaluated by K+-induced relaxation. L-NAME or endothelium removal were not able to block the increase in aortic functional Na+-K+ ATPase activity, however, TEA (5 mM) restored this increase to the control level. We also found that estrogen deficiency increased superoxide anion production and reduced nitric oxide release in aortic ring from ovariectomized animals. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals due to the activation of K+ channels and increased Na+-K+ ATPase activity.

1. Introduction The beneficial effects of estrogen in the cardiovascular system led to the hypothesis that estrogen is cardio protective [1–3]. Over a decade ago, it was surprising when several large prospective studies found that estrogen replacement therapy was not able to ameliorate cardiovascular outcome, even when the “timing hypothesis” was considered [4,5]. On the other hand, other studies have suggested beneficial effects of hormone replacement therapy and have reported vascular protective effects of female sex hormones [6,7]. The endothelium maintains a balance between vasoconstriction and vasodilatation [8] and the decrease in estrogen levels could contribute to endothelial dysfunction after the onset of menopause [9]. Estrogen deficiency increases oxygen reactive species formation and exacerbates the development of hypertension [10].

⁎

Estrogen plays a role in regulating endothelial nitric oxide synthase expression and induces nitric oxide release (NO) [11,12]. The total amount of NO is higher in premenopausal women than in men and estrogen appears to be responsible for the gender differences in endothelial NO release [13,14]. NO produces hyperpolarization of the vascular smooth muscle cell, it also activates potassium channels responsible for regulating vascular function and stimulates Na+-K+ ATPase as well [15–17]. Furthermore, Na+/K+-ATPase maintains cellular membrane potential and regulates vascular tone and it is important for membrane repolarization. Thus, alterations in the Na+/ K+-ATPase could worsen cardiovascular outcome [18]. Potassium channels play a key role to maintain resting membrane potential [19] and its activation leads to hyperpolarization and decreases voltage-gated L-type Ca2 + channels activity [19,20]. In vascular smooth muscle cells, Kv channels are activated by membrane

Corresponding author at: Programa de Pós-Graduação em Ciências Fisiológicas, CCS/UFES, Av. Marechal Campos, 1468, Maruípe, 29043-900, Vitoria, ES, Brazil. E-mail address: [email protected] (R.F. Ribeiro Junior).

http://dx.doi.org/10.1016/j.vph.2017.09.003 Received 8 June 2016; Received in revised form 9 September 2017; Accepted 9 September 2017 1537-1891/ © 2017 Published by Elsevier Inc.

Please cite this article as: Ribeiro, R.F., Vascular Pharmacology (2017), http://dx.doi.org/10.1016/j.vph.2017.09.003

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integrity of the endothelium. After a 45-min washout period, concentration-response curves to acetylcholine or phenylephrine were determined. Aortic rings were pre-contracted with phenylephrine at a concentration that produced ∼50% of the contraction to KCl (75 mM) in each case or 60 mM KCl, and the concentration–response curves to ACh (0.1 nM–300 μM) were determined. Single curves were performed in each segment. The role of NO in the relaxation induced by ACh was analyzed by incubating the vessels with NG-nitro-L-arginine methyl ester (L-NAME, 100 μM, nonspecific NOS inhibitor) for 30 min before phenylephrine or KCl administration. The contribution of K+ channels to ACh-induced relaxation was assessed in aortas previously incubated for 30 min with the K+ channel blockers tetraethylammonium (TEA, 5 mM, non-selective blocker of K+ channels), 4-aminopyridine (4-AP, 5 mM, Kv blocker), iberiotoxin (IbTX, 30 nM, selective BKCa blocker), apamin (0.5 μM, selective blocker of small-conductance Ca2 +-activated K+ channels—SKCa) and charybdotoxin (ChTX, 0.1 μM, blocker of KCa and Kv). The relaxing effect of NS1619 (100 μM, an activator of BKCa) and NS309 (3 μM Ca2 +-activator of SKCa and IKCa types) was evaluated in phenylephrine-contracted aortic rings. In some experiments, the concentration–response curves to sodium nitroprusside (SNP, 0.01 nM–0.3 μM) were performed in segments contracted with phenylephrine at a concentration that produced ∼50% of the contraction to KCl in each case. The role of the Kv and BKCa channels in the SNP-induced relaxation was analyzed by incubating the vessels with 4-AP and IbTX, respectively, for 30 min before phenylephrine administration. The functional activity of the Na+/K+-ATPase was measured in segments from SHAM, OVX and OVX + E2 rats using K+-induced relaxation, as described previously [16,25]. After a 30-min equilibration period in normal Krebs, the preparations were incubated for 30 min in K+-free Krebs. The vessels were subsequently pre-contracted with phenylephrine, and once a plateau was attained, the KCl concentration was increased stepwise (1, 2, 5 and 10 mM) with each step lasting for 2.5 min. After a washout period, the preparations were incubated with 100 μM ouabain (OUA) for 30 min to inhibit sodium pump activity, and the K+-induced relaxation curve was repeated. To study the influence of the endothelium, the involvement of K+ channels in OUA-sensitive Na+/K+-ATPase functional activity and the participation of nitric oxide, the endothelium was mechanically removed and rings were incubated with TEA (5 mM) or NG-nitro-L-arginine methyl ester (LNAME, 100 μM, nonspecific NOS inhibitor) for 30 min before phenylephrine administration, respectively.

depolarization [19] and BKCa are voltage and calcium-regulated potassium channels [19], therefore, BKCa strongly regulates vascular tone in resistance arteries whilst aortic tone is strongly dependent on the activity of Kv channels [21]. The involvement of potassium channels in cardiovascular diseases depends on the vascular bed and its impairment may lead to vasoconstriction [22]. The literature demonstrates that estrogen deficiency induces endothelial dysfunction [23] but the role of potassium channels in estrogen deficient rats has not been investigated. The goal of the present study was to evaluate potassium channels and Na+/K+-ATPase activation in estrogen deficient rats. Therefore, we evaluated the effects of estrogen deficiency on the following: 1) reactive oxygen species (ROS) production; 2) involvement of the NO pathway in Na+/K+-ATPase functional activity; and 3) participation of potassium channels in the relaxation induced by acetylcholine and sodium nitroprusside. 2. Materials and methods 2.1. Animals and treatment Two months old female Wistar rats (200–220 g) were used for these studies. The care and use of laboratory animals were in accordance with the NIH guidelines, and all experiments were conducted in compliance with the guidelines for biomedical research as stated by the Brazilian Societies of Experimental Biology and were approved by the Institutional Ethics Committee of the Health Science Center of Vitória (CEUA-EMESCAM 004/2007). All rats had free access to water and were fed with rat chow ad libitum. The rats were divided into 3 groups: OVX (ovariectomy), OVX + E2 (ovariectomy with estrogen replacement) and SHAM. Rats underwent bilateral ovariectomy and estrogen replacement as described previously [1,2,24]. Briefly, a dorsal midline skin incision was made under anesthesia caudal to the posterior border of the ribs. The posterior abdominal muscle wall was dissected, the abdominal cavity was opened and the ovary was gently exteriorized and removed. The uterine horn was returned into the abdomen. The skin incision was closed with sterile nylon sutures, and the process was repeated on the other side. After, rats were assigned randomly to receive treatment with estrogen replacement (OVX + E2, 1 mg/kg once a week i.m.) or placebo and the last group underwent a SHAM operation and served as normal controls. 2.2. Vascular reactivity measurements

2.3. In situ detection of vascular O2%− production

Vascular reactivity in aortic rings was studied at 60 days after surgery. At the end of the treatment, the animals were anesthetized with pentobarbital (35 mg/kg, intraperitoneal) and killed by exsanguination. The thoracic aortas were carefully dissected out, and the fat and connective tissue were removed. For the vascular reactivity experiments, the aortas were divided into cylindrical segments 4 mm in length. In addition, at the time of sacrifice, adequacy of the ovariectomy was determined grossly by the absence of ovarian tissue and marked atrophy of the uterus. We also determined the weight of the entire animal, as well as the weight of the uterus. The aortic segments were mounted between two parallel wires in organ baths containing Krebs-Henseleit solution (in mM: 124 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 0.01 EDTA, 23 NaHCO3) at 37 °C and gassed with 95% O2–5% CO2 (pH 7.4). The arterial segments were stretched to an optimal resting tension of 1 g. Isometric tension was recorded using a force transducer (TSD125C, CA, USA) connected to an acquisition system (MP100A, BIOPAC System, Inc., Santa Barbara, USA). After a 45 min equilibration period, all aortic rings were initially exposed twice to 75 mM KCl. The first exposure checks their functional integrity, and the second exposure assesses the maximal tension. Next, endothelial integrity was tested with acetylcholine (ACh, 10 μM) in segments previously contracted with phenylephrine (1 μM). A relaxation equal to or > 90% was considered demonstrative of the functional

The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate O2%− production in situ, as previously described [2,16]. Hydroethidine freely permeates cells and is oxidized in the presence of O2− to ethidium bromide, which is trapped by intercalation with DNA. Ethidium bromide is excited at 546 nm and has an emission spectrum of 610 nm. Frozen tissue segments were cut into 10-μm-thick sections and placed on a glass slide. Serial sections were equilibrated under identical conditions for 30 min at 37 °C in Krebs–HEPES buffer (in mM: 130 NaCl, 5.6 KCl, 2 CaCl2, 0.24 MgCl2, 8.3 HEPES, and 11 glucose, pH = 7.4). Fresh buffer containing DHE (2 μM) was applied topically to each tissue section, covered with a cover slip, incubated for 30 min in a light-protected humidified chamber at 37 °C, and then viewed with an inverted fluorescence microscope (NIKON Eclipse TieS, × 40 objective) using the same imaging settings. Fluorescence was detected with a 568nm long-pass filter. For quantification, eight frozen tissue segments per animal were sampled for each experimental condition and averaged. The mean fluorescence densities in the target region were calculated. 2.4. Immunofluorescence Aortic 2

segments

were

fixed

with

4%

phosphate-buffered

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and the concentration of agonist that produced 50% of the maximal response (log EC50) were calculated using non-linear regression analysis (GraphPad Prism, GraphPad Software, Inc., San Diego, CA). The sensitivities of the agonists were expressed as pD2 (− log EC50). To compare the effects of OUA on the relaxation induced by K+, some results were expressed as the differences in the area under the concentration response curves (dAUC) for the control and experimental groups. The differences were analyzed using one or two-way ANOVA followed by a Bonferroni test. P < 0.05 was considered to be significant.

paraformaldehyde (pH = 7.4) for 1 h and washed in three changes of phosphate-buffered saline solution (PBS 0.1 M, pH = 7.4). After cleaning, arterial segments were placed in PBS containing 30% sucrose (as a cryoprotectant) for 2 h. Segments were then transferred to a cryomold containing Tissue Tek OCT embedding medium for 20 min (Sakura Finetek Europe, The Netherlands) and frozen in a beaker of isopentane that had been cooled in liquid nitrogen. Tissue was kept at − 70 °C until the day of the experiments. Frozen transverse sections (10 μm) were cut onto gelatine-coated slides and air-dried for at least 60 min. After blockade, sections were incubated with a polyclonal antibody against p22phox (1:100, Santa Cruz Biotechnology) in PBS containing 3% bovine serum albumin overnight in a humid box. After washing, rings were incubated with the secondary antibody goat antirabbit IgG conjugated to Alexa 594 (Molecular Probes, Leiden, The Netherlands) at a dilution 1:200 for a further 1 h at 37 °C in a humid box. After washing, immunofluorescent signals were viewed using an Nikon microscope (×40). Alexa 594 labelled antibody was visualized by excitation at 543 nm and detection at 600–700 nm. The specificity of the immune staining was evaluated by omission of the primary antibody and processing as above. Under these conditions, no staining was observed in the vessel wall of all groups.

4. Drugs and reagents l-phenylephrine hydrochloride, acetylcholine chloride, SNP, sodium pentobarbital, ouabain, L-NAME, TEA, 4-AP,IbTX, CbTX and apamin were purchased from Sigma-Aldrich (St. Louis, USA). The salts and reagents used were of analytical grade from Sigma-Aldrich and Merck (Darmstadt, Germany). 5. Results 5.1. Participation of potassium channels on acetylcholine-induced relaxation after ovariectomy and estrogen replacement

2.5. Nitric oxide release Ovariectomy and estrogen replacement did not affect the contractile response to KCl (SHAM: 3.49 ± 0.05 g, n = 15; OVX: 3.51 ± 0.14 g, n = 15; OVX + E2: 3.46 ± 0.17 g, n = 15; P > 0.05). Ovariectomy and estrogen replacement did not change the relaxation induced by acetylcholine in aortic rings pre-contracted with phenylephrine (Fig. 1A, Table 1). However, in arteries pre-contracted with 60 mM KCl, the relaxation induced by ACh was reduced in the SHAM group (Rmax for phenylephrine pre-contraction: 95.62 ± 0.09%, n = 30; for KCl pre-contraction: 62.07 ± 0.83%,n = 9, P < 0.05), OVX group (Rmax for phenylephrine pre-contraction: 89.90 ± 0.09%, n = 15; for KCl pre-contraction: 26.54 ± 1.83%, n = 9, P < 0.05), and OVX + E2 group (Rmax for PHE pre-contraction: 90.93 ± 0.01%, n = 21; for KCl pre-contraction: 52.12 ± 0.29% n = 9, P < 0.05. In KCl pre-contracted aortic vessels, the reduction of ACh-induced relaxation was greater in ovariectomized rats, suggesting greater contribution of hyperpolarizing mechanisms in this group (Fig. 1B). The participation of NO in the ACh-induced relaxation was investigated using L-NAME (100 μM), which was added before phenylephrine or high K+. Under these conditions, the ACh-induced relaxation was negligible in arteries from all groups pre-contracted with either phenylephrine (Fig. 1C and D, Table 1) or KCl (Fig. 1D), indicating that NO accounted for most of the endothelium-dependent relaxation. Then, we evaluated the effects of K+ channel blockers on the basal tone (in vessels pre-contracted with phenylephrine) and in the AChinduced relaxation. TEA (5 mM), a nonselective K+ channel blocker, increased the basal tone in all groups, but these effects were greater in ovariectomized rats (Fig. 2A, Table 1), suggesting a relevant role for potassium channels in controlling arterial tone. In addition, TEA reduced the relaxation induced by ACh in aortic segments pre-contracted with phenylephrine from SHAM, OVX and OVX + E2 groups (Fig. 2B, Table 1). However, this reduction was greater in aorta from ovariectomized rats. Moreover, estrogen replacement restored the relaxation induced by ACh in aortic rings after incubation with TEA (Fig. 2B, Table 1). To evaluate the participation of Kv channels, we used 4-AP (5 mM), a specific inhibitor of Kv channels. This drug increased the basal tone in all groups in vessels pre-contracted with phenylephrine, but these effects were greater in the ovariectomized rats (Fig. 2A, Table 1), suggesting a relevant role for Kv channels in controlling arterial tone. Besides, 4-AP reduced the relaxation induced by ACh in aortic segments from SHAM, OVX and OVX + E2 groups (Fig. 2C, Table 1). However, this reduction was greater in aorta from ovariectomized rats. Moreover, estrogen replacement restored the relaxation induced by ACh in aortic

NO production was determined using 4,5-diaminofluoresceindiacetate (DAF-2). Aortic rings were dissected and embedded in freezing medium. Transverse cryostat sections (10 μm) were collected on glass slides and incubated at 37 °C with 8 μM DAF-2 in phosphate buffer (0.1 M) containing CaCl2 (0.45 mM). After 30 min, digital images were collected on an epifluorescence microscope(Nikon) and analyzed by measuring the mean optical density of the fluorescence. Pictures were taken from all rings before DAF-2 was incubated to rule out auto fluorescence. 2.6. Western blot analyses Western blot was performed as previously described [26,27]. Proteins from homogenized arteries (20 or 50 μg) were separated by 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes that were incubated overnight at 4 °C with the following primary antibodies: rabbit primary antibody against SK3 (1:500, Santa Cruz), rabbit primary antibody against KV 1.5 (1:1000, Chemicon) or mouse primary antibody against maxi-K (1:500, BD Transduction Laboratories, Lexington, KY, USA), anti-eNOS (1:1000, BD Transduction Laboratories, Lexington, KY, USA), anti-Cu/Zn SOD (1:2000, Sigma-Aldrich, Germany), anti-Mn SOD (1:2000, Millipore), anti-NOX-1 (1:2000, Sigma-Aldrich, USA). After washing, membranes were incubated with anti-mouse (1:5000; Cell Signalling, USA) or anti-rabbit (1:5000; Cell Signalling, USA) antibodies conjugated to horseradish peroxidase. After thorough washing, immunocomplexes were detected using an enhanced horseradish peroxidase/chemiluminescence system (ECL Prime, Amersham International, Little Chalfont, UK) and film (Hyperfilm ECL International). Signals on the immunoblots were quantified with the National Institutes of Health Image V1.56 software. The same membranes were used to determine α-actin expression using a mouse monoclonal antibody to α-actin (1:1,000,000; Sigma-Aldrich, USA) or to total β-actin expression (1:4000, Santa Cruz). 3. Statistical analyses All values are expressed as the mean ± standard error of the mean (SEM). Contractile responses to phenylephrine were expressed as a percentage of the maximal response induced by 75 mM KCl. Vasodilator responses to ACh or SNP were expressed as the percentage of relaxation of the previous contraction. For each concentration-response curve, the maximal effect (Rmax) 3

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Fig. 1. Concentration–response curves to acetylcholine (ACh) in aortic rings from SHAM, OVX and OVX + E2 groups pre-contracted with phenylephrine (a) or (b) 60 mmol/LKCl in the presence or absence of L-NAME (100 μM) (c and d). The results are expressed as the means ± SEM.*P < 0.05 vs SHAM by two-way ANOVA followed by a Bonferroni test. The number of animals used is indicated in parenthesis.

rings after incubation with 4-AP. To confirm this finding, we developed western blots for Kv1.5 and Kv2.1 and we found that ovariectomized animals present higher levels of Kv2.1 when compared to SHAM animals. Moreover, estrogen replacement therapy was able to restore this value to the control level (Fig. 10E and F). To evaluate the role of BKCa channels, the aortic rings were precontracted with phenylephrine and incubated with the selective BKCa blocker (IbTX), the selective SKCa blocker (apamin), the KCa and Kv blocker (ChTX) and co-incubation IbTX + apamin. The three calciumactivated potassium channels inhibitors reduced the relaxation induced by ACh in aortic segments from all groups (Fig. 3A, B and C, Table 1). However, this effect was not different among experimental groups. In addition, the co-incubation of IbTX plus apamin reduced the relaxation induced by ACh in aortic segments from all groups, but this reduction was greater in aorta from ovariectomized rats (Fig. 3D, Table 1). As shown in Fig. 5A, NS1619 (100 μM), an activator of BKCa channels, induced relaxation in aortas contracted with phenylephrine. This response was greater in aortic rings from ovariectomized animals when compared to control or estrogen replacement. When we evaluated the protein expression for BKCa we found that ovariectomy did not affect the protein expression, although estrogen replacement therapy increased the protein expression of BKCa in ovariectomized animals

(Fig. 10G). We also used NS309 (3 μM), an activator of SKCa and IKCa channels. NS309 induced relaxation in aortas contracted with phenylephrine. This response was greater in aortic rings from ovariectomized animals when compared to control or estrogen replacement (Fig. 5B). We also detected the protein expression for SK3 and we did not find any difference among groups, this result suggests that the activity might be increased in the ovariectomized group (Fig. 10H). We next evaluated phenylephrine-induced contraction in aortas from control, ovariectomized and estrogen replacement rats. As shown in Fig. 5C, the response to phenylephrine is increased in ovariectomized animals when compared to control and estrogen replacement. Then, we analyzed the participation of these K+ channels in the relaxation induced by the NO donor, sodium nitroprusside. The endothelium-independent relaxation induced by SNP in arteries pre-contracted with phenylephrine was similar in all groups (Fig. 4A, Table 2). After IbTX and 4-AP incubation, there was a decrease in the relaxation induced by SNP in all groups (Fig. 4B and C, Table 2) but this decrease was greater in the ovariectomized group. Moreover, estrogen replacement restored the relaxation induced by ACh in aortic rings after incubation with 4-AP and IbTX.

Table 1 Effects of NG-nitro-L-arginine methyl ester (L-NAME),tetraethylammonium (TEA),4-aminopyridine (4-AP), iberiotoxin (IbTX), apamin andcharybdotoxin (ChTX) on the vascular responses to acetylcholine (Rmax and pD2) in phenylephrinecontracted aortas from SHAM, OVX and OVX + E2 groups. SHAM

Control L-NAME TEA 4-AP IbTX Apamin ChTX AP + IbTX

OVX

Rmax

pD2

95.62 ± 2.6 1.03 ± 0.5 58.99 ± 4.84 85.30 ± 5.10 80.33 ± 4.87 77.83 ± 3.95 72.79 ± 4.47 98.39 ± 1.16

7.15 7.93 5.84 5.49 6.89 6.86 6.34 7.00

± ± ± ± ± ± ± ±

0.10 0.53 0.17# 0.12 0.29 0.21 0.20 0.14

OVX + E2

Rmax

pD2

89.31 ± 1.7 0.55 ± 0.5 8.59 ± 3.06* 37.84 ± 5.49* 80.49 ± 4.95 83.57 ± 2.49 76.90 ± 7.18 74.09 ± 3.02*

7.59 7.93 6.66 5.86 6.42 6.98 6.41 6.57

± ± ± ± ± ± ± ±

0.07 0.09 1.35 0.19 0.25 0.16 0.20 0.20

Rmax

pD2

90.23 ± 1.3 0.03 ± 0.01 44.68 ± 4.2# 83.01 ± 5.82# 82.54 ± 3.04 79.47 ± 2.54 75.42 ± 2.37 88.49 ± 1.38#

7.35 7.25 5.55 5.67 6.31 6.45 6.21 6.75

± ± ± ± ± ± ± ±

0.08 0.09 0.18 0.15 0.19 0.15 0.17 0.11

The results are expressed as the means ± SEM of the number of animals shown in Figs. 1–3. Rmax, maximal effect; pD2, − log one-half Rmax. P < 0.05 vs. SHAM animals (*) and OVX animals (#). One-way ANOVA followed by a Bonferroni test.

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Fig. 2. The contractile effect induced by TEA (5 mM) and 4aminopyridine (4-AP, 5 mM) in aortas from SHAM, OVX and OVX + E2 groups in vessels pre-contracted with phenylephrine (a). The results are expressed as the means ± SEM P < 0.05 vs. SHAM TEA and #P < 0.05 vs. SHAM 4-AP by one-way ANOVA. The effect of TEA (5 mM) (b) and 4-aminopyridine (4-AP, 5 mM) (c) on the concentration-response curves to acetylcholine (ACh) in aortic segments from SHAM, OVX and OVX + E2 groups. *P < 0.05 vs SHAM, #P < 0.05 vs OVX by two-way ANOVA followed by a Bonferroni test. The number of animals used is indicated in parenthesis.

5.2. Effects of ovariectomy and estrogen replacement on Na+/K+-ATPase activity

Fig. 7A demonstrates that, after endothelial removal; K+-induced relaxation was reduced in the SHAM and OVX + E2 groups. After incubating the rings with OUA, the KCl-induced relaxation was reduced in the aortic rings from all groups. We next evaluated the role of NO on Na+-K+ ATPase activity, rings were incubated with the nitric oxide synthase inhibitor L-NAME 100 μM. L-NAME was able to reduce K+induced relaxation in SHAM and OVX + E2 groups, demonstrating that NO blockade does not affect K+-induced relaxation in ovariectomized animals. The K+ channel blocker TEA (5 mM) did not modify the relaxation induced by K+ in the SHAM and OVX + E2 groups. However, K+-induced relaxation was reduced in the ovariectomized group, demonstrating that potassium channels affect Na+/K+ ATPase activity. Similarly, after co-incubation with OUA (100 μM), the KCl-induced relaxation was not different compared to ouabain alone inall experimental groups (Fig. 7C).

Fig. 6 shows functional Na+/K+ ATPase activity, as evaluated by K -induced relaxation in aortic rings from SHAM controls, OVX and OVX + E 2 groups. As expected, after incubating the rings with OUA (100 μM), the K+-induced relaxation was reduced in all experimental groups. The difference between the groups on the functional Na+/K+ ATPase activity was analyzed using the differences in the area under the curve (dAUC) with and without OUA. The dAUC was higher in ovariectomized rats (SHAM: 75,62 ± 3.50%, n = 8; OVX: 88,83 ± 3.25* %, n = 8; OVX + E2: 73,82 ± 4.48%, n = 8; *P < 0.05), suggesting that functional Na+/K+ ATPase has a higher activity in ovariectomized rats (Fig. 6A–D). Then, to investigate the involvement of endothelial modulation on Na+/K+-ATPase activity, the endothelium was mechanically removed. +

Fig. 3. The effect of iberiotoxin (IbTX, 30 nM) (a), apamin (0.5 μM) (b), charybdotoxin (ChTX, 0.1 μM) (c) and iberiotoxin (IbTX, 30 nM) plus apamin (0.5 μM) (d) on the concentration-response curve to acetylcholine (ACh) in aortic rings pre-contracted with phenylephrine from SHAM, OVX and OVX + E2 groups. The results are expressed as the means ± SEM. *P < 0.05 vs SHAM by two-way ANOVA followed by a Bonferroni test. The number of animals used is indicated in parenthesis.

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Fig. 4. Concentration–response curves to sodium nitroprusside (SNP) in aortic rings from SHAM, OVX and OVX + E2 groups pre-contracted with phenylephrine (a) in the presence or absence of iberiotoxin (IbTX, 30 nM) (b) and 4-aminopyridine (4-AP, 5 mM) (c) The results are expressed as the means ± SEM.*P < 0.05 vs SHAM by twoway ANOVA followed by a Bonferroni test. The number of animals used is indicated in parentheses.

5.3. Effects of ovariectomy and estrogen replacement on oxidative stress

6. Discussion

The basal O2− production in the aortas from ovariectomized animals was greater than that from the SHAM animals and estrogen replacement was able to restore to control levels (Fig. 8A–C). To investigate the source of superoxide anion production, we developed western blots for NOX-1 and immunofluorescence for p22phox. Our results show that NOX-1 protein levels are higher in ovariectomized animals compared to SHAM and estrogen replacement restored the protein amount to SHAM levels (Fig. 10). The immunofluorescence also shows that p22phox is increased in ovariectomized animals (Fig. 9). We also quantified two antioxidant enzymes, Cu-Zn-SOD and Mn-SOD, which are a superoxide anion scavenger and are located in two different compartments within the cell. Our results show that both SOD in the mitochondria and in the cytosol, are increased after ovariectomy and estrogen replacement restored them to SHAM levels (Fig. 10).

The results presented in this study show that ovariectomy increased free radical production, decreased NO release after 8 weeks of estrogen deficiency and did not change the relaxation induced by acetylcholine and sodium nitroprusside. In addition, our results indicate that Kv and BKCa as well as Na+-K+ ATPase activity are increased in ovariectomized animals after 8 weeks of estrogen deficiency. It is well known that estrogen protects the endothelium and smooth muscle cells against oxidative damage and it increases NO release [10,28]. Based on epidemiological, clinical and experimental studies, the loss of estrogen has detrimental effects on the cardiovascular system and triggers endothelial dysfunction induced by oxidative stress in women and animals [23,29,30]. In experimental models, it is recognized that the loss of estrogen induces endothelial dysfunction due to NADPH activation and increased oxygen reactive species formation [10], leading to sustained COX-2 derived prostanoids release [23], which in turn increases the response to phenylephrine, this increased response to phenylephrine can be explained by the increase in superoxide production and COX-2 derived prostanoids [10,23,31–33]. Our results confirm previous findings in the literature that after 8 weeks of estrogen deprivation, there is an increase in superoxide anion production most likely due to increased p22phox and NOX-1 protein expression. We also evaluated the protein expression for SOD and the results suggest that this increase in protein expression could be a compensatory mechanism to increased levels of superoxide anion production. We also found that phenylephrine induced-contraction is also increased in ovariectomized animals although the response to acetylcholine did not change after 8 weeks. This result corroborates our previous findings

5.4. Effects of ovariectomy and estrogen replacement on nitric oxide release Because eNOS alterations can produce endothelial dysfunction, the expression of this protein was measured. Ovariectomy reduced eNOS protein expression in aortas after 8 weeks and estrogen replacement was not different compared to the SHAM group (Fig. 10A). To confirm this result, we also measured in situ NO release by incubating rat aorta rings with 4,5-diaminofluorescein, a NO fluorescent probe. The result indicates that NO release is reduced in ovariectomized animals when compared to SHAM rats. Moreover, estrogen restored NO release to the SHAM levels (Fig. 8D–F).

Table 2 Effects of 4-aminopyridine (4-AP) and iberiotoxin (IbTX) on the vascular response to sodium nitroprusside (Rmax and pD2) in phenylephrine pre-contracted aortas from SHAM, OVX and OVX + E2 groups. SHAM

Control 4-AP IbTX

OVX

OVX + E2

Rmax

pD2

Rmax

pD2

Rmax

pD2

100 ± 0 88.98 ± 2.79 98.10 ± 0.76

7.59 ± 0.14 7.09 ± 0.16 6.86 ± 0.13

100 ± 0 60.32 ± 2.72* 90.27 ± 1.28*

7.54 ± 0.17 6.54 ± 0.14 5.88 ± 0.23*

99.49 ± 0.50 90.14 ± 0.93# 99.17 ± 1.46#

7.67 ± 0.13 6.77 ± 0.21 6.89 ± 0.17#

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Fig. 5. Relaxation to 100 μM NS1619 (a) and 3 μM to NS309 (b) of phenylephrine-contracted aortic rings and phenylephrine induced contraction (c) from SHAM, OVX and OVX + E2 animals. The results are expressed as the means ± SEM.*P < 0.05 vs SHAM, #P < 0.05 vs OVX by two-way ANOVA followed by a Bonferroni test. The number of animals used is indicated in parentheses.

concentration suggesting that it is mediated, in part, by K+ channels activation. However, this reduction was greater in arteries from ovariectomized animals. Estrogen can decrease calcium influx and/or stimulate calcium efflux through the membrane or it can also open potassium channels. Potassium efflux through the membrane repolarizes membrane potential, thereby closing voltage-dependent calcium channels [34–36]. It has been also demonstrated that estrogen enhances the large-

that after 8 weeks of ovariectomy, the response to phenylephrine-induced contraction increases and the response to acetylcholine remains unchanged [23]. To further investigate the mechanisms involved in the maintenance of acetylcholine-induced relaxation, we pre-contracted the vessels with high extracellular potassium (60 mM) and then the concentration–response curve to acetylcholine was performed. Acetylcholine induced-relaxation was partially blocked by high K+

Fig. 6. Potassium-induced relaxation in aortic rings from SHAM, OVX and OVX + E2 groups, previously incubated in a K+-free medium and contracted with phenylephrine before and after incubation with 100 μM ouabain (a, b and c). The inset shows differences in area under the concentration-response curves (dAUC) between groups. The results are expressed as the means ± SEM.⁎P < 0.05 vs SHAM, #P < 0.05 vs OVX by ANOVA followed by a Bonferroni test. Number of animals used is indicated in parentheses.

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Fig. 7. The effects of endothelium removal (a), NG-nitro-Larginine methyl ester (L-NAME, 100 μM) (b) and tetraethylammonium (TEA, 5 mM) (c) on potassium-induced relaxation in the aortic rings from SHAM, OVX and OVX + E2 groups, previously incubated in a K+-free medium and contracted with phenylephrine before and after incubation with 100 μM ouabain. The results are expressed as the means ± SEM.⁎P < 0.05 vs SHAM, #P < 0.05 vs OVX by ANOVA followed by a Bonferroni test. The number of animals used is indicated in parentheses.

suggest a greater contribution of potassium channels on basal tone and ACh-induced relaxation after 8 weeks of estrogen deficiency. Potassium channels participate in a different way in cardiovascular diseases such as hypertension [39], atherosclerosis [19] and stroke [40], therefore, we investigated the participation of different potassium channels on basal tone regulation and in NO mediated ACh-induced relaxation. The literature demonstrates that in aortic vessels, the basal tone is highly dependent on Kv activity [41] and our results indicate that Kv are more activated in ovariectomized animals and have a greater contribution in regulating the basal tone in these animals because 4-aminopyridine, a selective Kv blocker, induced a greater increase in basal tone and reduced the relaxation induced by ACh to a greater extent in preparations from ovariectomized animals. We also detected higher levels of Kv2.1 protein expression in ovariectomized animals. We next analyzed the contribution of BKCa on ACh-induced

conductance, calcium- and voltage-activated potassium (BKCa) channel in coronary myocytes and BKCa channel is an important mediator of estrogen-induced coronary relaxation [36,37]. However, our results indicate that estrogen deficiency stimulates BKCa and Kv channels in ovariectomized animals, suggesting a compensatory mechanism to prevent endothelial dysfunction in this premenopausal model, even though, the protein levels of BKCa were found increased in the estrogen replacement group. This data corroborates previous finding in the literature showing that estrogen regulates the α subunit of BKCa [38]. Firstly, TEA was used to evaluate the overall contribution of potassium channels to the basal tone and in ACh-induced relaxation. TEA had a greater effect on basal tone in preparations from the ovariectomized group compared to the SHAM group, TEA also reduced the relaxation induced by ACh but this effect was more effective in the ovariectomized group when compared to the SHAM group; these results

Fig. 8. Vascular superoxide anion production in segments of aorta from SHAM (a), OVX (b) and OVX + E2 (c) groups. Representative fluorescent photomicrographs of inverted microscopic arterial sections labelled with the oxidative dye hydroethidine and vascular superoxide anion quantification. Vascular nitric oxide release in segments of aorta from SHAM (d), OVX (e) and OVX + E2 (f) groups. Representative fluorescent photomicrographs of inverted microscopic arterial sections labelled with DAF-2 and vascular nitric oxide quantification. The results are expressed as the means ± SEM. *P < 0.05 vs SHAM, #P < 0.05 vs OVX by ANOVA followed by a Bonferroni test. The number of animals used is indicated in parentheses.

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as a homeostatic mechanism to regulate resting membrane potential during enhanced oxidative stress in disease states [45]. A possible explanation for the results from the apamin and IbTX association is that the higher total amount of intracellular Ca2 + after BKCa blockade contributes to the activation of SK in aortas from ovariectomized animals. The residual relaxation to acetylcholine in high-K+ pre-contracted vessels was abolished by L-NAME, which indicates an additional effect of NO, independent of potassium channel activation, on acetylcholineinduced relaxation in these vessels. The role of endothelial NO in modulating vascular tone in conductance arteries is well established [16,46,47] and it seems that the acetylcholine-induced relaxation in the aorta was entirely dependent on nitric oxide release because L-NAME abolished the response to acetylcholine in vessels pre-contracted with phenylephrine and high potassium as well. It is well known that Kv and BKCa channels are present in the vascular smooth muscle [17], therefore we investigated the endothelium-independent relaxation induced by sodium nitroprusside. After IbTX or 4-AP incubation, there was a greater decrease in the relaxation induced by sodium nitroprusside in aortic rings from ovariectomized animals, suggesting that these channels participate in nitric oxide-induced relaxation to a greater extent in ovariectomized animals, although, we cannot rule out alterations in nitric oxide-derived cGMPdependent mechanisms after 8 weeks of estrogen deficiency. Nitric oxide produces vasodilatation of the vascular smooth muscle cells and it could also stimulate Na+/K+-ATPase activity. Na+/K+ATPase activation is an important mechanism that contributes to the maintenance of vascular tone and membrane potential in vascular smooth muscle cells [15,18,48,49] and changes in its activity have been associated with abnormalities in endothelium-dependent vasodilatation. Our results suggest that Na+/K+-ATPase activity is increased in ovariectomized animals. Interestingly, endothelial removal or the nitric oxide synthase blocker, L-NAME, were not able to inhibit the increase in activity. On the other hand, the nonselective blocker of potassium channels, TEA, was able to prevent this increase in Na+-K+ ATPase activity, suggesting a similar action between potassium channels and Na+/K+-ATPase activity in the ovariectomized group. We also observed that estrogen replacement had a larger impact on endothelial removal or LNAME incubation in OVX + E2 group. This finding could be due to differences in the expression of Na+-K+ ATPase alpha-2 subunit. It is well described that estradiol regulates Na+-K+ ATPase alpha-2 subunit and it is dose dependent [50] and we also know that the Na+-K+ ATPase alpha-2 subunit is more sensitive to ouabain [51]. The limitations of the present investigation need to be addressed. First, the reduction in NO release when we incubated the aortic rings with DAF-2 could be attributed to a decrease in fluorescence of the internal elastic lamina, although images were taken from all aortic rings before DAF-2 was incubated to rule out auto-fluorescence. Secondly, the protein expression for BKCa in the ovariectomized animals is the same as compared to the SHAM group. In addition, the estrogen replacement group showed higher values of the BKCa α-subunit. Further experiments will be necessary to elucidate this issue. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals after 8 weeks due to the activation of K+ channels and increased Na+/K+ ATPase activity.

Fig. 9. Representative photomicrographs of p22phox immunofluorescence (arrow) in aortic rings from SHAM (a), OVX (b) and OVX + E2 (c) groups.

relaxation [22,42–44]. Our results show that iberiotoxin alone, a selective BKCa blocker, did not modify the basal tone in all groups, suggesting that this channel equally contributes to the basal tone in preparations from all groups. Regarding the involvement of BKCa channels on ACh-induced relaxation, our results demonstrate that iberiotoxin alone did not modify the relaxation induced by acetylcholine in OVX animals when compared to control, suggesting that estrogen deficiency did not lead to the activation of this channel in the endothelium-dependent relaxation induced by acetylcholine, however, when we incubated iberiotoxin plus apamin, we observed a decrease in ACh-induced relaxation, suggesting that both BKCa and SKCa could be more activated in the ovariectomized group. To confirm this hypothesis, we incubated rings with the BKCa activator (NS1619) and the SKCa/IKCa activator (NS309). Our results confirmed that these channels have a greater contribution in ovariectomized animals. It is possible that BKCa is being activated by reactive oxygen species in the ovariectomized group. Current data on the effect of oxidative stress on BKCa channel activity in smooth muscle suggest that O2%− and H2O2 enhance BKCa channel activity, whereas ONOO%− decreases it. These differential responses depending on the free radical involved may serve

Sources of funding This study was supported by grants from CAPES; CNPq (455294/ 2014-3); and FAPES/FUNCITEC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Fig. 10. Densitometric analysis of the Western blot for eNOS (a), NOX-1 (b), Cu/Zn-SOD (c), Mn-SOD (d), Kv 2.1 (e), Kv 1.5 (f), maxi-K α (g) and SK3 (h) protein expression in aortic artery from SHAM, OVX and OVX + E2 groups. Number of animals used is indicated in parenthesis. *P < 0.05vs. SHAM, #P < 0.05 vs. OVX by One-way ANOVA followed by a Bonferroni test. The number of animals used is indicated in parentheses.

Author contribution

Conflict of interest

Rogério Faustino Ribeiro Júnior, Jonaina Fiorim, Vinicius Bermond Marques, Karoline de Sousa Ronconi, Tatiani Botelho and Marcella Grando performed the experiments, analyzed the data, discussed the results and wrote the paper, Lusiane Bendhack, Dalton Valentim Vassallo and Ivanita Stefanon were involved in discussing the results and writing the paper.

The authors declare that there are no conflicts of interest related to this work. The results are expressed as the means ± SEM of the number of animals shown in Fig. 4. Rmax, maximal effect; pD2, − log one-half Rmax. P < 0.05 vs. SHAM animals (*) and OVX animals (#). One-way ANOVA followed by a Bonferroni test.

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813–821. [27] A.A. Fernandes, R.F. Ribeiro Jr., V.G. de Moura, F.D. Siman, F.M. Dias, J. Zoghaib, et al., SERCA-2a is involved in the right ventricular function following myocardial infarction in rats, Life Sci. 124 (2015 Mar 1) 24–30. [28] R.E. White, Estrogen and vascular function, Vasc. Pharmacol. 38 (2) (2002 Feb) 73–80. [29] A. Virdis, L. Ghiadoni, S. Pinto, M. Lombardo, F. Petraglia, A. Gennazzani, et al., Mechanisms responsible for endothelial dysfunction associated with acute estrogen deprivation in normotensive women, Circulation 101 (19) (2000 May 16) 2258–2263. [30] G. Mercuro, S. Zoncu, F. Saiu, M. Mascia, G.B. Melis, G.M. Rosano, Menopause induced by oophorectomy reveals a role of ovarian estrogen on the maintenance of pressure homeostasis, Maturitas 47 (2) (2004 Feb 20) 131–138. [31] L.M. Yung, W.T. Wong, X.Y. Tian, F.P. Leung, L.H. Yung, Z.Y. Chen, et al., Inhibition of renin-angiotensin system reverses endothelial dysfunction and oxidative stress in estrogen deficient rats, PLoS One 6 (3) (2011 Mar 29) e17437. [32] S. Wassmann, A.T. Baumer, K. Strehlow, E.M. van, C. Grohe, K. Ahlbory, et al., Endothelial dysfunction and oxidative stress during estrogen deficiency in spontaneously hypertensive rats, Circulation 103 (3) (2001 Jan 23) 435–441. [33] C.M. Wong, X. Yao, Au CL, S.Y. Tsang, K.P. Fung, I. Laher, et al., Raloxifene prevents endothelial dysfunction in aging ovariectomized female rats, Vasc. Pharmacol. 44 (5) (2006 May) 290–298. [34] S.L. Stice, S.P. Ford, J.P. Rosazza, D.E. Van Orden, Role of 4-hydroxylated estradiol in reducing Ca2 + uptake of uterine arterial smooth muscle cells through potentialsensitive channels, Biol. Reprod. 36 (2) (1987 Mar) 361–368. [35] J.K. Crews, R.A. Khalil, Antagonistic effects of 17 beta-estradiol, progesterone, and testosterone on Ca2 + entry mechanisms of coronary vasoconstriction, Arterioscler. Thromb. Vasc. Biol. 19 (4) (1999 Apr) 1034–1040. [36] Y.S. Prakash, A.A. Togaibayeva, M.S. Kannan, V.M. Miller, L.A. Fitzpatrick, G.C. Sieck, Estrogen increases Ca2 + efflux from female porcine coronary arterial smooth muscle, Am. J. Phys. 276 (3 Pt 2) (1999 Mar) H926–H934. [37] R.E. White, D.J. Darkow, J.L. Lang, Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism, Circ. Res. 77 (5) (1995 Nov) 936–942. [38] K. Jamali, B.R. Naylor, M.J. Kelly, O.K. Ronnekleiv, Effect of 17beta-estradiol on mRNA expression of large-conductance, voltage-dependent, and calcium-activated potassium channel alpha and beta subunits in guinea pig, Endocrine 20 (3) (2003 Apr) 227–237. [39] G.E. Callera, A. Yogi, R.C. Tostes, L.V. Rossoni, L.M. Bendhack, Ca2 +-activated K+ channels underlying the impaired acetylcholine-induced vasodilation in 2K-1C hypertensive rats, J. Pharmacol. Exp. Ther. 309 (3) (2004 Jun) 1036–1042. [40] J. Ledoux, M.E. Werner, J.E. Brayden, M.T. Nelson, Calcium-activated potassium channels and the regulation of vascular tone, Physiology (Bethesda) 21 (2006 Feb) 69–78. [41] P. Tammaro, A.L. Smith, S.R. Hutchings, S.V. Smirnov, Pharmacological evidence for a key role of voltage-gated K+ channels in the function of rat aortic smooth muscle cells, Br. J. Pharmacol. 143 (2) (2004 Sep) 303–317. [42] A. Cheong, K. Quinn, A.M. Dedman, D.J. Beech, Activation thresholds of K(V), BK andCl(Ca) channels in smooth muscle cells in pial precapillary arterioles, J. Vasc. Res. 39 (2) (2002 Mar) 122–130. [43] B. Eichhorn, D. Dobrev, Vascular large conductance calcium-activated potassium channels: functional role and therapeutic potential, Naunyn Schmiedeberg's Arch. Pharmacol. 376 (3) (2007 Nov) 145–155. [44] J. Ledoux, D.M. Gee, N. Leblanc, Increased peripheral resistance in heart failure: new evidence suggests an alteration in vascular smooth muscle function, Br. J. Pharmacol. 139 (7) (2003 Aug) 1245–1248. [45] Y. Liu, D.D. Gutterman, Oxidative stress and potassium channel function, Clin. Exp. Pharmacol. Physiol. 29 (4) (2002 Apr) 305–311. [46] L. Urakami-Harasawa, H. Shimokawa, M. Nakashima, K. Egashira, A. Takeshita, Importance of endothelium-derived hyperpolarizing factor in human arteries, J. Clin. Invest. 100 (11) (1997 Dec 1) 2793–2799. [47] M. Feletou, P.M. Vanhoutte, EDHF: an update, Clin. Sci. (Lond.) 117 (4) (2009 Aug) 139–155. [48] V.M. Bolotina, S. Najibi, J.J. Palacino, P.J. Pagano, R.A. Cohen, Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle, Nature 368 (6474) (1994 Apr 28) 850–853. [49] M.P. Blaustein, Physiological effects of endogenous ouabain: control of intracellular Ca2 + stores and cell responsiveness, Am. J. Phys. 264 (6 Pt 1) (1993 Jun) C1367–C1387. [50] J. Palacios, E.T. Marusic, N.C. Lopez, M. Gonzalez, L. Michea, Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors, Am. J. Physiol. Heart Circ. Physiol. 286 (5) (2004 May) H1793–H1800. [51] M. Juhaszova, M.P. Blaustein, Na+ pump low and high ouabain affinity alpha subunit isoforms are differently distributed in cells, Proc. Natl. Acad. Sci. U. S. A. 94 (5) (1997 Mar 4) 1800–1805.

References [1] R.F. Ribeiro Jr., F.F. Potratz, B.M. Pavan, L. Forechi, F.L. Lima, J. Fiorim, et al., Carvedilol prevents ovariectomy-induced myocardial contractile dysfunction in female rat, PLoS One 8 (1) (2013) e53226. [2] R.F. Ribeiro Jr., B.M. Pavan, F.F. Potratz, J. Fiorim, M.R. Simoes, F.M. Dias, et al., Myocardial contractile dysfunction induced by ovariectomy requires AT1 receptor activation in female rats, Cell. Physiol. Biochem. 30 (1) (2012) 1–12. [3] R.L. dos Santos, F.B. da Silva, R.F. Ribeiro Jr., I. Stefanon, Sex hormones in the cardiovascular system, Horm. Mol. Biol. Clin. Invest. 18 (2) (2014 May) 89–103. [4] J.E. Rossouw, G.L. Anderson, R.L. Prentice, A.Z. LaCroix, C. Kooperberg, M.L. Stefanick, et al., Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial, JAMA 288 (3) (2002 Jul 17) 321–333. [5] M.G. Shlipak, L.A. Chaput, E. Vittinghoff, F. Lin, V. Bittner, R.H. Knopp, et al., Lipid changes on hormone therapy and coronary heart disease events in the Heart and Estrogen/progestin Replacement Study (HERS), Am. Heart J. 146 (5) (2003 Nov) 870–875. [6] J.M. Orshal, R.A. Khalil, Gender, sex hormones, and vascular tone, Am. J. Phys. Regul. Integr. Comp. Phys. 286 (2) (2004 Feb) R233–R249. [7] F. Grodstein, M.J. Stampfer, J.E. Manson, G.A. Colditz, W.C. Willett, B. Rosner, et al., Postmenopausal estrogen and progestin use and the risk of cardiovascular disease, N. Engl. J. Med. 335 (7) (1996 Aug 15) 453–461. [8] M. Feletou, Endothelium-dependent hyperpolarization and endothelial dysfunction, J. Cardiovasc. Pharmacol. (2015 Nov 26). [9] M.A. Maturana, M.C. Irigoyen, P.M. Spritzer, Menopause, estrogens, and endothelial dysfunction: current concepts, Clinics (Sao Paulo) 62 (1) (2007 Feb) 77–86. [10] A.P. Dantas, M.C. Franco, M.M. Silva-Antonialli, R.C. Tostes, Z.B. Fortes, D. Nigro, et al., Gender differences in superoxide generation in microvessels of hypertensive rats: role of NAD(P)H-oxidase, Cardiovasc. Res. 61 (1) (2004 Jan 1) 22–29. [11] K.H. Kim, J.R. Bender, Rapid, estrogen receptor-mediated signaling: why is the endothelium so special? Sci. STKE 2005 (288) (2005 Jun 14) e28. [12] F.M. Dias, R.F. Ribeiro Jr., A.A. Fernandes, J. Fiorim, T.C. Travaglia, D.V. Vassallo, et al., Na+ K+-ATPase activity and K+ channels differently contribute to vascular relaxation in male and female rats, PLoS One 9 (9) (2014) e106345. [13] P. Forte, B.J. Kneale, E. Milne, P.J. Chowienczyk, A. Johnston, N. Benjamin, et al., Evidence for a difference in nitric oxide biosynthesis between healthy women and men, Hypertension 32 (4) (1998 Oct) 730–734. [14] K. Kauser, G.M. Rubanyi, Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae, Am. J. Phys. 267 (6 Pt 2) (1994 Dec) H2311–H2317. [15] S. Gupta, C. McArthur, C. Grady, N.B. Ruderman, Stimulation of vascular Na(+)-K (+)-ATPase activity by nitric oxide: a cGMP-independent effect, Am. J. Phys. 266 (5 Pt 2) (1994 May) H2146–H2151. [16] J. Fiorim, R.F. Ribeiro Jr., B.F. Azevedo, M.R. Simoes, A.S. Padilha, I. Stefanon, et al., Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats, Toxicol. Appl. Pharmacol. 262 (1) (2012 Ju1 1) 22–31. [17] M. Feletou, Calcium-activated potassium channels and endothelial dysfunction: therapeutic options? Br. J. Pharmacol. 156 (4) (2009 Feb) 545–562. [18] J. Marin, J. Redondo, Vascular sodium pump: endothelial modulation and alterations in some pathological processes and aging, Pharmacol. Ther. 84 (3) (1999 Dec) 249–271. [19] M.T. Nelson, J.M. Quayle, Physiological roles and properties of potassium channels in arterial smooth muscle, Am. J. Phys. 268 (4 Pt 1) (1995 Apr) C799–C822. [20] M. Feletou, P.M. Vanhoutte, Endothelium-dependent hyperpolarizations: past beliefs and present facts, Ann. Med. 39 (7) (2007) 495–516. [21] A.M. Briones, A.S. Padilha, A.L. Cogolludo, M.J. Alonso, D.V. Vassallo, F. PerezVizcaino, et al., Activation of BKCa channels by nitric oxide prevents coronary artery endothelial dysfunction in ouabain-induced hypertensive rats, J. Hypertens. 27 (1) (2009 Jan) 83–91. [22] E.A. Ko, J. Han, I.D. Jung, W.S. Park, Physiological roles of K+ channels in vascular smooth muscle cells, J. Smooth Muscle Res. 44 (2) (2008 Apr) 65–81. [23] P.R. Bianchi, B.P. Gumz, K. Giuberti, I. Stefanon, Myocardial infarction increases reactivity to phenylephrine in isolated aortic rings of ovariectomized rats, Life Sci. 78 (8) (2006 Jan 18) 875–881. [24] A.S. Paigel, R.F. Ribeiro Jr., A.A. Fernandes, G.P. Targueta, D.V. Vassallo, I. Stefanon, Myocardial contractility is preserved early but reduced late after ovariectomy in young female rats, Reprod. Biol. Endocrinol. 9 (2011) 54. [25] L.V. Rossoni, M. Salaices, J. Marin, D.V. Vassallo, M.J. Alonso, Alterations in phenylephrine-induced contractions and the vascular expression of Na+,K+-ATPase in ouabain-induced hypertension, Br. J. Pharmacol. 135 (3) (2002 Feb) 771–781. [26] A.A. Fernandes, T.O. Faria, R.F. Ribeiro Jr., G.P. Costa, B. Marchezini, E.A. Silveira, et al., A single resistance exercise session improves myocardial contractility in spontaneously hypertensive rats, Braz. J. Med. Biol. Res. 48 (9) (2015 Sep)

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