Avaliação da participação da Na+K+ - ATPase e dos canais para K+ na reatividade de anéis isolados de aorta de ratos após infarto agudo do miocárdio
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2011-10-18
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Dias, Fernanda Moura Vargas
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Universidade Federal do Espírito Santo
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Introduction: Na+K + -ATPase and K + channels are essential for the regulation of membrane potential in the vascular smooth muscle cells (VSMC). The activation or inhibition of these channels by neural, humoral and local vasoactive factors enable the physiological modulation of vascular tone. Studies show that the genesis of changes in vascular reactivity found in diseases such as hypertension and diabetes may be related to increased oxidative stress and alteration of NO bioavailability that influence the Na+K + -ATPase and K + channels of the VSM . Although several studies demonstrate changes in vascular reactivity after myocardial infarction (MI), no study to date evaluated the involvement of Na+K + -ATPase and K + channels in the alterations of vascular reactivity that occur in a chronic phase after MI, in rats with similar scar area (SA), with and without signs of heart failure (HF). Objective: To evaluate the functional activity of the Na+-K + -ATPase ouabain (OUA)-sensitive and K + channels in aortic rings of rats, after MI, with similar SA, with and without signs of HF. Materials and Methods: 89 male Wistar rats (220-360 g) were divided into: Sham operation (Sham, n = 37), animals after MI without signs of HF (Inf, n = 27) with signs of HF (HF, n = 25). The MI was surgically induced by occlusion of the left anterior coronary artery. 30 days after MI, animals were weighed, anesthetized (urethane, 1.2 g / kg, ip), catheterized and hemodynamic measurements were performed. The animals were sacrificed and the mass data were evaluated by the ratio of wet mass of the right ventricle (RV/BW), left ventricle (LV/BW) and lung (Lung/BW) by body mass (BW) of each animal. The aortic rings were removed, superfused with Krebs solution and aerated with carbogen mixture. The functional activity of the Na+-K + - ATPase OUA-sensitive was investigated by the relaxation induced by potassium (0- 10 mM) before and after OUA incubation, in rings with intact (E+) and removed endothelium (E-), and after L-NAME incubation. The participation of K+ channels in vascular reactivity was performed using the acetylcholine (ACh) induced relaxation after L-NAME and K+ channels blockers incubation: tetra-ethylammonium (TEA, nonselective blockers of K+ channels), Aminopiridin (4-AP, voltage-dependent K+ channels inhibitor - Kv), Iberiotoxin (blocker of Ca2+ channels sensitive of largeconductance - BKCa), Apamina (blocker of Ca2+ channels sensitive of smallconductance - SKCa) and the association of iberiotoxin and apamin. We performed the evaluation of α1 Na+-K + -ATPase and eNOS protein expression by Western blot. The production of superoxide anion (O2 .- ) "in situ" was performed by the fluorescence produced by oxidation of dihidroethidium (DHE). The SA was assessed by counting of the points on graph paper (planimetry) and histology with picrosirius red staining. We carried out evaluation of sensitivity (pEC50) and maximal response (Rmax) to ACh induced relaxation. To compare results between groups was performed ANOVA 1 and 2 ways, post hoc tests Tukey, Bonfferroni and Student t. Values were considered significant for *P < 0.05. The experimental protocols were approved (005/2007) by the Ethics Committee on Animal Use (CEUA-EMESCAM). Results: There were no differences between the SA (Inf: 33.67 ± 1.62, HF: 38.7 ± 2.45%), body mass (Sham: 322 ± 7; Inf: 341 ± 5, HF: 337 ± 7 g) and the ratio LV/BW (Sham: 2.12 ± 0.03; Inf: 2.19 ± 0.05, IC: 2.27 ± 0.07 mg/ g) between groups. However, there were increased in the RV/BW (Sham: 0.64 ± 0.09; Inf: 0.74 ± 0.04, HF: 1.25 ± 0.08*+ mg/g; *+P <0.01), Lung/BW (Sham: 4.41 ± 0.35, Inf: 3.91 ± 0.48, HF: 8.34 ± 0.69*+ mg/ g *+P< 0.01) and LVEDP (Sham: 5.26 ± 0.52, Inf: 7.20 ± 0.64, IC: 19.65 ± 2.13*+ ; *+P < 0.05) in the IC group when compared with Sham and Inf. The K + -induced relaxation was higher in Inf compared to Sham and HF animals. However, the endothelium removal, as well as the L-NAME or TEA incubation were able to abolish the differences between groups. In IC group K + -induced relaxation was increased in the presence of OUA compared with Sham. Removal of the endothelium and incubation with TEA were able to abolish the differences between the Sham and HF groups, but the LNAME incubation did not. In the HF group compared with Sham and Inf, L-NAME incubation decreased the K + -induced relaxation. After incubation with TEA, Rmax and the sensitivity of aortic rings to ACh Inf and HF decreased compared with Sham. The dAUC performed with the curves of ACh before and after incubation with 4-AP was higher in Inf animals compared with Sham and HF. Although the blocking with Apamin or Iberiotoxin has not caused the differences between the dAUC of groups, the double block resulted in the dAUC higher in HF group compared with Sham and Inf. There was no difference in protein expression of α1 Na+K + -ATPase between groups. However, the α1 Na+-K + -ATPase, p-eNOS and eNOS protein expression was diminished in the HF animals. In addition, a higher production of O2 .- in the Inf group compared with Sham and HF. Conclusions: In a chronic phase after experimental MI in rats with similar AI, with and without signs of HF, differences exist in mechanisms that induce vascular relaxation characterization of two distinct groups with functional responses. Among the mechanisms involved are increasing the participation of K+ channels and oxidative stress after MI, as well as decreased expression of eNOS and p-eNOS in the HF group of animals. Moreover, it is suggested that the Inf animals Kv channels are contributing more to the vascular relaxation, while the HF animals, seem to depend on the channels of KCa. Differences in vascular relaxation, according to the protocols used in this study, no relation to the change in function and expression of Na+K +- ATPase between the groups.
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DIAS, F. M. V., Avaliação da participação da Na+K+ - ATPase e dos canais para K+ na reatividade de anéis isolados de aorta de ratos após infarto agudo do miocárdio. 2011. Tese (Doutorado em Ciências Fisiológicas) - Universidade Federal do Espírito Santo, Centro de Ciências da Saúde, Vitória, 2011.