ORIGINAL ARTICLE
Ouabain Induces Nitric Oxide Release by a PI3K/ Akt-dependent Pathway in Isolated Aortic Rings From Rats With Heart Failure Fabiana D. M. Siman, PhD,* Edna A. Silveira, PhD,* Aurélia A. Fernandes, PhD,* Ivanita Stefanon, PhD,* Dalton V. Vassallo, PhD,*† and Alessandra S. Padilha, PhD*
Background: Ouabain occurs in nanomolar concentrations in myocardial infarction and heart failure (HF). However, the effects of ouabain in vascular function in HF conditions were not investigated yet. Therefore, we analyzed the effects of acute administration of 3 nM ouabain in isolated aortic rings from rats with HF 4 weeks after myocardial infarction.
Methods and Results: Rats were submitted to sham operation or coronary artery occlusion. In HF rats, left ventricular positive and negative derivatives of intraventricular pressure reduced and left ventricular end diastolic pressure increased. Phenylephrine responses increased in HF rings when compared with controls. Ouabain incubation for 45 minutes reduced phenylephrine-induced contraction in both groups. Endothelial removal increased more phenylephrine response in ouabain-treated rings of sham rats. Ouabain potentiated the effect of L-NAME in both groups but more in sham rats. Wortmannin increased the phenylephrine response only in HF rings. The effect of tetraethylammonium was potentiated by ouabain only in HF rings. Ouabain increased phenylephrine-stimulated nitric oxide production in rings from both groups but increased the activation of Akt only in vessels from HF rats. Conclusions: Results demonstrate that low ouabain concentration can decrease vascular reactivity of aortic rings from HF rats. Ouabain was able to increase nitric oxide production in HF rats by triggering a signal transduction PI3K/Akt-dependent pathway and increasing an endothelium-hyperpolarizing factor release. Key Words: ouabain, heart failure, vascular reactivity, nitric oxide, PI3K/Akt pathway (J Cardiovasc Pharmacol Ô 2015;65:28–38)
INTRODUCTION Ouabain is an endogenous cardiac glycoside produced in the central nervous system,1,2 in the adrenal cortex3 and in Received for publication March 10, 2014; accepted August 1, 2014. From the *Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, Brazil; and †Department of Physiological Sciences, EMESCAM, Vitoria, Brazil. Supported by grants from CNPq, CAPES, and FAPES/FUNCITEC. The authors report no conflicts of interest. Reprints: Alessandra S. Padilha, PhD, Department of Physiological Sciences, Federal University of Espirito Santo, Av. Marechal Campos, 1468, Maruípe, 29043-090 Vitoria, ES, Brazil (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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the heart4 in response to volume expansion, angiotensin II, and/or adrenocorticotropic hormone stimulation.5–7 It has been implicated in the control of many functions involving the cardiovascular and nervous systems,6–9 as control of blood pressure and sodium balance.10,11 Alternately, the increase of endogenous ouabain (EO) has been associated to the pathogenesis of various disorders, including hypertension,5,9,10 diabetes,12 myocardial infarction (MI),13 and congestive heart failure (HF).14,15 Moreover, it also increases cellular proliferation and differentiation of the heart16 and vascular smooth muscle,17 which could contribute to the maintenance of hypertension. However, the pathophysiologic role for elevated levels of EO in these diseases is still under debate. Several studies have suggested that EO may have a primary role in causing cardiac dysfunction and failure.14,18–20 In a study in patients with idiopathic dilated cardiomyopathy, the high circulating levels of EO identified in those individuals predisposed to progress more rapidly to HF.14 Classically, ouabain inhibits Na+, K+ATPase activity by binding preferentially to the a-2 and a-3 isoforms of the a-subunit in rodents, in the plasmerosome region.21,22 This inhibition increases the Na+ concentration in the plasmerosome region, which reduces the activity of the Na+/Ca2+ exchanger (NCX) and, consequently, increases the amount of activator Ca2+ for contraction.22–24 As a result, on activation intracellular calcium concentration increases, and consequently, an increase in the vascular tone and/or positive inotropic effects occur. However, ouabain been shown to have variable vascular effects on different blood vessels, that could be attributed, at least in part, to the length of treatment, doses used, and the kind of artery (conductance or resistance). Thus, it can increase25,26 or decrease vascular contractile responses27,28 in resistance and conductance arteries, respectively. It has been reported that the decrease in vascular contractile responses in conductance arteries, specifically in aorta, induced by ouabain could be related the release nitric oxide (NO) by the endothelium.27 In fact, ouabain-induced hypertension has been accompanied by the endothelial NO-induced modulation of vasoconstrictor responses in the aorta.27 Other studies have also demonstrated that ouabain enhances basal NO release by human umbilical cord endothelial cells29 and porcine carotid artery endothelium.30 Eva et al29 reported that nanomolar concentrations of ouabain stimulate NO release by triggering J Cardiovasc Pharmacol ä Volume 65, Number 1, January 2015
J Cardiovasc Pharmacol ä Volume 65, Number 1, January 2015
a signal transduction pathway, including the activation of phosphoinositide-3 kinase (PI3K) and the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). These observations prompted us to investigate if the endothelial NO-induced modulation of vasoconstrictor responses induced by ouabain might be altered in conductance arteries in HF rats.
METHODS Animals Studies were performed on male Wistar rats (220–240 g). All experiments were conducted in compliance with the guidelines for biomedical research as stated by the Brazilian Societies of Experimental Biology and approved by the Institutional Committee of Ethics on Animal Research (CEUA-UFES 028/ 2012). All rats had free access to water and were fed rat chow ad libitum.
Myocardial Infarction Myocardial infarction was induced by ligation of the coronary artery as described previously.31 Briefly, animals were anesthetized with ketamine (50 mg/kg; Agener, MG, Brazil) and xylazine (10 mg/kg; Bayer, SP, Brazil). A thoracotomy at the fourth intercostal space was used to quickly expose the heart. The left coronary artery was permanently occluded ;2 mm from its origin with a 6-0 mononylon suture. The heart was rapidly returned to its position, and the thorax was closed. Mechanical respiratory support was used in those animals that did not recover spontaneous respiration. A group of animals was submitted to a sham operation in which all surgical procedures were performed as described, except that the coronary suture was not tied. After recovery, the animals were kept in collective cages at the animal facility. From the total number of 26 rats that were MI operated. Seventeen of the 26 rats (70%) survived the entire 4-week period and were included for analysis. All the 26 animals survived the sham surgery procedure.
Hemodynamic Measurements Four weeks after surgery, the animals were anesthetized with urethane (1.2 g/kg, intraperitoneally) and, after loss of the righting reflex, the right common carotid artery was dissected to insert a fluid-filled polyethylene catheter (P50) connected to a blood pressure transducer (TSD 104A; Biopac System, Goleta, CA). The catheter was then advanced into the left ventricle (LV). The following measurements were performed: systolic and diastolic blood pressure, left ventricular systolic pressure, left ventricular end diastolic pressure (LVEDP), heart rate, and positive and negative first derivatives of intraventricular pressure (dP/dt+ and dP/dt2, respectively). After hemodynamic recordings, the heart and lungs were removed. The right ventricle (RV) and LV were separated, rinsed in a physiological solution (0.9% NaCl), blotted, and weighed. Scar tissue was separated from the remaining LV muscle as previously described.32 The scar and muscle areas were determined by planimetry, and the infarct area was expressed as the percentage of LV surface covered by the scar Ó 2014 Lippincott Williams & Wilkins
Ouabain Induces NO Release in HF Rats
tissue. HF was defined using 3 criteria as follow: lung/body weight (BW) ratio greater than lung/BWsham (approximately 2-fold),33 RV hypertrophy (RV/BW) proportional to lung/ BW,34 and LVEDP greater than 15 mm Hg.34,35
Vascular Reactivity Studies After the hemodynamic measurements, rats were killed by exsanguination. The thoracic aortas were carefully dissected and separated from the connective tissue. For the reactivity experiments, the aortas were divided into cylindrical segments 4 mm in length. Segments of thoracic aorta were mounted in an isolated tissue chamber containing Krebs–Henseleit solution (in millimolar: NaCl 118; KCl 4.7; NaHCO3 23; CaCl2 2.5; KH2PO4 1.2; MgSO4 1.2; glucose 11, and EDTA 0.01), gassed with 95% O2 and 5% CO2 and maintained at a resting tension of 1 g at 378C. Isometric tension was recorded using an isometric force transducer (TSD125C; CA) connected to an acquisition system (MP100; Biopac Systems Inc, Santa Barbara, CA). After a 45-minute equilibration period, all aortic rings were initially exposed twice to 75 mM KCl, first to check their functional integrity and again to assess the maximal tension that could be developed. Afterward, endothelial integrity was tested with acetylcholine (10 mM) in segments that were previously contracted with phenylephrine (1 mM). After a washout period (30 minutes), the aortic rings were incubated with ouabain (3 nM) or not (control) for 45 minutes. Then, concentration–response curves to phenylephrine (0.1 nM–0.3 mM) were determined. The ouabain concentration for these experiments was chosen based on previous reports that demonstrated nanomolar levels of circulating ouabain in the plasma of patients with cardiac dysfunction.36,37 As the experiments were performed in isolated aortic rings, the addition of exogenous ouabain sought to mimic the vascular effects of circulating ouabain in rats with HF. The influence of the endothelium on the response to phenylephrine in the presence or absence of ouabain (3 nM) was investigated after its mechanical removal by rubbing the lumen with a needle. The absence of endothelium was confirmed by the inability of 10 mM acetylcholine to induce relaxation. The role of endothelial-derived vasoactive factors on the phenylephrine-elicited contractile response was investigated. The effects of the following drugs were evaluated: (1) a nonspecific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 100 mM); (2) the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (0.1 mM); and (3) the potassium (K+) channel blocker tetraethylammonium (TEA, 2 mM, nonselective blocker of K+ channels). These drugs were added 45 minutes before examining the concentration– response curves to phenylephrine in the presence or absence of ouabain.
Immunodetection of Akt To confirm the involvement of the PI3K/Akt pathway in oubain-induced NO release, Akt expression and phosphorylated-Ser473 Akt levels were determined in homogenates from aortic segments (those used for the reactivity www.jcvp.org |
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TABLE 1. Morphometric and Hemodynamic Parameters of Sham and HF Rats Sham (N = 26) BW, g LV, mg LV/BW, mg/g RV, mg RV/BW, mg/g Lung, mg Lung/BW, mg/g Infarct area, % Aortic SP, mm Hg Aortic DP, mm Hg MBP, mm Hg LVSP, mm Hg LVEDP, mm Hg LV + dP/dT, mm Hg/s LV 2 dP/dT, mm Hg/s Heart rate, beats per minute
351 785 2.24 213 0.6 1832 5.25
6 6 6 6 6 6 6
4.6 11.1 0.02 5.05 0.01 50.8 0.13
107 70 87 111 3.6 5554 26038 346
6 6 6 6 6 6 6 6
3.2 3.1 3.6 3.6 0.5 481 334 13.2
HF (N = 18) 314 822 2.62 410 1.3 3198 10.1 36 104 76.4 92 110 18.4 3977 24918 368
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
9.3* 37.1 0.1* 24.7* 0.07* 106* 0.4* 1.5 2.9 2.2 3.3 3.6 1.3* 269* 196* 10.5
Values are mean values 6 SEM. Values in parentheses represent the number of animals used in that analysis. *t test: P , 0.05 versus sham. SP, systolic pressure; DP, diastolic pressure; MBP, mean blood pressure; LVSP, left ventricle systolic pressure; dP/dt, maximal rate of pressure rise.
experiments) that had been preincubated with or without ouabain (for 45 minutes). Thus, after the reactivity experiment, arteries were rapidly frozen in liquid nitrogen and kept at 2708C until the day of analysis. Proteins from homogenized arteries (80 mg) were separated by 7.5% SDS-PAGE and then transferred to nitrocellulose membranes. Next, the membrane was incubated with rabbit polyclonal antibody for Akt (1:500; Santa Cruz Biotechnology) and phosphorylated-Ser473 Akt (1:500; Santa Cruz Biotechnology). After washing, the membranes were incubated with anti-rabbit (1:7500; StressGen, Victoria, Canada) immunoglobulin antibody conjugated to horseradish peroxidase. After thorough washing, immunocomplexes were detected using an enhanced horseradish peroxidase/luminal chemiluminescence system (ECL Plus; Amersham International, Little Chalfont, United Kingdom) and film (Hyperfilm ECL International). Signals on the immunoblot were quantified with the National Institutes of Health Image V1.56 computer program. The same membrane was used to determine a-actin expression using a mouse monoclonal antibody (1:5000; Sigma).
ouabain (3 nM) for 45 minutes at 378C. Concentration– response curve to phenylephrine (0.1 nM–0.3 mM) was performed, and the medium was collected to measure stimulated NO production. The fluorescence of the medium was measured at room temperature using a spectrofluorometer (BioTek Synergy 2 Instruments, BioTek’s Gen 5 Software) with excitation wavelength set at 492 nm and emission wavelength at 515 nm. Blank measurement samples were similarly collected but without arteries to subtract background emission. The amount of NO released was expressed as arbitrary units per milligram tissue.
Data Analyses and Statistics
All values are expressed as the mean 6 SEM. Contractile responses was expressed as a percentage of the maximal response induced by 75 mM KCl. For each concentration– response curve, the maximal effect (Rmax) and the concentration of agonist that produced 50% of the maximal response (log EC50) were calculated using nonlinear regression analysis (GraphPad Prism; GraphPad Software Inc, San Diego, CA). The sensitivities of the agonists were expressed as pD2 (2log EC50). Rmax was expressed as a percentage of contraction to KCl 75 mmol/L. To compare the effect of drugs on the response to phenylephrine in aortic rings from both groups, some results were expressed as differences of the area under the concentration–response curves (dAUC) in control and experimental situations. AUCs were calculated from individual concentration–response curve plots; the differences were expressed as a percentage of the AUC of the control situation. The results of the Akt expression and phosphorylatedSer473 Akt level analysis were expressed as the ratio between phosphoAkt/Akt. The results were expressed as the mean 6 SEM of the number of rats indicated; differences were analyzed using Student’s t test or 2-way analysis of variance followed by a Tukey’s test. P , 0.05 was considered significant.
Drugs and Reagents Ouabain, L-phenylephrine hydrochloride, L-NAME, acetylcholine chloride, wortmannin, and tetraethylammonium were purchased from Sigma–Aldrich (St Louis, MO). Salts and reagents used were of analytical grade from Sigma– Aldrich and Merck (Darmstadt, Germany). Wortmannin was dissolved in dimethyl sulfoxide (DMSO) and diluted in deionized water. All other drugs were dissolved and diluted in deionized water. At the final bath concentration used (0.005%), DMSO vehicle had no effect on contractile responses.
NO Release NO release was measured as previously described.37 The segments of the thoracic aorta artery were dissected and equilibrated for 30 minutes in HEPES buffer (in mmol$L21: 119 NaCl; 20 HEPES; 1.2 CaCl2; 4.6 KCl; 1 MgSO4; 0.4 KH2PO4; 5 NaHCO3; 5.5 glucose; 0.15 NaH2PO4; pH 7.4) at 378C. The arteries were then incubated with the fluorescent probe 4,5-diaminofluorescein (2 mmol$L21) for 30 minutes, and the medium was collected to measure basal NO release. Then, the aortic rings were incubated in the presence or absence of
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RESULTS Global Parameters and Hemodynamics The global morphological and hemodynamic characteristics of HF and sham groups are described in Table 1. BW was lower in the HF group compared with the sham group. As expected, the lung/BW and RV/BW ratios were increased in the HF rats compared with the sham controls (Table 1). Concerning hemodynamic parameters, no differences were Ó 2014 Lippincott Williams & Wilkins
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FIGURE 1. Concentration–response curves to phenylephrine in aortic rings of HF and sham rats (A). Effect of ouabain (Oua) incubation on the contraction induced by phenylephrine on the aortic rings of sham (B) and HF (C) rats. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
observed in arterial pressure or left ventricular systolic pressure among groups (Table 1). However, LV function was affected in the HF rats, with a reduction of inotropic indexes of contractility, the +dP/dt and 2dP/dt, and an elevation of the LVEDP (Table 1).
Vascular Reactivity Study The maximal response to 75 mM KCl in segments with endothelium (sham: 2.37 6 0.06 g, n = 26; HF: 2.26 6 0.08 g; n = 18; P . 0.05) and after endothelial removal (sham: 2.61 6 0.25 g, n = 6; HF: 2.1 6 0.3 g; n = 7; P . 0.05) was
similar in HF and sham groups. Ach-induced (10 mM) relaxation was also similar in aortic rings from HF and sham rats (sham: 88.25 6 1.60%, n = 25; HF: 88.96 6 2.47%, n = 16). Contractile responses induced by phenylephrine were increased in aortic rings from HF rats compared with sham (Rmax: sham: 104 6 3.04%; HF: 119 6 3.7%; P , 0.05) (Fig. 1A). However, acute ouabain incubation promoted a reduction in the phenylephrine-induced contraction in both groups (Figs. 1B, C; Table 2). To analyze the role of the endothelium in phenylephrine responses, experiments were performed in the absence of endothelium. Endothelial removal left-shifted
TABLE 2. Effect of Endothelial Denudation on the Vascular Response to Phenylephrine (Emax and pD2) in the Presence and Absence of Ouabain (Oua) on the Aortic Rings of Sham and HF Rats pD2
Emax, %
Sham E+ E2 Oua E+ Oua E2
5.98 7.73 5.68 7.61
6 6 6 6
0.17 (25) 0.28 (6)* 0.20 (26) 0.1 (6)‡
HF 6.39 7.9 5.91 7.69
6 6 6 6
0.19 (16) 0.3 (7)† 0.13† (18) 0.15 (7)§
Sham 104.4 131.5 94.4 131.3
6 6 6 6
3.04 (25) 3.0 (6) 3.16 (26)* 6.8 (6)‡
HF 119 135.7 108 123.9
6 6 6 6
3.7 (16)* 7.7 (7) 3.76 (18) 5.6 (7)
Values are means 6SEM; the number of animals is indicated in parentheses. *P , 0.05 versus sham E+ (t test). †P , 0.05 versus HF E+ (t test). ‡P , 0.05 versus sham Oua E+ (t test). §P , 0.05 versus HF Oua E+ (t test). Emax expressed as a percentage of contraction to KCl 75 mmol/L. E2, endothelium denuded; E+, endothelium intact; Emax, maximum response; pD2, negative logarithm of concentration producing 50% of maximum response.
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FIGURE 2. Effect of endothelial removal on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain (E). Concentration–response curve to phenylephrine of aortic rings from sham and HF rats after endothelial removal. Inset shows differences in the area under the concentration–response curve (dAUC) in endothelium-denuded and intact segments in the presence (ouabain) and absence (control) of ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
the concentration–response curves to phenylephrine in aortic segments from both groups, but this effect was greater in the ouabain-treated rings of sham rats, as shown by the dAUCs (Figs. 2A, B; Table 2). Therefore, after endothelial removal, the phenylephrine responses were similar in aortic rings from HF and sham rats (Fig. 2E). This suggests an endothelial modulation of the phenylephrine contractile response. To assess the contribution of endothelium-derived NO in phenylephrine responses induced by ouabain, arteries were incubated with an NOS inhibitor (L-NAME, 100 mM). The incubation with the NOS inhibitor L-NAME (Fig. 3; Table 3) left-shifted the concentration–response curves to phenylephrine
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in aortic segments from HF and sham groups, but this effect was greater in sham rats (Table 3, Figs. 3A, B). These results demonstrate that the negative modulation of the phenylephrine response by NO was reduced in the aortic rings from HF rats. Moreover, in the presence of ouabain, the L-NAME effect was potentiated in both groups, as shown by the dAUCs (Figs. 3B, D). These results indicate that ouabain incubation increased NO bioavailability in both the sham and HF groups. To confirm this increased NO bioavailability, NO release was measured by the fluorescent probe 4,5-diaminofluorescein in segments of the thoracic aorta, in the presence and absence of ouabain. Ouabain increased Ó 2014 Lippincott Williams & Wilkins
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FIGURE 3. Effect of L-NAME on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain. Inset shows differences in area under the concentration–response curve (dAUC) of aortic rings in the presence and absence L-NAME under control conditions and after acute incubation with ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
phenylephrine-stimulated NO release in the aortic rings of both the sham and HF rats (Fig. 4). To analyze whether ouabain stimulates NO production by the PI3K/Akt-dependent pathway, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin was used.
In isolated aortic rings from sham rats, the response to phenylephrine remained unaltered after wortmannin incubation, in the presence and absence of ouabain. In rings of HF rats, in the absence of ouabain, the phenylephrine response was not modified after wortmannin incubation (Fig. 5C).
TABLE 3. Effect of L-NAME on the Vascular Response to Phenylephrine (Emax and pD2) in the Presence and Absence of Ouabain (Oua) on the Aortic Rings of Sham and HF Rats pD2 Sham Control L-NAME Ouabain control Ouabain L-NAME
6.37 6 0.08 6.91 6 0.1* 6.3 6 0.11
Emax, % HF
Sham
HF
6.54 6 0.08 90 6 3.94 112 6 5.43 7.1 6 0.11* 133 6 6.28* 129 6 6.33 6.31 6 0.09 86 6 5.77 106 6 7.13
6.83 6 0.06* 7.13 6 0.06†
136 6 5.8†
136 6 4.62†
Emax expressed as a percentage of contraction to KCl 75 mmol/L. Values are means 6 SEM. *P , 0.05 versus control (t test). †P , 0.05 versus ouabain control (t test). Note that L-NAME has controls. Emax, maximum response; pD2, negative logarithm of concentration producing 50% of maximum response.
Ó 2014 Lippincott Williams & Wilkins
FIGURE 4. Stimulated NO production by aortic rings from sham and HF rats in the presence (ouabain) and absence (control) of ouabain. The results are expressed as the mean 6 SEM. Number of animals used was between 6 and 9. Student’s t test. *P , 0.05 versus controls. www.jcvp.org |
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FIGURE 5. Effect of wortmannin on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
determined the Akt and phosphorylated-Ser473 Akt protein levels by Western blot. We observed that ouabain increased the activation of Akt in the vessels of HF rats, as shown by the phospho/total Akt ratio, suggesting the participation of PI3K/ Akt signaling (Fig. 6). Therefore, these results demonstrated that ouabain increased NO release in both groups, but by different
However, in the ouabain-treated rings of HF rats, the incubation with wortmannin resulted in a left shift in the concentration– response curves to phenylephrine, reducing pD2 (Fig. 5; Table 4). These results suggest that ouabain might stimulate NO production by the PI3K/Akt-dependent pathway in HF rats. Because PI3K activates Akt by phosphorylation at Ser473 and Akt activates eNOS by phosphorylation at Ser1177, we
TABLE 4. Effect of Wortmannin and TEA on the Vascular Response to Phenylephrine (Emax and pD2) in the Presence and Absence of Ouabain (Oua) on the Aortic Rings of Sham and HF Rats pD2
Emax, %
Sham Control Wortmannin Ouabain control Ouabain wortmannin Control TEA Ouabain control Ouabain TEA
6.25 6.19 6.36 6.19 5.5 5.84 5.18 6.23
6 6 6 6 6 6 6 6
0.08 0.16 0.13 0.17 0.12 0.08* 0.11 0.10‡
HF 6.62 6.76 6.35 7.18 5.55 6.05 5.22 6.28
6 6 6 6 6 6 6 6
0.05 0.11 0.12 0.14* 0.13 0.13† 0.10 0.06‡
Sham 107 111 90 101 100 143 90 134
6 6 6 6 6 6 6 6
3.79 4.72 4.72 5.56 6.12 8.35† 6.27 5.35‡
HF 111 105 102 115 110 126 107 136
6 6 6 6 6 6 6 6
5.55 6.81 5.22 6.72 6.45 7.17 8.84 2.78‡
Emax expressed as a percentage of contraction to KCl 75 mmol/L. Values are mean values 6 SEM. *P , 0.05 versus ouabain control (t test). †P , 0.05 versus control (t test). ‡P , 0.05 versus ouabain control (t test). Note that wortmannin and TEA have controls. Emax, maximum response; pD2, negative logarithm of concentration producing 50% of maximum response.
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FIGURE 6. Densitometric analysis of a Western blot for Akt and phosphorylated-Ser473 Akt protein expression by aortic rings cultured in the absence (control) of ouabain and after acute incubation with ouabain. Representative blots are also shown. Number of animals used was between 6 and 8.*P , 0.05 versus HF control by Student’s t test.
mechanisms. Although, in the sham group, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin did not affect the concentration–response curves to phenylephrine, in the HF group, we observed evidence indicating the PI3K/Akt pathway involvement. Because NO can open K+ channels,38 we determined the role of K+ channels in ouabain-induced phenylephrine responses. Arteries were incubated with TEA, a nonselective K+ channel blocker. TEA potentiated the vasoconstrictor response induced by phenylephrine in aortic segments from either group, but this effect was greater in sham rats (Table 4; Figs. 7A, C). Moreover, in the presence of ouabain, the TEA effect was greater in the rings of HF rats, as shown by the dAUC values (Fig. 7B, D; Table 4). These results suggest that ouabain’s effects on phenylephrine-induced contraction in aortic segments from the HF group involve the participation of K+ channels.
DISCUSSION AND CONCLUSIONS
Our study shows, for the first time, that ouabain can modify vascular function in rats with HF. Our results demonstrate that ouabain decreases contractile responses to phenylephrine in sham and HF rats, most likely because of the increased bioavailability of NO. However, ouabain acts through different mechanisms in both groups (sham and HF rats). In the sham rats, ouabain acts by a mechanism independent of the PI3K/Akt pathway, whereas in the HF rats, ouabain increases NO production by a mechanism Ó 2014 Lippincott Williams & Wilkins
Ouabain Induces NO Release in HF Rats
dependent on PI3K/Akt pathways and on potassium channel activation. Evidence suggests that EO may have a primary role in causing cardiac dysfunction and failure, by inducing increasing in left ventricular mass and enhance apoptosis, suggesting a possible association between ouabain and the pathogenesis of HF.14,18–20 Although previous studies have shown that ouabain could modify the vascular function of the ouabaininduced hypertensive rats,8,39–41 the role of this endogenous compound on the vascular function of the HF rats had not yet been investigated. In this study, we observed a reduction in aortic ring reactivity to phenylephrine after ouabain incubation in both the sham and HF groups. This observation is accordance with other reports that demonstrated a reduction vascular reactivity in aortic segments after ouabain incubation.27,42 In aortic rings, ouabain has been related with decrease in vascular contractile responses, which could be related the release NO by endothelium.27,42 However, ouabain might increase vascular contractile responses in resistance arteries.25,26 These contradictory effects could be related, at least in part, to different blood vessels used in these studies. As showed in Figure 2, the endothelial modulation was increased after ouabain incubation only in isolated aortic rings from sham rats. This effect was not observed in HF rats, likely because these rats already present endothelial dysfunction.35,43 Although some reports35,43 have shown that HF rats have endothelial dysfunction, the increase in phenylephrine contractile response in aortic segments from HF rats when compared with sham (Fig. 1A) is not sufficient to confirm the endothelial dysfunction. Moreover, after endothelial removal, the phenylephrine contractile responses in segments from HF and sham rats were similar (Fig. 2E), suggesting a modulating by endothelial factors. Previous reports demonstrated that the endothelial dysfunction and an increased vasoconstrictor response are present in HF dogs.35,43,44 Furthermore, previous studies demonstrated an increase of vascular reactivity to vasoconstrictor agents in aortic rings of HF rats.45,46 This response probably was due to decrease in the NO basal release.45 In fact, the involvement of NO in the contractile response to phenylephrine is reduced in HF rats, as demonstrated by dAUC% (results obtained with L-NAME; Figs. 3A, C; sham: 74.5 6 8.6 vs. HF: 28.3 6 10.8, t test: P , 0.05). Therefore, in accordance with this result, we suggest that in aortic segments of HF rats, the NO-induced modulation of phenylephrine vasoconstrictor responses was reduced. Moreover, when analyzed NO release by fluorescent reagent 4,5-diaminofluorescein (DAF) method, we observed a tendency to NO reduce (P = 0.08) in aortic rings of HF rats. However, other reports demonstrated a reduction phenylephrine contractile response associated with increase of NO release in the tail vascular bed from HF rats after 30 days MI.34,47 These discrepancies about NO bioavailability might result from different studied vessels. However, the association of L-NAME plus ouabain, amplified the contractile response to phenylephrine, but this effect was similar in sham and HF groups. In addition, direct measurements of phenylephrinewww.jcvp.org |
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FIGURE 7. Effect of tetraethylammonium (TEA) on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain. Inset shows differences in area under the concentration–response curve (dAUC) of aortic rings in the presence and absence of TEA under control conditions and after acute incubation with ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
stimulated NO release by diaminofluorescein confirm this increase. Together, these results confirm that ouabain can stimulate NO release. Previous studies have shown that ouabain-induced hypertension is accompanied by increased NO bioavailability, which decreases vascular reactivity in resistance and conductance arteries.27,39,42 Furthermore, it has been previously reported that ouabain, acutely administered, produces an increase of basal NO release.29,30,48 Several pathways may be involved in the stimulation of NO production by ouabain. Eva et al29 observed, in human umbilical cord endothelial cells, that the binding of ouabain to a sodium pump could result in the activation of the proliferation and survival pathways involving PI3K, Akt activation, eNOS stimulation, and NO production. The activation of PI3K/Akt by ouabain has also been demonstrated in rat cardiac myocytes49 and in opossum kidney proximal tubule cells.50 To analyze whether ouabain could stimulate NO production by the PI3K/Akt-dependent pathway, wortmannin, a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, was used. Wortmannin enhanced the contractile response to phenylephrine exclusively in the ouabain-treated rings of HF rats. Additionally, Akt phosphorylation was increased in aortic rings from HF rats after ouabain incubation. These results suggest a potential
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involvement of the PI3K/Akt pathway in the production of NO induced by ouabain in the aortic rings of HF rats. Thus, ouabain is able to increase NO production in HF rats, in a manner different from controls. In addition to NO, K+ channels in vascular smooth muscle cells might also implicated in the reduction of ouabain-induced vascular responses. Briones et al51 demonstrated that chronic ouabain treatment increases the activation of large conductance calcium-activated potassium channels (BKCa) currents by NO in coronary artery smooth muscle cells. Thus, the increased NO observed in this study could open K+ channels and contribute to the negative modulation of ouabain-induced phenylephrine contraction. We demonstrated that TEA, a K+ channel blocker, potentiated the response to phenylephrine exclusively in the ouabain-treated rings of HF rats. Together, these findings suggest that NO may be activating K+ channels in vascular smooth muscle cells, contributing to a reduction of ouabain-induced phenylephrine contraction in HF rats. Thus, previous studies have demonstrated that acute or chronic ouabain administration induces the release of an endothelium-derived relaxing factor, which appears to open KCa channels.27,52 In summary, these results demonstrated, for the first time, that low concentration of ouabain could release NO in isolated aortic rings of HF rats by triggering signal transduction through the PI3K/Akt-dependent pathway. Ó 2014 Lippincott Williams & Wilkins
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Ouabain Induces NO Release in HF Rats
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endothelium of the tail vascular bed of spontaneously hypertensive rats. J Cardiovasc Pharmacol. 2004;44:372–380. Pulgar VM, Jeffers AB, Rashad HM, et al. Increased constrictor tone induced by ouabain treatment in rats. J Cardiovasc Pharmacol. 2013;62: 174–183. Rossoni LV, Salaices M, Miguel M, et al. Ouabain-induced hypertension is accompanied by increases in endothelial vasodilator factors. Am J Heart Circ Physiol. 2002;283:H2110–H2118. Xavier FE, Salaices M, Márquez-Rodas I, et al. Neurogenic nitric oxide release increases in mesenteric arteries from ouabain hypertensive rats. J Hypertens. 2004;22:949–957. Eva A, Kirch U, Scheiner-Bobis G. Signaling pathways involving the sodium pump stimulate NO production in endothelial cells. Biochim Biophys Acta. 2006;1758:1809–1814. Xie J, Wang Y, Summer WR, et al. Ouabain enhances basal release of nitric oxide from carotid artery. Am J Med Sci. 1993;305:157–162. Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res. 1979;44:503–512. Mill JG, Stefanon I, Leite CM, et al. Changes in performance of the surviving myocardium after left ventricular infarction in rats. Cardiovasc Res. 1990;24:748–753. Francis J, Weiss RM, Wei SG, et al. Progression of heart failure after myocardial infarction in the rat. Am J Physiol Reg Int Comp Physiol. 2001;281:R1734–R1745. Pereira RB, Sartório CL, Vassallo DV, et al. Differences in tail vascular bed reactivity in rats with and without heart failure following myocardial infarction. J Pharmacol Exp Ther. 2005;312:1321–1325. Teerlink JR, Clozel M, Fischli W, et al. Temporal evolution of endothelial dysfunction in a rat model of chronic heart failure. J Am Coll Cardiol. 1993;22:615–620. Gottlieb SS, Rogowski AC, Weinberg M, et al. Elevated concentrations of ouabain in patients with congestive heart failure. Circulation. 1992;86: 420–425. Martín MC, Balfagón G, Minoves N, et al. Androgen eprivation increases neuronal nitric oxide metabolism and its vasodilator effect in rat mesenteric arteries. Nitric Oxide. 2005;12:163–176. Bolotina VM, Najibi S, Palacino JJ, et al. Nitric oxide directly activates calcium-dependent potassium channel in vascular smooth muscle. Nature. 1994;368:850–853. Padilha AS, Peçanha FM, Vassallo DV, et al. Ouabain treatment changes the role of endothelial factors in rat resistance arteries. Eur J Pharmacol. 2008;600:110–116. Xavier FE, Davel APC, Fukuda LE, et al. Chronic ouabain treatment exacerbates blood pressure elevation in spontaneously hypertensive rats: the role of vascular mechanisms. J Hypertens. 2009;27:1233–1242. Wenceslau CF, Davel AP, Xavier FE, et al. Long-term ouabain treatment impairs vascular function in resistance arteries. J Vasc Res. 2011;48: 316–326. Xavier FE, Rossoni LV, Alonso MJ, et al. Ouabain-induced hypertension alters the participation of endothelial factors in alpha adrenergic responses differently in rat resistance and conductance mesenteric arteries. Br J Pharmacol. 2004;143:215–225. Bauersachs J, Widder JD. Endothelial dysfunction in heart failure. Pharmac Rep. 2008;60:119–126. Elsner D, Müntze A, Kromer EP, et al. Systemic vasoconstriction induced by inhibition of nitric oxide synthesis is attenuated in conscious dogs with heart failure. Cardiovasc Res. 1991;25:438–440. Teerlink JR, Gray GA, Clozel M, et al. Increased vascular responsiveness to norepinephrine in rats with heart failure is endothelium dependent. Dissociation of basal and stimulated nitric oxide release. Circulation. 1994;89:393–401. Miller AA, Megson IL, Gray GA. Inducible nitric oxide synthasederived superoxide contributes to hypereactivity in small mesenteric arteries from a rat model of chronic heart failure. Br J Pharmacol. 2000;131:29–36. Faria Tde O, Baldo MP, Simões MR, et al. Body weight loss after myocardial infarction in rats as a marker of early heart failure development. Arch Med Res. 2011;42:274–280. Dong XH, Komiyama Y, Nishimura N, et al. Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells. Clin Exp Pharmacol Physiol. 2004;31: 276–283.
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49. Liu L, Zhao X, Pierre SV, et al. Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes. Am J Physiol Cell Physiol. 2007;293:C1489–C1497. 50. Khundmiri SJ, Amin V, Henson J, et al. Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway. Am J Physiol Cell Physiol. 2007; 293:C1171–C1180.
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J Cardiovasc Pharmacol ä Volume 65, Number 1, January 2015 51. Briones AM, Padilha AS, Cogolludo AL, et al. Activation of BKCa channels by nitric oxide prevents coronary artery endothelial dysfunction in ouabain-induced hypertensive rats. J Hypertens. 2009;27: 83–91. 52. Rossoni LV, Cunha V, França A, et al. The influence of nanomolar ouabain on vascular pressor responses in modulated by the endothelium. J Cardiovasc Pharmacol. 1999;34:887–892.
Ó 2014 Lippincott Williams & Wilkins
Ouabain Induces Nitric Oxide Release by a PI3K/ Akt-dependent Pathway in Isolated Aortic Rings From Rats With Heart Failure Fabiana D. M. Siman, PhD,* Edna A. Silveira, PhD,* Aurélia A. Fernandes, PhD,* Ivanita Stefanon, PhD,* Dalton V. Vassallo, PhD,*† and Alessandra S. Padilha, PhD*
Background: Ouabain occurs in nanomolar concentrations in myocardial infarction and heart failure (HF). However, the effects of ouabain in vascular function in HF conditions were not investigated yet. Therefore, we analyzed the effects of acute administration of 3 nM ouabain in isolated aortic rings from rats with HF 4 weeks after myocardial infarction.
Methods and Results: Rats were submitted to sham operation or coronary artery occlusion. In HF rats, left ventricular positive and negative derivatives of intraventricular pressure reduced and left ventricular end diastolic pressure increased. Phenylephrine responses increased in HF rings when compared with controls. Ouabain incubation for 45 minutes reduced phenylephrine-induced contraction in both groups. Endothelial removal increased more phenylephrine response in ouabain-treated rings of sham rats. Ouabain potentiated the effect of L-NAME in both groups but more in sham rats. Wortmannin increased the phenylephrine response only in HF rings. The effect of tetraethylammonium was potentiated by ouabain only in HF rings. Ouabain increased phenylephrine-stimulated nitric oxide production in rings from both groups but increased the activation of Akt only in vessels from HF rats. Conclusions: Results demonstrate that low ouabain concentration can decrease vascular reactivity of aortic rings from HF rats. Ouabain was able to increase nitric oxide production in HF rats by triggering a signal transduction PI3K/Akt-dependent pathway and increasing an endothelium-hyperpolarizing factor release. Key Words: ouabain, heart failure, vascular reactivity, nitric oxide, PI3K/Akt pathway (J Cardiovasc Pharmacol Ô 2015;65:28–38)
INTRODUCTION Ouabain is an endogenous cardiac glycoside produced in the central nervous system,1,2 in the adrenal cortex3 and in Received for publication March 10, 2014; accepted August 1, 2014. From the *Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, Brazil; and †Department of Physiological Sciences, EMESCAM, Vitoria, Brazil. Supported by grants from CNPq, CAPES, and FAPES/FUNCITEC. The authors report no conflicts of interest. Reprints: Alessandra S. Padilha, PhD, Department of Physiological Sciences, Federal University of Espirito Santo, Av. Marechal Campos, 1468, Maruípe, 29043-090 Vitoria, ES, Brazil (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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the heart4 in response to volume expansion, angiotensin II, and/or adrenocorticotropic hormone stimulation.5–7 It has been implicated in the control of many functions involving the cardiovascular and nervous systems,6–9 as control of blood pressure and sodium balance.10,11 Alternately, the increase of endogenous ouabain (EO) has been associated to the pathogenesis of various disorders, including hypertension,5,9,10 diabetes,12 myocardial infarction (MI),13 and congestive heart failure (HF).14,15 Moreover, it also increases cellular proliferation and differentiation of the heart16 and vascular smooth muscle,17 which could contribute to the maintenance of hypertension. However, the pathophysiologic role for elevated levels of EO in these diseases is still under debate. Several studies have suggested that EO may have a primary role in causing cardiac dysfunction and failure.14,18–20 In a study in patients with idiopathic dilated cardiomyopathy, the high circulating levels of EO identified in those individuals predisposed to progress more rapidly to HF.14 Classically, ouabain inhibits Na+, K+ATPase activity by binding preferentially to the a-2 and a-3 isoforms of the a-subunit in rodents, in the plasmerosome region.21,22 This inhibition increases the Na+ concentration in the plasmerosome region, which reduces the activity of the Na+/Ca2+ exchanger (NCX) and, consequently, increases the amount of activator Ca2+ for contraction.22–24 As a result, on activation intracellular calcium concentration increases, and consequently, an increase in the vascular tone and/or positive inotropic effects occur. However, ouabain been shown to have variable vascular effects on different blood vessels, that could be attributed, at least in part, to the length of treatment, doses used, and the kind of artery (conductance or resistance). Thus, it can increase25,26 or decrease vascular contractile responses27,28 in resistance and conductance arteries, respectively. It has been reported that the decrease in vascular contractile responses in conductance arteries, specifically in aorta, induced by ouabain could be related the release nitric oxide (NO) by the endothelium.27 In fact, ouabain-induced hypertension has been accompanied by the endothelial NO-induced modulation of vasoconstrictor responses in the aorta.27 Other studies have also demonstrated that ouabain enhances basal NO release by human umbilical cord endothelial cells29 and porcine carotid artery endothelium.30 Eva et al29 reported that nanomolar concentrations of ouabain stimulate NO release by triggering J Cardiovasc Pharmacol ä Volume 65, Number 1, January 2015
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a signal transduction pathway, including the activation of phosphoinositide-3 kinase (PI3K) and the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). These observations prompted us to investigate if the endothelial NO-induced modulation of vasoconstrictor responses induced by ouabain might be altered in conductance arteries in HF rats.
METHODS Animals Studies were performed on male Wistar rats (220–240 g). All experiments were conducted in compliance with the guidelines for biomedical research as stated by the Brazilian Societies of Experimental Biology and approved by the Institutional Committee of Ethics on Animal Research (CEUA-UFES 028/ 2012). All rats had free access to water and were fed rat chow ad libitum.
Myocardial Infarction Myocardial infarction was induced by ligation of the coronary artery as described previously.31 Briefly, animals were anesthetized with ketamine (50 mg/kg; Agener, MG, Brazil) and xylazine (10 mg/kg; Bayer, SP, Brazil). A thoracotomy at the fourth intercostal space was used to quickly expose the heart. The left coronary artery was permanently occluded ;2 mm from its origin with a 6-0 mononylon suture. The heart was rapidly returned to its position, and the thorax was closed. Mechanical respiratory support was used in those animals that did not recover spontaneous respiration. A group of animals was submitted to a sham operation in which all surgical procedures were performed as described, except that the coronary suture was not tied. After recovery, the animals were kept in collective cages at the animal facility. From the total number of 26 rats that were MI operated. Seventeen of the 26 rats (70%) survived the entire 4-week period and were included for analysis. All the 26 animals survived the sham surgery procedure.
Hemodynamic Measurements Four weeks after surgery, the animals were anesthetized with urethane (1.2 g/kg, intraperitoneally) and, after loss of the righting reflex, the right common carotid artery was dissected to insert a fluid-filled polyethylene catheter (P50) connected to a blood pressure transducer (TSD 104A; Biopac System, Goleta, CA). The catheter was then advanced into the left ventricle (LV). The following measurements were performed: systolic and diastolic blood pressure, left ventricular systolic pressure, left ventricular end diastolic pressure (LVEDP), heart rate, and positive and negative first derivatives of intraventricular pressure (dP/dt+ and dP/dt2, respectively). After hemodynamic recordings, the heart and lungs were removed. The right ventricle (RV) and LV were separated, rinsed in a physiological solution (0.9% NaCl), blotted, and weighed. Scar tissue was separated from the remaining LV muscle as previously described.32 The scar and muscle areas were determined by planimetry, and the infarct area was expressed as the percentage of LV surface covered by the scar Ó 2014 Lippincott Williams & Wilkins
Ouabain Induces NO Release in HF Rats
tissue. HF was defined using 3 criteria as follow: lung/body weight (BW) ratio greater than lung/BWsham (approximately 2-fold),33 RV hypertrophy (RV/BW) proportional to lung/ BW,34 and LVEDP greater than 15 mm Hg.34,35
Vascular Reactivity Studies After the hemodynamic measurements, rats were killed by exsanguination. The thoracic aortas were carefully dissected and separated from the connective tissue. For the reactivity experiments, the aortas were divided into cylindrical segments 4 mm in length. Segments of thoracic aorta were mounted in an isolated tissue chamber containing Krebs–Henseleit solution (in millimolar: NaCl 118; KCl 4.7; NaHCO3 23; CaCl2 2.5; KH2PO4 1.2; MgSO4 1.2; glucose 11, and EDTA 0.01), gassed with 95% O2 and 5% CO2 and maintained at a resting tension of 1 g at 378C. Isometric tension was recorded using an isometric force transducer (TSD125C; CA) connected to an acquisition system (MP100; Biopac Systems Inc, Santa Barbara, CA). After a 45-minute equilibration period, all aortic rings were initially exposed twice to 75 mM KCl, first to check their functional integrity and again to assess the maximal tension that could be developed. Afterward, endothelial integrity was tested with acetylcholine (10 mM) in segments that were previously contracted with phenylephrine (1 mM). After a washout period (30 minutes), the aortic rings were incubated with ouabain (3 nM) or not (control) for 45 minutes. Then, concentration–response curves to phenylephrine (0.1 nM–0.3 mM) were determined. The ouabain concentration for these experiments was chosen based on previous reports that demonstrated nanomolar levels of circulating ouabain in the plasma of patients with cardiac dysfunction.36,37 As the experiments were performed in isolated aortic rings, the addition of exogenous ouabain sought to mimic the vascular effects of circulating ouabain in rats with HF. The influence of the endothelium on the response to phenylephrine in the presence or absence of ouabain (3 nM) was investigated after its mechanical removal by rubbing the lumen with a needle. The absence of endothelium was confirmed by the inability of 10 mM acetylcholine to induce relaxation. The role of endothelial-derived vasoactive factors on the phenylephrine-elicited contractile response was investigated. The effects of the following drugs were evaluated: (1) a nonspecific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 100 mM); (2) the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (0.1 mM); and (3) the potassium (K+) channel blocker tetraethylammonium (TEA, 2 mM, nonselective blocker of K+ channels). These drugs were added 45 minutes before examining the concentration– response curves to phenylephrine in the presence or absence of ouabain.
Immunodetection of Akt To confirm the involvement of the PI3K/Akt pathway in oubain-induced NO release, Akt expression and phosphorylated-Ser473 Akt levels were determined in homogenates from aortic segments (those used for the reactivity www.jcvp.org |
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TABLE 1. Morphometric and Hemodynamic Parameters of Sham and HF Rats Sham (N = 26) BW, g LV, mg LV/BW, mg/g RV, mg RV/BW, mg/g Lung, mg Lung/BW, mg/g Infarct area, % Aortic SP, mm Hg Aortic DP, mm Hg MBP, mm Hg LVSP, mm Hg LVEDP, mm Hg LV + dP/dT, mm Hg/s LV 2 dP/dT, mm Hg/s Heart rate, beats per minute
351 785 2.24 213 0.6 1832 5.25
6 6 6 6 6 6 6
4.6 11.1 0.02 5.05 0.01 50.8 0.13
107 70 87 111 3.6 5554 26038 346
6 6 6 6 6 6 6 6
3.2 3.1 3.6 3.6 0.5 481 334 13.2
HF (N = 18) 314 822 2.62 410 1.3 3198 10.1 36 104 76.4 92 110 18.4 3977 24918 368
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
9.3* 37.1 0.1* 24.7* 0.07* 106* 0.4* 1.5 2.9 2.2 3.3 3.6 1.3* 269* 196* 10.5
Values are mean values 6 SEM. Values in parentheses represent the number of animals used in that analysis. *t test: P , 0.05 versus sham. SP, systolic pressure; DP, diastolic pressure; MBP, mean blood pressure; LVSP, left ventricle systolic pressure; dP/dt, maximal rate of pressure rise.
experiments) that had been preincubated with or without ouabain (for 45 minutes). Thus, after the reactivity experiment, arteries were rapidly frozen in liquid nitrogen and kept at 2708C until the day of analysis. Proteins from homogenized arteries (80 mg) were separated by 7.5% SDS-PAGE and then transferred to nitrocellulose membranes. Next, the membrane was incubated with rabbit polyclonal antibody for Akt (1:500; Santa Cruz Biotechnology) and phosphorylated-Ser473 Akt (1:500; Santa Cruz Biotechnology). After washing, the membranes were incubated with anti-rabbit (1:7500; StressGen, Victoria, Canada) immunoglobulin antibody conjugated to horseradish peroxidase. After thorough washing, immunocomplexes were detected using an enhanced horseradish peroxidase/luminal chemiluminescence system (ECL Plus; Amersham International, Little Chalfont, United Kingdom) and film (Hyperfilm ECL International). Signals on the immunoblot were quantified with the National Institutes of Health Image V1.56 computer program. The same membrane was used to determine a-actin expression using a mouse monoclonal antibody (1:5000; Sigma).
ouabain (3 nM) for 45 minutes at 378C. Concentration– response curve to phenylephrine (0.1 nM–0.3 mM) was performed, and the medium was collected to measure stimulated NO production. The fluorescence of the medium was measured at room temperature using a spectrofluorometer (BioTek Synergy 2 Instruments, BioTek’s Gen 5 Software) with excitation wavelength set at 492 nm and emission wavelength at 515 nm. Blank measurement samples were similarly collected but without arteries to subtract background emission. The amount of NO released was expressed as arbitrary units per milligram tissue.
Data Analyses and Statistics
All values are expressed as the mean 6 SEM. Contractile responses was expressed as a percentage of the maximal response induced by 75 mM KCl. For each concentration– response curve, the maximal effect (Rmax) and the concentration of agonist that produced 50% of the maximal response (log EC50) were calculated using nonlinear regression analysis (GraphPad Prism; GraphPad Software Inc, San Diego, CA). The sensitivities of the agonists were expressed as pD2 (2log EC50). Rmax was expressed as a percentage of contraction to KCl 75 mmol/L. To compare the effect of drugs on the response to phenylephrine in aortic rings from both groups, some results were expressed as differences of the area under the concentration–response curves (dAUC) in control and experimental situations. AUCs were calculated from individual concentration–response curve plots; the differences were expressed as a percentage of the AUC of the control situation. The results of the Akt expression and phosphorylatedSer473 Akt level analysis were expressed as the ratio between phosphoAkt/Akt. The results were expressed as the mean 6 SEM of the number of rats indicated; differences were analyzed using Student’s t test or 2-way analysis of variance followed by a Tukey’s test. P , 0.05 was considered significant.
Drugs and Reagents Ouabain, L-phenylephrine hydrochloride, L-NAME, acetylcholine chloride, wortmannin, and tetraethylammonium were purchased from Sigma–Aldrich (St Louis, MO). Salts and reagents used were of analytical grade from Sigma– Aldrich and Merck (Darmstadt, Germany). Wortmannin was dissolved in dimethyl sulfoxide (DMSO) and diluted in deionized water. All other drugs were dissolved and diluted in deionized water. At the final bath concentration used (0.005%), DMSO vehicle had no effect on contractile responses.
NO Release NO release was measured as previously described.37 The segments of the thoracic aorta artery were dissected and equilibrated for 30 minutes in HEPES buffer (in mmol$L21: 119 NaCl; 20 HEPES; 1.2 CaCl2; 4.6 KCl; 1 MgSO4; 0.4 KH2PO4; 5 NaHCO3; 5.5 glucose; 0.15 NaH2PO4; pH 7.4) at 378C. The arteries were then incubated with the fluorescent probe 4,5-diaminofluorescein (2 mmol$L21) for 30 minutes, and the medium was collected to measure basal NO release. Then, the aortic rings were incubated in the presence or absence of
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RESULTS Global Parameters and Hemodynamics The global morphological and hemodynamic characteristics of HF and sham groups are described in Table 1. BW was lower in the HF group compared with the sham group. As expected, the lung/BW and RV/BW ratios were increased in the HF rats compared with the sham controls (Table 1). Concerning hemodynamic parameters, no differences were Ó 2014 Lippincott Williams & Wilkins
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Ouabain Induces NO Release in HF Rats
FIGURE 1. Concentration–response curves to phenylephrine in aortic rings of HF and sham rats (A). Effect of ouabain (Oua) incubation on the contraction induced by phenylephrine on the aortic rings of sham (B) and HF (C) rats. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
observed in arterial pressure or left ventricular systolic pressure among groups (Table 1). However, LV function was affected in the HF rats, with a reduction of inotropic indexes of contractility, the +dP/dt and 2dP/dt, and an elevation of the LVEDP (Table 1).
Vascular Reactivity Study The maximal response to 75 mM KCl in segments with endothelium (sham: 2.37 6 0.06 g, n = 26; HF: 2.26 6 0.08 g; n = 18; P . 0.05) and after endothelial removal (sham: 2.61 6 0.25 g, n = 6; HF: 2.1 6 0.3 g; n = 7; P . 0.05) was
similar in HF and sham groups. Ach-induced (10 mM) relaxation was also similar in aortic rings from HF and sham rats (sham: 88.25 6 1.60%, n = 25; HF: 88.96 6 2.47%, n = 16). Contractile responses induced by phenylephrine were increased in aortic rings from HF rats compared with sham (Rmax: sham: 104 6 3.04%; HF: 119 6 3.7%; P , 0.05) (Fig. 1A). However, acute ouabain incubation promoted a reduction in the phenylephrine-induced contraction in both groups (Figs. 1B, C; Table 2). To analyze the role of the endothelium in phenylephrine responses, experiments were performed in the absence of endothelium. Endothelial removal left-shifted
TABLE 2. Effect of Endothelial Denudation on the Vascular Response to Phenylephrine (Emax and pD2) in the Presence and Absence of Ouabain (Oua) on the Aortic Rings of Sham and HF Rats pD2
Emax, %
Sham E+ E2 Oua E+ Oua E2
5.98 7.73 5.68 7.61
6 6 6 6
0.17 (25) 0.28 (6)* 0.20 (26) 0.1 (6)‡
HF 6.39 7.9 5.91 7.69
6 6 6 6
0.19 (16) 0.3 (7)† 0.13† (18) 0.15 (7)§
Sham 104.4 131.5 94.4 131.3
6 6 6 6
3.04 (25) 3.0 (6) 3.16 (26)* 6.8 (6)‡
HF 119 135.7 108 123.9
6 6 6 6
3.7 (16)* 7.7 (7) 3.76 (18) 5.6 (7)
Values are means 6SEM; the number of animals is indicated in parentheses. *P , 0.05 versus sham E+ (t test). †P , 0.05 versus HF E+ (t test). ‡P , 0.05 versus sham Oua E+ (t test). §P , 0.05 versus HF Oua E+ (t test). Emax expressed as a percentage of contraction to KCl 75 mmol/L. E2, endothelium denuded; E+, endothelium intact; Emax, maximum response; pD2, negative logarithm of concentration producing 50% of maximum response.
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FIGURE 2. Effect of endothelial removal on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain (E). Concentration–response curve to phenylephrine of aortic rings from sham and HF rats after endothelial removal. Inset shows differences in the area under the concentration–response curve (dAUC) in endothelium-denuded and intact segments in the presence (ouabain) and absence (control) of ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
the concentration–response curves to phenylephrine in aortic segments from both groups, but this effect was greater in the ouabain-treated rings of sham rats, as shown by the dAUCs (Figs. 2A, B; Table 2). Therefore, after endothelial removal, the phenylephrine responses were similar in aortic rings from HF and sham rats (Fig. 2E). This suggests an endothelial modulation of the phenylephrine contractile response. To assess the contribution of endothelium-derived NO in phenylephrine responses induced by ouabain, arteries were incubated with an NOS inhibitor (L-NAME, 100 mM). The incubation with the NOS inhibitor L-NAME (Fig. 3; Table 3) left-shifted the concentration–response curves to phenylephrine
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in aortic segments from HF and sham groups, but this effect was greater in sham rats (Table 3, Figs. 3A, B). These results demonstrate that the negative modulation of the phenylephrine response by NO was reduced in the aortic rings from HF rats. Moreover, in the presence of ouabain, the L-NAME effect was potentiated in both groups, as shown by the dAUCs (Figs. 3B, D). These results indicate that ouabain incubation increased NO bioavailability in both the sham and HF groups. To confirm this increased NO bioavailability, NO release was measured by the fluorescent probe 4,5-diaminofluorescein in segments of the thoracic aorta, in the presence and absence of ouabain. Ouabain increased Ó 2014 Lippincott Williams & Wilkins
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Ouabain Induces NO Release in HF Rats
FIGURE 3. Effect of L-NAME on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain. Inset shows differences in area under the concentration–response curve (dAUC) of aortic rings in the presence and absence L-NAME under control conditions and after acute incubation with ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
phenylephrine-stimulated NO release in the aortic rings of both the sham and HF rats (Fig. 4). To analyze whether ouabain stimulates NO production by the PI3K/Akt-dependent pathway, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin was used.
In isolated aortic rings from sham rats, the response to phenylephrine remained unaltered after wortmannin incubation, in the presence and absence of ouabain. In rings of HF rats, in the absence of ouabain, the phenylephrine response was not modified after wortmannin incubation (Fig. 5C).
TABLE 3. Effect of L-NAME on the Vascular Response to Phenylephrine (Emax and pD2) in the Presence and Absence of Ouabain (Oua) on the Aortic Rings of Sham and HF Rats pD2 Sham Control L-NAME Ouabain control Ouabain L-NAME
6.37 6 0.08 6.91 6 0.1* 6.3 6 0.11
Emax, % HF
Sham
HF
6.54 6 0.08 90 6 3.94 112 6 5.43 7.1 6 0.11* 133 6 6.28* 129 6 6.33 6.31 6 0.09 86 6 5.77 106 6 7.13
6.83 6 0.06* 7.13 6 0.06†
136 6 5.8†
136 6 4.62†
Emax expressed as a percentage of contraction to KCl 75 mmol/L. Values are means 6 SEM. *P , 0.05 versus control (t test). †P , 0.05 versus ouabain control (t test). Note that L-NAME has controls. Emax, maximum response; pD2, negative logarithm of concentration producing 50% of maximum response.
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FIGURE 4. Stimulated NO production by aortic rings from sham and HF rats in the presence (ouabain) and absence (control) of ouabain. The results are expressed as the mean 6 SEM. Number of animals used was between 6 and 9. Student’s t test. *P , 0.05 versus controls. www.jcvp.org |
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FIGURE 5. Effect of wortmannin on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
determined the Akt and phosphorylated-Ser473 Akt protein levels by Western blot. We observed that ouabain increased the activation of Akt in the vessels of HF rats, as shown by the phospho/total Akt ratio, suggesting the participation of PI3K/ Akt signaling (Fig. 6). Therefore, these results demonstrated that ouabain increased NO release in both groups, but by different
However, in the ouabain-treated rings of HF rats, the incubation with wortmannin resulted in a left shift in the concentration– response curves to phenylephrine, reducing pD2 (Fig. 5; Table 4). These results suggest that ouabain might stimulate NO production by the PI3K/Akt-dependent pathway in HF rats. Because PI3K activates Akt by phosphorylation at Ser473 and Akt activates eNOS by phosphorylation at Ser1177, we
TABLE 4. Effect of Wortmannin and TEA on the Vascular Response to Phenylephrine (Emax and pD2) in the Presence and Absence of Ouabain (Oua) on the Aortic Rings of Sham and HF Rats pD2
Emax, %
Sham Control Wortmannin Ouabain control Ouabain wortmannin Control TEA Ouabain control Ouabain TEA
6.25 6.19 6.36 6.19 5.5 5.84 5.18 6.23
6 6 6 6 6 6 6 6
0.08 0.16 0.13 0.17 0.12 0.08* 0.11 0.10‡
HF 6.62 6.76 6.35 7.18 5.55 6.05 5.22 6.28
6 6 6 6 6 6 6 6
0.05 0.11 0.12 0.14* 0.13 0.13† 0.10 0.06‡
Sham 107 111 90 101 100 143 90 134
6 6 6 6 6 6 6 6
3.79 4.72 4.72 5.56 6.12 8.35† 6.27 5.35‡
HF 111 105 102 115 110 126 107 136
6 6 6 6 6 6 6 6
5.55 6.81 5.22 6.72 6.45 7.17 8.84 2.78‡
Emax expressed as a percentage of contraction to KCl 75 mmol/L. Values are mean values 6 SEM. *P , 0.05 versus ouabain control (t test). †P , 0.05 versus control (t test). ‡P , 0.05 versus ouabain control (t test). Note that wortmannin and TEA have controls. Emax, maximum response; pD2, negative logarithm of concentration producing 50% of maximum response.
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FIGURE 6. Densitometric analysis of a Western blot for Akt and phosphorylated-Ser473 Akt protein expression by aortic rings cultured in the absence (control) of ouabain and after acute incubation with ouabain. Representative blots are also shown. Number of animals used was between 6 and 8.*P , 0.05 versus HF control by Student’s t test.
mechanisms. Although, in the sham group, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin did not affect the concentration–response curves to phenylephrine, in the HF group, we observed evidence indicating the PI3K/Akt pathway involvement. Because NO can open K+ channels,38 we determined the role of K+ channels in ouabain-induced phenylephrine responses. Arteries were incubated with TEA, a nonselective K+ channel blocker. TEA potentiated the vasoconstrictor response induced by phenylephrine in aortic segments from either group, but this effect was greater in sham rats (Table 4; Figs. 7A, C). Moreover, in the presence of ouabain, the TEA effect was greater in the rings of HF rats, as shown by the dAUC values (Fig. 7B, D; Table 4). These results suggest that ouabain’s effects on phenylephrine-induced contraction in aortic segments from the HF group involve the participation of K+ channels.
DISCUSSION AND CONCLUSIONS
Our study shows, for the first time, that ouabain can modify vascular function in rats with HF. Our results demonstrate that ouabain decreases contractile responses to phenylephrine in sham and HF rats, most likely because of the increased bioavailability of NO. However, ouabain acts through different mechanisms in both groups (sham and HF rats). In the sham rats, ouabain acts by a mechanism independent of the PI3K/Akt pathway, whereas in the HF rats, ouabain increases NO production by a mechanism Ó 2014 Lippincott Williams & Wilkins
Ouabain Induces NO Release in HF Rats
dependent on PI3K/Akt pathways and on potassium channel activation. Evidence suggests that EO may have a primary role in causing cardiac dysfunction and failure, by inducing increasing in left ventricular mass and enhance apoptosis, suggesting a possible association between ouabain and the pathogenesis of HF.14,18–20 Although previous studies have shown that ouabain could modify the vascular function of the ouabaininduced hypertensive rats,8,39–41 the role of this endogenous compound on the vascular function of the HF rats had not yet been investigated. In this study, we observed a reduction in aortic ring reactivity to phenylephrine after ouabain incubation in both the sham and HF groups. This observation is accordance with other reports that demonstrated a reduction vascular reactivity in aortic segments after ouabain incubation.27,42 In aortic rings, ouabain has been related with decrease in vascular contractile responses, which could be related the release NO by endothelium.27,42 However, ouabain might increase vascular contractile responses in resistance arteries.25,26 These contradictory effects could be related, at least in part, to different blood vessels used in these studies. As showed in Figure 2, the endothelial modulation was increased after ouabain incubation only in isolated aortic rings from sham rats. This effect was not observed in HF rats, likely because these rats already present endothelial dysfunction.35,43 Although some reports35,43 have shown that HF rats have endothelial dysfunction, the increase in phenylephrine contractile response in aortic segments from HF rats when compared with sham (Fig. 1A) is not sufficient to confirm the endothelial dysfunction. Moreover, after endothelial removal, the phenylephrine contractile responses in segments from HF and sham rats were similar (Fig. 2E), suggesting a modulating by endothelial factors. Previous reports demonstrated that the endothelial dysfunction and an increased vasoconstrictor response are present in HF dogs.35,43,44 Furthermore, previous studies demonstrated an increase of vascular reactivity to vasoconstrictor agents in aortic rings of HF rats.45,46 This response probably was due to decrease in the NO basal release.45 In fact, the involvement of NO in the contractile response to phenylephrine is reduced in HF rats, as demonstrated by dAUC% (results obtained with L-NAME; Figs. 3A, C; sham: 74.5 6 8.6 vs. HF: 28.3 6 10.8, t test: P , 0.05). Therefore, in accordance with this result, we suggest that in aortic segments of HF rats, the NO-induced modulation of phenylephrine vasoconstrictor responses was reduced. Moreover, when analyzed NO release by fluorescent reagent 4,5-diaminofluorescein (DAF) method, we observed a tendency to NO reduce (P = 0.08) in aortic rings of HF rats. However, other reports demonstrated a reduction phenylephrine contractile response associated with increase of NO release in the tail vascular bed from HF rats after 30 days MI.34,47 These discrepancies about NO bioavailability might result from different studied vessels. However, the association of L-NAME plus ouabain, amplified the contractile response to phenylephrine, but this effect was similar in sham and HF groups. In addition, direct measurements of phenylephrinewww.jcvp.org |
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FIGURE 7. Effect of tetraethylammonium (TEA) on the concentration–response curve to phenylephrine of aortic rings from sham and HF rats in the presence (B–D) and absence (A–C) of ouabain. Inset shows differences in area under the concentration–response curve (dAUC) of aortic rings in the presence and absence of TEA under control conditions and after acute incubation with ouabain. The results are expressed as the mean 6 SEM. *P , 0.05 by Student’s t test.
stimulated NO release by diaminofluorescein confirm this increase. Together, these results confirm that ouabain can stimulate NO release. Previous studies have shown that ouabain-induced hypertension is accompanied by increased NO bioavailability, which decreases vascular reactivity in resistance and conductance arteries.27,39,42 Furthermore, it has been previously reported that ouabain, acutely administered, produces an increase of basal NO release.29,30,48 Several pathways may be involved in the stimulation of NO production by ouabain. Eva et al29 observed, in human umbilical cord endothelial cells, that the binding of ouabain to a sodium pump could result in the activation of the proliferation and survival pathways involving PI3K, Akt activation, eNOS stimulation, and NO production. The activation of PI3K/Akt by ouabain has also been demonstrated in rat cardiac myocytes49 and in opossum kidney proximal tubule cells.50 To analyze whether ouabain could stimulate NO production by the PI3K/Akt-dependent pathway, wortmannin, a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, was used. Wortmannin enhanced the contractile response to phenylephrine exclusively in the ouabain-treated rings of HF rats. Additionally, Akt phosphorylation was increased in aortic rings from HF rats after ouabain incubation. These results suggest a potential
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involvement of the PI3K/Akt pathway in the production of NO induced by ouabain in the aortic rings of HF rats. Thus, ouabain is able to increase NO production in HF rats, in a manner different from controls. In addition to NO, K+ channels in vascular smooth muscle cells might also implicated in the reduction of ouabain-induced vascular responses. Briones et al51 demonstrated that chronic ouabain treatment increases the activation of large conductance calcium-activated potassium channels (BKCa) currents by NO in coronary artery smooth muscle cells. Thus, the increased NO observed in this study could open K+ channels and contribute to the negative modulation of ouabain-induced phenylephrine contraction. We demonstrated that TEA, a K+ channel blocker, potentiated the response to phenylephrine exclusively in the ouabain-treated rings of HF rats. Together, these findings suggest that NO may be activating K+ channels in vascular smooth muscle cells, contributing to a reduction of ouabain-induced phenylephrine contraction in HF rats. Thus, previous studies have demonstrated that acute or chronic ouabain administration induces the release of an endothelium-derived relaxing factor, which appears to open KCa channels.27,52 In summary, these results demonstrated, for the first time, that low concentration of ouabain could release NO in isolated aortic rings of HF rats by triggering signal transduction through the PI3K/Akt-dependent pathway. Ó 2014 Lippincott Williams & Wilkins
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