DOI 10.1515/hmbci-2013-0058 Horm Mol Biol Clin Invest 2014; 18(2): 113–122
Ana Paula Davel*, Gisele Kruger Couto, Camilla Ferreira Wenceslau, Emilia Cristina Peres, Fabiano Elias Xavier and Luciana Venturini Rossoni
Enhanced Na+, K+-ATPase activity and endothelial modulation decrease phenylephrine-induced contraction in aorta from ouabain-treated normotensive and hypertensive rats Abstract Aim: The purpose of this study was to compare the effect of long-term ouabain treatment on the vascular reactivity and Na+, K+-ATPase activity of a conductance artery from normotensive and hypertensive rats. Methods: Male Wistar rats were treated with ouabain (~8.0 µg/day, subcutaneously) or vehicle for 5 and 20 weeks, and spontaneously hypertensive rats (SHRs) for 5 weeks. Vasoconstrictor response to phenylephrine (10−10 to 10−4 M) and relaxation curves to KCl (1–10 mM) were analyzed in thoracic aorta. The effects of endothelial removal, L-NAME (100 μM), and indomethacin (10 μM) were used to evaluate the endothelial, nitric oxide (NO), and cyclooxygenase (COX) modulation of phenylephrine response, respectively. Protein expression of endothelial and neuronal NO synthase (NOS) and COX-2 were also investigated. Results: The phenylephrine-induced contraction was reduced, whereas the relaxation to KCl was enhanced in the aorta of ouabain-treated Wistar rats and SHRs. In both strains, endothelial modulation of α-adrenergic response was enhanced, related to an increased NO and reduced COX-derived vasoconstrictor factor modulation. Aortas from 20-week ouabain-treated Wistar rats showed reduced COX-2 and enhanced eNOS protein expression. In SHRs, 5-week ouabain treatment reduced COX-2 and increased nNOS protein expression. Conclusions: The results suggest that long-term ouabain treatment reduces the α-adrenergic response of aorta from normotensive rats and SHRs, associated with an increase of NO synthesis, reduced COX-2-derived vasoconstrictor factors, and enhanced ouabain-sensitive Na+, K+-ATPase activity. These aortic mechanisms could be adjustments to the elevated blood pressure induced by ouabain, even in the presence of preexisting hypertension.
Keywords: aorta; cyclooxygenase-2; endothelium; Na+, K+-ATPase; ouabain. *Corresponding author: Ana Paula Davel, Institute of Biology, Department of Structural and Functional Biology, Building J, Laboratory 04, State University of Campinas-UNICAMP, Rua Monteiro Lobato, 255, CEP 13083-862 Campinas, SP, Brazil, Phone: +55 19 35216189, Fax: +55 19 35216185, E-mail: [email protected] Gisele Kruger Couto, Camilla Ferreira Wenceslau, Emilia Cristina Peres and Luciana Venturini Rossoni: Institute of Biomedical Sciences, Department of Physiology and Biophysics, University of Sao Paulo, Sao Paulo, Brazil Fabiano Elias Xavier: Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Brazil
Introduction Ouabain (OUA) is an endogenous circulating Na+, K+ATPase inhibitor in humans and other mammals [1, 2]. This hormone is secreted into the brain and adrenal cortex and is found in plasma at a range of femtomolar to nanomolar concentrations [3–6]. Elevated circulating levels of OUA have been positively correlated to high blood pressure in patients with essential hypertension as well as in animal models of hypertension [5–7]. Moreover, it has been demonstrated that chronic administration of OUA to normotensive rats induces hypertension [2, 5, 8–15]. However, the mechanisms underlying the role of this hormone in the pathogenesis of hypertension are not completely elucidated. Changes produced by OUA in vascular function may contribute to elevated blood pressure [14–17], but also may play a compensatory role counteracting OUA-induced hypertension [8–13]. In line with the compensatory hypothesis, 5 weeks of treatment with OUA decreases phenylephrine-induced contraction in aorta and superior mesenteric artery from Wistar rats [8, 9, 18]. These alterations were related to an increase in the endothelium-derived
114 Davel et al.: Chronic ouabain in endothelial modulation of the aorta relaxing factors nitric oxide (NO) and hyperpolarizing factor. In addition, it was accompanied by an increased OUA-sensitive Na+, K+-ATPase activity [8]. It is interesting to note that 5-week OUA treatment did not change the α-adrenergic-induced contraction in mesenteric resistance arteries from Wistar rats [10, 14]. However, more prolonged (20 weeks) treatment with OUA in Wistar rats was able to increase the noradrenaline-induced contraction in mesenteric resistance arteries [14]. This effect was associated with an increase in cyclooxygenase-2 (COX-2)-derived prostaglandins, oxidative stress, and reduced NO bioavailability [14]. Taken together, these results strongly indicate the time-dependent effects of OUA on the vascular adjustments in small vessels. However, regardless of the observed effects in response to 5-week OUA treatment in conductance arteries [8, 9, 18], at the present, there are no published studies analyzing the effects of OUA for a longer period in these vessels. Some studies have demonstrated that hypertensive rats are more sensitive to the vascular effects of acute and chronic OUA than normotensive animals [19–22]. In this sense, 5 weeks of OUA administration resulted in an additional increase of blood pressure in spontaneously hypertensive rats (SHRs), associated with higher contractile response of tail artery to phenylephrine compared with untreated SHRs [23]. This effect was related to a reduced modulation of endothelial-derived NO and hyperpolarization factor, as well to an increase in COX-2-derived vasoconstrictor prostanoids [23]. By contrast, in tail arteries from Wistar rats, the phenylephrine-induced contraction remained unmodified after 5 weeks treatment with OUA [8, 9]. Therefore, these results indicate that the vascular effects of OUA treatment, besides depending on the exposure time and the vessel type, can be magnified and/or found early due to preexisting hypertension. Thus, the purpose of the present study was to analyze the effect of 5 or 20 weeks treatment with OUA on phenylephrineinduced contraction in aorta from SHRs and Wistar rats. The role of endothelial-derived factors in the aortic contractile responses and the OUA-sensitive Na+, K+-ATPase activity were also investigated.
Materials and methods Animals Six-week-old male Wistar rats and SHRs were obtained from colonies maintained at the Animal House of the Institute of Biomedical Sciences of University of Sao Paulo. Rats were housed in constant room
temperature, humidity, and light cycles (12:12 h light-dark), with free access to standard rat chow and tap water. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and with the guidelines of the Committee on Care and Use of Laboratory Animal Resources of the Institute of Biomedical Sciences of the University of Sao Paulo. Animals were anesthetized with a mixture of ketamine, xylazine, and acetopromazin (64.9, 3.2, and 0.78 mg/kg, respectively, intraperitoneally) to implant subcutaneously a controlled time-released pellet (Innovative Research of America, Sarasota, FL, USA) containing OUA (0.5 mg) or vehicle. Then, the rats received vehicle or OUA (8.0 μg/day) for 5 or 20 weeks [8–11, 14, 23].
Vascular reactivity After 5 or 20 weeks of treatment with OUA, rats were anaesthetized with a ketamine, xylazine, and acepromazine mixture and killed by exsanguination. The thoracic aorta was dissected, cleaned of surrounding connective and adipose tissue, and cut into rings 4 mm in length as previously described [24]. The endothelial layer was mechanically removed in certain segments by rubbing the lumen with a needle. The rings were mounted in an isolated organ bath system containing Krebs-Henseleit bicarbonate buffer (in mmol/L: NaCl 118; KCl 4.7; NaHCO3 25; CaCl2-2H2O 2.5; KH2PO4 1.2; MgSO4-7H2O 1.2; glucose 11 and EDTA 0.01; at 37°C and pH 7.4). Thoracic aorta segments were subjected to tension of 1.0 g, during a 45-min equilibration period. Isometric tension was recorded using an isometric force transducer (TRI 210; Letica, Barcelona, Spain) connected to an acquisition system (MP100; Biopac Systems, Santa Barbara, CA, USA). After 45 min of stabilization, aortas were exposed twice to KCl (75 mmol/L) to assess maximum contractility. Afterward, the integrity of the endothelium was determined by the acetylcholineinduced relaxation (10 μmol/L) in segments precontracted with phenylephrine (~10 μmol/L) at a concentration producing 50%–70% of the contraction induced by KCl (75 mmol/L). The endothelium was considered intact if the aortic ring relaxed > 80% to acetylcholine, whereas endothelial denudation was confirmed by
Ana Paula Davel*, Gisele Kruger Couto, Camilla Ferreira Wenceslau, Emilia Cristina Peres, Fabiano Elias Xavier and Luciana Venturini Rossoni
Enhanced Na+, K+-ATPase activity and endothelial modulation decrease phenylephrine-induced contraction in aorta from ouabain-treated normotensive and hypertensive rats Abstract Aim: The purpose of this study was to compare the effect of long-term ouabain treatment on the vascular reactivity and Na+, K+-ATPase activity of a conductance artery from normotensive and hypertensive rats. Methods: Male Wistar rats were treated with ouabain (~8.0 µg/day, subcutaneously) or vehicle for 5 and 20 weeks, and spontaneously hypertensive rats (SHRs) for 5 weeks. Vasoconstrictor response to phenylephrine (10−10 to 10−4 M) and relaxation curves to KCl (1–10 mM) were analyzed in thoracic aorta. The effects of endothelial removal, L-NAME (100 μM), and indomethacin (10 μM) were used to evaluate the endothelial, nitric oxide (NO), and cyclooxygenase (COX) modulation of phenylephrine response, respectively. Protein expression of endothelial and neuronal NO synthase (NOS) and COX-2 were also investigated. Results: The phenylephrine-induced contraction was reduced, whereas the relaxation to KCl was enhanced in the aorta of ouabain-treated Wistar rats and SHRs. In both strains, endothelial modulation of α-adrenergic response was enhanced, related to an increased NO and reduced COX-derived vasoconstrictor factor modulation. Aortas from 20-week ouabain-treated Wistar rats showed reduced COX-2 and enhanced eNOS protein expression. In SHRs, 5-week ouabain treatment reduced COX-2 and increased nNOS protein expression. Conclusions: The results suggest that long-term ouabain treatment reduces the α-adrenergic response of aorta from normotensive rats and SHRs, associated with an increase of NO synthesis, reduced COX-2-derived vasoconstrictor factors, and enhanced ouabain-sensitive Na+, K+-ATPase activity. These aortic mechanisms could be adjustments to the elevated blood pressure induced by ouabain, even in the presence of preexisting hypertension.
Keywords: aorta; cyclooxygenase-2; endothelium; Na+, K+-ATPase; ouabain. *Corresponding author: Ana Paula Davel, Institute of Biology, Department of Structural and Functional Biology, Building J, Laboratory 04, State University of Campinas-UNICAMP, Rua Monteiro Lobato, 255, CEP 13083-862 Campinas, SP, Brazil, Phone: +55 19 35216189, Fax: +55 19 35216185, E-mail: [email protected] Gisele Kruger Couto, Camilla Ferreira Wenceslau, Emilia Cristina Peres and Luciana Venturini Rossoni: Institute of Biomedical Sciences, Department of Physiology and Biophysics, University of Sao Paulo, Sao Paulo, Brazil Fabiano Elias Xavier: Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Brazil
Introduction Ouabain (OUA) is an endogenous circulating Na+, K+ATPase inhibitor in humans and other mammals [1, 2]. This hormone is secreted into the brain and adrenal cortex and is found in plasma at a range of femtomolar to nanomolar concentrations [3–6]. Elevated circulating levels of OUA have been positively correlated to high blood pressure in patients with essential hypertension as well as in animal models of hypertension [5–7]. Moreover, it has been demonstrated that chronic administration of OUA to normotensive rats induces hypertension [2, 5, 8–15]. However, the mechanisms underlying the role of this hormone in the pathogenesis of hypertension are not completely elucidated. Changes produced by OUA in vascular function may contribute to elevated blood pressure [14–17], but also may play a compensatory role counteracting OUA-induced hypertension [8–13]. In line with the compensatory hypothesis, 5 weeks of treatment with OUA decreases phenylephrine-induced contraction in aorta and superior mesenteric artery from Wistar rats [8, 9, 18]. These alterations were related to an increase in the endothelium-derived
114 Davel et al.: Chronic ouabain in endothelial modulation of the aorta relaxing factors nitric oxide (NO) and hyperpolarizing factor. In addition, it was accompanied by an increased OUA-sensitive Na+, K+-ATPase activity [8]. It is interesting to note that 5-week OUA treatment did not change the α-adrenergic-induced contraction in mesenteric resistance arteries from Wistar rats [10, 14]. However, more prolonged (20 weeks) treatment with OUA in Wistar rats was able to increase the noradrenaline-induced contraction in mesenteric resistance arteries [14]. This effect was associated with an increase in cyclooxygenase-2 (COX-2)-derived prostaglandins, oxidative stress, and reduced NO bioavailability [14]. Taken together, these results strongly indicate the time-dependent effects of OUA on the vascular adjustments in small vessels. However, regardless of the observed effects in response to 5-week OUA treatment in conductance arteries [8, 9, 18], at the present, there are no published studies analyzing the effects of OUA for a longer period in these vessels. Some studies have demonstrated that hypertensive rats are more sensitive to the vascular effects of acute and chronic OUA than normotensive animals [19–22]. In this sense, 5 weeks of OUA administration resulted in an additional increase of blood pressure in spontaneously hypertensive rats (SHRs), associated with higher contractile response of tail artery to phenylephrine compared with untreated SHRs [23]. This effect was related to a reduced modulation of endothelial-derived NO and hyperpolarization factor, as well to an increase in COX-2-derived vasoconstrictor prostanoids [23]. By contrast, in tail arteries from Wistar rats, the phenylephrine-induced contraction remained unmodified after 5 weeks treatment with OUA [8, 9]. Therefore, these results indicate that the vascular effects of OUA treatment, besides depending on the exposure time and the vessel type, can be magnified and/or found early due to preexisting hypertension. Thus, the purpose of the present study was to analyze the effect of 5 or 20 weeks treatment with OUA on phenylephrineinduced contraction in aorta from SHRs and Wistar rats. The role of endothelial-derived factors in the aortic contractile responses and the OUA-sensitive Na+, K+-ATPase activity were also investigated.
Materials and methods Animals Six-week-old male Wistar rats and SHRs were obtained from colonies maintained at the Animal House of the Institute of Biomedical Sciences of University of Sao Paulo. Rats were housed in constant room
temperature, humidity, and light cycles (12:12 h light-dark), with free access to standard rat chow and tap water. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and with the guidelines of the Committee on Care and Use of Laboratory Animal Resources of the Institute of Biomedical Sciences of the University of Sao Paulo. Animals were anesthetized with a mixture of ketamine, xylazine, and acetopromazin (64.9, 3.2, and 0.78 mg/kg, respectively, intraperitoneally) to implant subcutaneously a controlled time-released pellet (Innovative Research of America, Sarasota, FL, USA) containing OUA (0.5 mg) or vehicle. Then, the rats received vehicle or OUA (8.0 μg/day) for 5 or 20 weeks [8–11, 14, 23].
Vascular reactivity After 5 or 20 weeks of treatment with OUA, rats were anaesthetized with a ketamine, xylazine, and acepromazine mixture and killed by exsanguination. The thoracic aorta was dissected, cleaned of surrounding connective and adipose tissue, and cut into rings 4 mm in length as previously described [24]. The endothelial layer was mechanically removed in certain segments by rubbing the lumen with a needle. The rings were mounted in an isolated organ bath system containing Krebs-Henseleit bicarbonate buffer (in mmol/L: NaCl 118; KCl 4.7; NaHCO3 25; CaCl2-2H2O 2.5; KH2PO4 1.2; MgSO4-7H2O 1.2; glucose 11 and EDTA 0.01; at 37°C and pH 7.4). Thoracic aorta segments were subjected to tension of 1.0 g, during a 45-min equilibration period. Isometric tension was recorded using an isometric force transducer (TRI 210; Letica, Barcelona, Spain) connected to an acquisition system (MP100; Biopac Systems, Santa Barbara, CA, USA). After 45 min of stabilization, aortas were exposed twice to KCl (75 mmol/L) to assess maximum contractility. Afterward, the integrity of the endothelium was determined by the acetylcholineinduced relaxation (10 μmol/L) in segments precontracted with phenylephrine (~10 μmol/L) at a concentration producing 50%–70% of the contraction induced by KCl (75 mmol/L). The endothelium was considered intact if the aortic ring relaxed > 80% to acetylcholine, whereas endothelial denudation was confirmed by
