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Endothelial hyperpolarizing factor increases acetylcholine-induced vasodilatation in pulmonary hypertensive broilers arterial rings
Research in Veterinary Science 92 (2012) 1–6
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Endothelial hyperpolarizing factor increases acetylcholine-induced vasodilatation in pulmonary hypertensive broilers arterial rings Diana I. Alvarez-Medina a, Aureliano Hernandez b, Camilo Orozco b,⇑ a b
Directora Unidad Municipal de Asistencia Técnica Agropecuaria, La Palma, Cundinamarca, Colombia Departamento de Ciencias para la Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Sede Bogotá, Colombia
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Article history: Received 16 June 2010 Accepted 5 February 2011
Keywords: Endothelial-derived hyperpolarizing factor Pulmonary hypertension Broiler Hypoxia
a b s t r a c t Pulmonary arterial hypertension (PAH) develops as result of imbalances between endothelium derived vasoconstrictors and vasodilators. Pulmonary hypertensive broiler chickens (PHBs) are deficient in NO production and endothelin-1 (ET-1) excess. With respect to prostacyclin, it appears that it does not alter vascular pulmonary tone in broilers. However, the role of Endothelium-Derived Hyperpolarizing Factor (EDHF) in PAH in broilers has not been determined. The possible involvement of EDHF in acetylcholine (Ach) induced vasodilatation was studied in pulmonary arterial rings taken from PHB and non-pulmonary hypertensive broilers (NPHBs). Ach induced higher vasodilatation in PHB than in NPHB. This dilatation seems to be directly related to the degree of PAH. Ach derived vasodilatation was inhibited, in PBH but not in NPHB, by blocking EDHF action with K+ or Apamin plus Charybdotoxin. It is proposed EDHF as an important vasodilator in the pulmonary arteries of PHB, which may play a compensatory role in PAH pathophysiology. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Genetically predisposed broiler chickens develop hypoxic pulmonary arterial hypertension (PAH), mostly in commercial operations located above 1500 m above sea level (Cueva et al., 1974; Hernández, 1987). Genetically predisposed subjects (Huchzermeyer et al., 1987) have a pathological exaggerated response to hypoxia (that is PAH). The latter, eventually leads to insufficiency of the right ventricle to pump returning venous blood to the lungs (Baghbanzadeh and Decuypere, 2008; Balog, 2003; Maxwell et al., 1990). From there, hypertrophy and hyperplasia ensue and the cardiac index value (CI = right ventricular weight/total ventricular mass weight) increases over 25%. This measure is a reliable parameter to indirectly classify non-hypertensive (CI less than 25%) or hypertensive chickens CI values above 25%; (Hernández, 1987; Julian, 1993). The pathological findings in PAH in broiler chickens are similar to those reported in PAH in humans, and, therefore, possibly the broiler chicken can be used as a model in this context. The morphological changes in PAH includes thickening of the medial layer of pulmonary blood vessels (Karamsetty et al., 1993) and nowadays, ⇑ Corresponding author. Address: Departamento de Ciencias para la Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 No. 26-85. Bogotá D.C. Colombia. Tel.: +57 1 3165000. E-mail address: [email protected] (C. Orozco). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.004
it is quite clear that molecules produced in the endothelium are partly responsible for smooth muscle contraction and growth of the medial layer of the pulmonary arterial tree, when the balance of endothelial produced contractors and dilators is disrupted which leads to endothelial malfunction (Adnot et al., 1991; Budhiraja et al., 2004; Thorin and Webb, 2009). In previous studies, endothelin-1 (ET-1), an important vasoconstrictor, was found to be upregulated in susceptible chickens (Gomez et al., 2007). In pulmonary hypertensive mammals and pulmonary hypertensive broiler chickens (PHBs), there is lesser expression of endothelial nitric oxide synthase (eNOS) (Moreno De Sandino and Hernández, 2003) and, therefore, a diminished endothelial derived vasodilating response to hypoxia (Gomez et al., 2007). Prostaglandin I2 (PGI2), of endothelial origin, has a vasodilating effect in mammals (Steudel et al., 1997) and blood flow resistance is lessened, which might reduce PAH (Robbins et al., 2001). Under normal conditions, in mammals, PGI2 synthase is expressed in major quantities in large blood vessels and it seems that in PHA, that characteristic is reversed, probably due to a more pronounced endothelial alterations there. This downregulated alteration of PGI2 function, induces greater vasoconstriction and tendency to intravascular coagulation (Wideman et al., 2005). In contrast, it has been proposed that in broiler chickens PGI2 and prostaglandin E2 (PGE2) probably lower pulmonary artery pressure indirectly by altering cardiac output and not through a direct action on blood vessels (Robbins et al., 2001; Lopes et al., 2002).
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It is well established that acetylcholine (Ach), and some of its agonists, could stimulate the synthesis of relaxing factors in the endothelium, and thus induce hyperpolarization of vascular smooth muscle cells. This hyperpolarizing effect is resistant to the opposing effects of NOS and cyclooxygenase inhibitors (Stebel and Wideman, 2008; Chen et al., 1988). Hence, it is likely that a third endothelial hyperpolarizing agent could have a role in the pathogenesis of PAH. Release of endothelial hyperpolarizing factor (EDHF) is initiated through activation of cholinergic muscarinic receptors, which are pharmacologically different from those responsible for NO release. The expression of M1 Ach receptors mediates EDHF liberation, whereas M2 and M3 receptors should do so for NO (Yanagisawa et al., 1998). Relaxation of vascular smooth muscle induced by EDHF is associated with K+ channels activation (Komori and Suzuki, 1987). Thus, EDHF could induce hyperpolarization by promoting the opening of various K+ channels, after Ca2+ or ATP activation. Distribution of these type of K+ channels could change within animal species, and also, according to the correspondent arterial system. For instance, in the dog´s pulmonary artery, EDHF action is associated with ATP dependent K+ channels, whereas in the rat´s mesenteric artery, the correspondent action is Ca2+ dependent (Garland et al., 1995; Doughty et al., 1999). The abovementioned characteristics, were demonstrated by EDHF inactivation with depolarizing agents (Gambone et al., 1997). The objective of the present study was to evaluate the possible participation of EDHF on endothelial dependent vasodilatation in pulmonary arterial rings and its possible intervention in the inherent mechanisms of PAH in broilers. It should be noted that the role of EDHF in pathogenesis of PAH in broiler chickens has not been previously reported.
2. Materials and methods 2.1. Vascular rings preparation Commercial male broiler chickens with ages ranging from 28 to 42 days, were obtained from a local commercial farm in Bogotá (Colombia; 2638 m above sea level). They were classified according to the so called cardiac index (CI = right ventricular weight/total ventricular mass weight (McCulloch and Randall, 1998; Alexander et al., 1960). That value in non-pulmonary hypertensive broilers (NPHBs) were under 25%, (average mean ± standard deviation values: 21.3 ± 1.8). PHB, correspondent values were 43.1 ± 6.6 (Hernández, 1987; Julian, 1993). Moreover, clinical and necropsy findings were taken into account as to discard subjects which had lesions and clinical signs of illnesses different from PAH. All broilers chickens were subjected to anesthesia with inhaled 8% isoflurane. Under deep anesthesia maintained with isoflurane (6%), death was provoked by incision in the aorta. The heart and lungs were immediately separated, the pulmonary artery devoid of surrounding fat, was placed in a Petri dish with a Krebs– Henseleit solution (119 mM NaCl, 4.7 mM KCl, 2.5 mM, CaCl21.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3 and 5.6 mM glucose), maintained at 42 °C and continuously bubbled with a gas mixture (5% CO2 and 95% O2). Three to 5 mm rings were taken at 2 mm distance from the pulmonary artery bifurcation (Alexander and Jensen, 1959). Each ring was fixed at opposing points from each other, using two stainless steel wires. One of them was secured within the rings bath and the other, to an isometric transducer (MLT0202, Panlab, Spain) connected to a dispositive for data acquisition (Power Lab, USA) which provided information directly to a computer. Every ring´s bath was maintained at 42 °C and contained 20 ml of Krebs-Henseleit solution with the abovementioned bubbling mixture. The pulmonary arterial rings were stabilized
for 30 min by Krebs-Henseleit solution changes every 5 min and 2 g basal tension. Ach, phenylephrine y SNAP were obtained from Sigma Aldrich (St. Louis, MO). NaHCO3, C6H12O6, KCl, CaCl2, NaCl, KH2PO4, MgSO4, and C6H8O6, from Merck Laboratories (USA). All concentrations of used solutions were expressed as final molar concentration in the organs bath. Employed reagents were diluted in bi-distilled water (MiliQ), except for SNAP which was diluted in DMSO. None of the abovementioned vehicles modified the level of contraction or dilatation of the arterial rings used. 3. Experimental protocols 3.1. Assay 1: Vascular response to phenylephrine and Ach with intact endothelium Following a period of stabilization, the first vascular contraction was induced by KCl (40 mM) to obtain maximum response for each vascular ring, which was taken as 100% for comparative purposes. After sequential washings with Krebs-Henseleit solution to obtain a basal tension of 0 g, the second contraction was obtained with phenylephrine (10 4 M) and no differences were found in the force of contraction between the two experimental groups. Following a maximal induced contraction with a 10 4 M phenylephrine, the vascular rings were exposed to increasing concentrations of Ach (10 7–10 2 M). Contractions induced by phenylephrine were expressed as a percentage of the maximum contraction quantified with the use of KCl. Ach induced relaxation was quantified as a percentage of the level of contraction acquired by the use of 10 4 M phenylephrine. 3.2. Assay 2: Vascular response to Ach in endothelium deprived vascular rings The endothelium was removed by rubbing (three times) the lining of the vascular rings with a cotton swab. A 100% contractile response was induced with 10 4 M phenylephrine. Following phenylephrine response stabilization, the vascular rings were challenged with increasing Ach concentrations (10 7–10 2 M). Results were expressed as percentage of relaxation induced by different Ach concentrations, in relation to the contraction induced by 10 4 M phenylephrine. 3.3. Assay 3: Vascular response to S-Nitroso-N-acetyl-D,Lpenicillamine (SNAP), a NO donor A contractile response was induced with 10 4 M phenylephrine. SNAP was then added to the bath (50 nM). Results were expressed in relation to phenylephrine induced contraction. 3.4. Assay 4: Vascular relaxation response to Ach, following constriction with KCl Vascular contraction was induced with KCl (20 mM). From there on, increasing concentrations of Ach were employed (10 7– 10 2 M) and the relaxation responses were expressed as a percentage of the abovementioned contraction. 3.5. Assay 5: Vascular response to Ach in presence of apamin and Charybdotoxin (K channel blockers) Apamin (50 nM) and Charybdotoxin (50 nM) were used 30 min before obtaining a contractile response induced with 10 4 M phenylephrine. Increasing Ach concentrations (10 7–10 2 M) were used
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to induce relaxation. Results were expressed in relation to the maximal contraction induced by 10 4 M phenylephrine. 3.6. Data analysis Data obtained for muscular contraction and the effect of each treatment were based on real time observations using Labchart V6 software for Windows XP and values were expressed as mean ± standard deviations and stored in digital format. Statistical analysis (t student) was doing using the Statview software. The differences were considered significant at P = 0.05. 4. Results 4.1. Vascular response to phenylphrine and Ach with intact endothelium 4.1.1. Contractile response of pulmonary arterial rings from PHB and NPHB to phenylephrine In order to determine possible differences in the contractile response of vascular rings, a concentration curve was prepared. Lower concentrations to 1 10 6 phenylephrine did not induce an increment in vascular tone in rings of PHB and NPHB. Only phenylephrine concentrations of 1 10 6,1 10 5,1 10 4 could induce contraction of vascular rings of both, non-hypertensive and hypertensive broilers, but no statistical differences were detected. Lower phenylephrine concentrations did not have any effect. Fifty percent was the highest level of contractile response in the studied broiler chickens, regardless of their health condition (Fig. 1). 4.1.2. Response of pre-contracted pulmonary artery rings from PHB and NPHB to Ach To determine possible differences in vasodilator responses due to a cholinergic stimulus, a correspondent response concentration curve was generated. The major vasodilator effect in NPHB was observed using a 1 10 7 M Ach concentration, obtained after precontraction with 1 10 4 M phenylephrine. More concentrated treatments did not enhance that response. In contrast, in vascular rings taken from PHB, vasodilatations occurred with concentrations ranging from 1 10 5 M to 1 10 2 M. However differences in relaxation (%) between PHB and NPHB were observed only at 10 4–10 2 M Ach concentration. (Fig. 2). 4.1.3. Response of arterial rings from chickens with varying CI value Given that Ach generates responses with statistical differences in rings from both, NPHB and PHB, we tried to establish if there was, in vascular rings from PHB animals, a possible relationship between the exaggerated response to cholinergic stimuli and the CI. To do that, PHB were divided in two subgroups: in the first one, chickens with CI values between 25 and 32, with no apparent le-
Fig. 2. Dose–response curve to Ach in phenylephrine pre-contracted rings (1 10– 4 M). Values are expressed as mean ± SD of relaxation induced by Ach; aaa, bbb p < 0.001. (a) Comparison of Ach-induced response in vascular rings of NPHB and PHB. (b) Comparison of Ach-induced responses in vascular rings of of NPHB and PHB, with or without endothelium.
Fig. 3. Response to Ach in arterial rings pre-contracted with phenylephrine 1 10– 4 M. Values are expressed as mean ± SD. cc p < 0.01 and aaa, bbb, ccc p < 0.001. (a) Comparison of Ach-induced responses in vascular rings from chickens with CI < 25 and chickens with CI between 25 and 32. (b) Comparison of Ach-induced response in vascular rings from chickens with CI < 25 and chickens with IC between 44 and 52. (c) Comparison of Ach-induced responses in vascular rings of chickens with IC between 25 and 32 and chicken with IC between 44 and 52.
sions different from augmentation of the right ventricle mass. In subgroup 2, animals with CI ranging from 44 to 52 and overt lesions: hydropericardium, ascites and fibrin deposits in the corporal cavity. The relaxing effect of increased concentrations of Ach on vascular rings of NPHB and PHB are shown in Fig. 3. Statistical differences were found between subgroups 1 and 2 and NPHB broilers. For subgroup 1, the difference was detected at 10 4 M concentration, whereas that was true for subgroup 2, from concentrations of 10 5 M. Furthermore, there was a greater degree of vasodilatation in vascular rings prepared from subgroup 2, than in those obtained from broilers in subgroup 1.
n=15
n=24
4.2. Vascular response to Ach in endothelium deprived vascular rings The same methodology described in the preceding paragraph, was employed in denuded endothelium arterial rings taken from NPHB and PHB broilers. The vasodilator response to Ach was abolished when the endothelium was denuded in vascular rings from all broilers used (Fig. 2). 4.3. Vascular response to SNAP, a NO donor
Fig. 1. Contraction level of pulmonary artery rings from hypertensive and nonhypertensive chickens in response to phenylephrine.
After verifying the involvement of vascular endothelium in the response to Ach, we intended to study the involvement of endothe-
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Fig. 4. Comparative response to NO donor, SNAP, in pulmonary arterial rings from hypertensives and non-hypertensives broilers. Values are expressed as mean ± SD of relaxation induced by NO donor (SNAP) in phenylephrine pre-contracted rings.
Fig. 6. Charybdotoxin and Apamin effect on Ach-induced relaxation in phenylephrine pre-contracted rings from hypertensives and non-hypertensive broilers. Values are expressed as mean ± SD. a p < 0.05. aaa p < 0.001.
phenylephrine. Thereafter, the rings were exposed to different Ach concentrations. Vascular rings from NPHB showed greater vasodilatations than those from PHB, at all Ach concentrations employed, as seen in Fig. 6. 5. Discussion
Fig. 5. Ach-induced relaxation in K pre-contracted (20 mM) pulmonary arterial rings from chicken broilers with or without signs of HPA. Values are expressed as mean ± SD. ⁄p < 0.05.
lium-derived vasodilator factors in this response. Fig. 4 shows the effect of different concentrations of NO donor in pulmonary vascular rings pre contracted with phenylephrine 1 10 4 M. The figure shows that there are no statistically significant differences between rings from PHB and NPHB, after exposure to different concentrations of NO donor SNAP. 4.4. Vascular relaxation response to Ach following constriction with KCl Having noted that arterial rings from hypertensive and nonhypertensive broiler chickens, respond similarly to the NO donor, and after considering that PGI2 and PGE2 did not induce decreased vascular resistance in broilers, we studied the possible involvement of the third vasodilator agent, EDHF in the vasodilatation initiated by Ach. To do this, we compared the ability of Ach to relax KCl (20 mM) pre contracted arterial rings, obtained from NPHB and PHB. Fig. 5 shows how under condition of high extracellular K concentration, the Ach-induced vasodilating responses were reversed, in contrast to results obtained when contraction was induced with phenylephrine. In this case, NPHB showed the highest vasodilatation induced by Ach. Furthermore, differences in the relaxation induced by all concentrations of Ach are statistically significant. 4.5. Vascular response to Ach in presence of apamin and charymbdotoxin (K channel blockers) To determine a possible participation of Ca2+ activated K+ channels in the process of vasodilatation, through a cell hyperpolarizing effect, apamin (50 nM) and Charybdotoxine (50 nM) were added to the baths using rings previously contracted with 1 10 4 M
It appears that this is the first study to compare the vascular rings response of PHB and NPHB to phenylephrine and Ach. No differences were found in the effect of phenylephrine between PHB and NPHB. Pulmonary hypertensive broilers showed greater vasodilatation, dependent on the presence of the endothelium, and activated by Ach in vascular rings previously contracted with the alpha-1 agonist phenylephrine. Most likely, the observed response is not due to greater numbers of muscarinic receptors in the endothelial cells in PHB, since low concentrations of Ach did not elicit different vascular responses as seen in the correspondent assays with vascular rings obtained from NPHB. Several researchers have proposed that the endothelial dependent pulmonary vasodilatation is controlled by NO, PGI2 and EDHF (Campbell and Harder, 2001; Dora, 2001; Feletou and Vanhoutte, 2000). Vasodilatation evoked by the hyperpolarizing factor, which is resistant to NOS and prostaglandins pharmacological inhibitors, represents a fraction of the total vascular relaxation induced by muscarine cholinergic receptors in the endothelium (Chen et al., 1988). EDHF action is mediated by K+ channels opening, which leads to cellular hyperpolarization by K+ leaking (Garland et al., 1995). Under conditions of high potassium concentration dependent vasocontraction, cell hyperpolarization is not allowed. Therefore, EDHF relaxing action is thus impeded (Fukao et al., 1995; Gerber et al., 1998). From there, it can be suggested that previously observed disappearance of vasodilatation in vascular rings of PHB broilers contracted with phenylephrine (in this case potassium induced contraction), is associated with inactivation of the EDHF mechanism. It can be also suggested that the relaxing effect of EDHF should play a major role in vasodilatation in the pulmonary arteries of PHB. Possibly, the augmented vasorelaxation observed in vascular rings from healthy chickens, could be explained from a previous report, that NPHB express more eNOS in the endothelium of pulmonary arterioles of NPHB, than PHB (Moreno De Sandino and Hernández, 2003). The abovementioned results, together with others obtained in different experimental animal models (Morio et al., 2007), could suggest that PHB develop adaptive responses to overcome pathogenic effects. The present findings could reinforce this statement, since broiler chickens with higher CI developed a more pronounced vasodilating response, possibly dependent on EDHF. EDHF func-
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tion, through K+ channels activation (either by Ca2+ or ATP), is modified in various manners, according to the animal species and specific vascular tree (Doughty et al., 1999; Gambone et al., 1997). Apamin and Charybdotoxine K+ channel blockers were presently employed, as to find the type of K+ channels involved in EDHF relaxation in the pulmonary artery rings from broiler chickens. The use of apamin and Charybdotoxine disrupted the previously observed differences in vascular rings of PHB. It means that those differences could be associated with the EDHF action, when there was a potassium induced contraction. Also, that in broiler chickens the hyperpolarizing activity is mediated through Ca2+ activated K+ channels. Likewise, the action of apamin and Charybdotoxine on vascular rings response, clearly showed NO vasodilating activity, this due to inability of prostacyclin to relax the arterial rings from broiler and the inctivación of EDHF. In vascular rings from NPHB there was greater dilatation than in those rings from PHB. This could imply that there was a major production of NO in healthy broilers, as in an earlier study (Moreno De Sandino and Hernández, 2003). In acute hypoxia, there is inhibition of opening of KCa and KATP channels, which elevates intracytoplasmic Ca2+ concentration and subsequent vasoconstriction, due to alterations in membrane action potential. During chronic hypoxia there is a reduction of genetic expression of K+ channels in arterial smooth muscle cells (Coppock et al., 2001; López-Barneo et al., 1988; Nelson and Quayle, 1995; Wang et al., 1997), which leads to enhanced contractility. However, contrasting present findings with abovementioned observations, it might be suggested that in chickens with PHB, an augmented EDHF function, accompanied by K+ channels enhanced activity, could represent a compensatory alternative to hypoxic deleterious effects on expression and activity of K+ channels. EDHF over expression could be facilitated by chronic exposure to NO synthases inhibitors (Brandes et al., 2000; Scotland et al., 2001). Moreover, it has been proposed that PHB have diminished quantities of the NO synthases (Brandes et al., 2000). If we take together the abovementioned findings, it can be explained why in the present study there was excess activity of EDHF, and therefore a possible compensatory role of this molecule in the pathogenesis of PAH. Given that ET-1 levels are elevated in PAH broilers (Gomez et al., 2007; Gomez et al., 2008), it can be suggested that the EDHF vasodilating effect can be cancelled by ET-1 excess. It is proposed EDHF as an important vasodilator in the pulmonary arteries of PHB, which may play a compensatory role against the deleterious effects of hypoxia or various vasoconstrictor factors like ET-1. The greater response to Ach in PHB with CI values ranging from 44 and 55, appears to be unexpected if data from previous studies are taken into account. In those works (Gomez et al., 2007; Gomez et al., 2008; Moreno De Sandino and Hernández, 2003), there was lesser amounts of eNOS in the endothelium of PHB, as compared to healthy ones. That would imply a lost of the principal endothelium derived factor, the NO, in PHB. It is considered that observed differences in vascular rings in NHPB and HPB are not necessary related to NOS activity. However, we studied NO participation on the observed response. Therefore present study was conducted to examine if the endothelium of PHB and NPHB produced vasodilation of similar magnitude and also to see if the observed response was not due to the excessive reaction of vascular rings of PHB to NO. Pulmonary artery rings of both, healthy and PHB, responded in a similar manner when subjected to SNAP, a NO donor. Perhaps the observed greater response to Ach in artery rings of PHB is not related to an exaggerated vasodilation to NO. Besides, in order to specifically see NO involvement in the Ach induced vasodilation in rings from PHB or NPHB, we decided to discard or inactivate the vasodilating action of endothelial derived prostacyclin and EDHF. It should be
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emphasized that prostacyclin and prostaglandin E2, do not modify vascular resistance in pulmonary blood vessels and arterial pulmonary rings (Wideman et al., 2005; Stebel and Wideman, 2008; Odom et al., 2004). Given that EDHF action was presently abolished by 2 different procedures, it is feasible to propose that data in Figs. 5 and 6 clearly show specifically the involvement of NO in the vasodilation of arterial rings from NPHB and PHB. Furthermore, we could discard NO participation in the presently observed differences in Ach induced. Hence, that inhibition of NOS should not probably deny or confirm the obtained. Hence, the possibility that, under the in vitro conditions of the present work, NO was probably not responsible for the observed difference. It is desirable to undertake future studies, as to widen our knowledge on the effect of nitric oxide arterial preparations. Financial support This work was made possible by funds given to C.O. by Dirección de Investigación, Sede Bogotá. Universidad Nacional de Colombia. Acknowledgements Doctors Nestor Guerrero, Alex Garcia (Pollo Fiesta) and Martha Pulido gave important support to bring about the present work, financially supported by División de Investigaciones, Sede Bogota, National University of Colombia. References Adnot, S., Raffestin, B., Eddhahibi, S., Braquet, P., Chabrier, P., 1991. Lost of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. Journal of Clinical Investigation 87, 155–162. Alexander, A.F., Jensen, R.G., 1959. Cardiac changes in cattle with high mountain (brisket) disease and in experimental cattle maintained at high altitudes. American Journal of Veterinary Research 20, 680–689. Alexander, A.F., Will, D.H., Grover, J.F., Reeves, J.T., 1960. Pulmonary hypertension and right ventricular hypertrophy in cattle at high altitude. American Journal of Veterinary Research 21, 199–204. Baghbanzadeh, A., Decuypere, E., 2008. Ascites syndrome in broilers: physiological and nutritional perspectives. Avian Pathology 37, 117–126. Balog, J.M., 2003. Ascites syndrome (pulmonary hypertension syndrome) in broiler chickens: Are we seeing the light at the end of the tunnel? Avian and Poultry Biology Reviews 14, 99–126. Brandes, R.P., Schmitz-Winnenthal, F.H., Feletou, M., Godecke, A., Huang, P.L., Vanhoutte, P.M., Busse, R., 2000. An endothelium-derived hyperpolarizing factor distinct from no and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial no synthase knockout mice. Proceedings of the National Academy of Sciences of the United States of America 97, 9747–9752. Budhiraja, R., Tuder, R.M., Hassoun, P.M., 2004. Endothelial dysfunction in pulmonary hypertension. Circulation 109, 159–165. Campbell, W.B., Harder, D.R., 2001. Prologue: EDHF–What is it? American Journal of Physiology. Heart and Circulatory Physiology 280, H2413–H2416. Chen, G., Suzuki, H., Weston, A.H., 1988. Acetylcholine releases endotheliumderived hyperpolarizing factor and EDRF from rat blood vessels. British Journal of Pharmacology 95, 1165–1174. Coppock, E.A., Martens, J.R., Tamkun, M.M., 2001. Molecular basis of hypoxiainduced pulmonary vasoconstriction: role of voltage-gated K+ channels. American Journal of Physiology Lung Cell Molecular Physiology 281, 1–12. Cueva, S., Sillau, H., Valenzuela, A., Ploog, H.P., 1974. High altitude induced pulmonary hypertension and right heart failure in chickens. Research in Veterinary Science 16, 370–374. Dora K.A., 2001 Cell–Cell communication in the vessel wall. Vascular Methods 6 43– 50. Doughty, J.M., Plane, F., Langton, P.D., 1999. Charybdotoxin and apamin block edhf in rat mesenteric artery if selectively applied to the endothelium. American Journal of Physiology Heart Circulatory Physiology 276, H1107–H1112. Feletou, M., Vanhoutte, P.M., 2000. Endothelium dependent hyperpolarization of vascular smooth muscle cells. Acta Pharmacologica Sinica 21, 1–18. Fukao, M., Hattori, Y., Kanno, M., Sakuma, I., Kitabatake, A., 1995. Evidence for selective inhibition by lysophosphatidylcholine of acetylcholine-induced endothelium-dependent hyperpolarization and relaxation in rat mesenteric artery. British Journal of Pharmacology 116, 1541–1543. Gambone, L.M., Murray, P.A., Flavahan, N.A., 1997. Synergistic interaction between endothelium-derived no and prostacyclin in pulmonary artery: Potential role for K1 ATP channels. British Journal of Pharmacology 121, 271–279.
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Moreno De Sandino M, Hernández A., 2003, nitric oxide synthase expression in the endothelium of pulmonary arterioles in normal and pulmonary hypertensive chickens subjected to chronic hypobaric hypoxia. Avian Disorder, (47) 1291– 1297. Morio, Y., Homma, N., Takahashi, H., Yamamoto, A., Nagaoka, T., Sato, K., Muramatsu, M., Fukuchi, Y., 2007. Activity of endothelium-derived hyperpolarizing factor is augmented in monocrotaline-induced pulmonary hypertension of rat lungs. Journal of Vascular Research 44, 325–335. Nelson, M.T., Quayle, J.B., 1995. Physiological roles and properties of potassium channels in arterial smooth muscle. American Journal of Physiology 268, 799– 822. Odom, T.W., Martinez-Lemus, L.A., Hester, R.K., Becker, E.J., Jeffrey, J.S., Meininger, G.A., Ramirez, G.A., 2004. In vitro hypoxia differentially affects constriction and relaxation responses of isolated pulmonary arteries from broiler and leghorn chickens. Poultry Science 83, 835–841. Robbins, I.M., Barst, R.J., Rubin, L.J., Gaine, S.P., Price, P.V., Morrow, J.D., Christman, B.W., 2001. Increased levels of prostaglandin D2 suggest macrophage activation in patients with primary pulmonary hypertension. Chest 120, 1639–1644. Scotland, R.S., Chauhan, S., Vallance, P.J., Ahluwalia, A., 2001. An endotheliumderived hyperpolarizing factor-like factor moderates myogenic constriction of mesenteric resistance arteries in the absence of endothelial nitric oxide synthase-derived nitric oxide. Hypertension 38, 833–839. Stebel, S., Wideman, R.F., 2008. Pulmonary hemodynamic responses to intravenous prostaglandin E2 in broiler chickens. Poultry Science 87, 138–145. Steudel, W., Ichinose, F., Huang, P.L., Hurford, W.E., Jones, R.C., Bevan, J.A., Fishman, M.C., Zapol, W.M., 1997. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circulation Research 81, 34–41. Thorin E, Webb DJ, 2009. Endothelium-derived endothelin-1. Pflugers Arch. Wang, J., Juhaszova, M., Rubin, L.J., Yuan, X.J., 1997. Hypoxia inhibits of voltagegated K+ channel a subunits in pulmonary artery smooth muscle cells. Journal of Clinical Investigation 100, 2347–2353. Wideman, R.F., CPAHman, M.E., Erf, G.F., 2005. Pulmonary and systemic hemodynamic responses to intravenous prostacyclin in broilers. Poultry Science 84, 442–453. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, Y., 1998. A novel potent vasonconstrictor peptide produced by vascular endothelial cells. Nature 332, 411–415.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Endothelial hyperpolarizing factor increases acetylcholine-induced vasodilatation in pulmonary hypertensive broilers arterial rings Diana I. Alvarez-Medina a, Aureliano Hernandez b, Camilo Orozco b,⇑ a b
Directora Unidad Municipal de Asistencia Técnica Agropecuaria, La Palma, Cundinamarca, Colombia Departamento de Ciencias para la Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Sede Bogotá, Colombia
a r t i c l e
i n f o
Article history: Received 16 June 2010 Accepted 5 February 2011
Keywords: Endothelial-derived hyperpolarizing factor Pulmonary hypertension Broiler Hypoxia
a b s t r a c t Pulmonary arterial hypertension (PAH) develops as result of imbalances between endothelium derived vasoconstrictors and vasodilators. Pulmonary hypertensive broiler chickens (PHBs) are deficient in NO production and endothelin-1 (ET-1) excess. With respect to prostacyclin, it appears that it does not alter vascular pulmonary tone in broilers. However, the role of Endothelium-Derived Hyperpolarizing Factor (EDHF) in PAH in broilers has not been determined. The possible involvement of EDHF in acetylcholine (Ach) induced vasodilatation was studied in pulmonary arterial rings taken from PHB and non-pulmonary hypertensive broilers (NPHBs). Ach induced higher vasodilatation in PHB than in NPHB. This dilatation seems to be directly related to the degree of PAH. Ach derived vasodilatation was inhibited, in PBH but not in NPHB, by blocking EDHF action with K+ or Apamin plus Charybdotoxin. It is proposed EDHF as an important vasodilator in the pulmonary arteries of PHB, which may play a compensatory role in PAH pathophysiology. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Genetically predisposed broiler chickens develop hypoxic pulmonary arterial hypertension (PAH), mostly in commercial operations located above 1500 m above sea level (Cueva et al., 1974; Hernández, 1987). Genetically predisposed subjects (Huchzermeyer et al., 1987) have a pathological exaggerated response to hypoxia (that is PAH). The latter, eventually leads to insufficiency of the right ventricle to pump returning venous blood to the lungs (Baghbanzadeh and Decuypere, 2008; Balog, 2003; Maxwell et al., 1990). From there, hypertrophy and hyperplasia ensue and the cardiac index value (CI = right ventricular weight/total ventricular mass weight) increases over 25%. This measure is a reliable parameter to indirectly classify non-hypertensive (CI less than 25%) or hypertensive chickens CI values above 25%; (Hernández, 1987; Julian, 1993). The pathological findings in PAH in broiler chickens are similar to those reported in PAH in humans, and, therefore, possibly the broiler chicken can be used as a model in this context. The morphological changes in PAH includes thickening of the medial layer of pulmonary blood vessels (Karamsetty et al., 1993) and nowadays, ⇑ Corresponding author. Address: Departamento de Ciencias para la Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 No. 26-85. Bogotá D.C. Colombia. Tel.: +57 1 3165000. E-mail address: [email protected] (C. Orozco). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.004
it is quite clear that molecules produced in the endothelium are partly responsible for smooth muscle contraction and growth of the medial layer of the pulmonary arterial tree, when the balance of endothelial produced contractors and dilators is disrupted which leads to endothelial malfunction (Adnot et al., 1991; Budhiraja et al., 2004; Thorin and Webb, 2009). In previous studies, endothelin-1 (ET-1), an important vasoconstrictor, was found to be upregulated in susceptible chickens (Gomez et al., 2007). In pulmonary hypertensive mammals and pulmonary hypertensive broiler chickens (PHBs), there is lesser expression of endothelial nitric oxide synthase (eNOS) (Moreno De Sandino and Hernández, 2003) and, therefore, a diminished endothelial derived vasodilating response to hypoxia (Gomez et al., 2007). Prostaglandin I2 (PGI2), of endothelial origin, has a vasodilating effect in mammals (Steudel et al., 1997) and blood flow resistance is lessened, which might reduce PAH (Robbins et al., 2001). Under normal conditions, in mammals, PGI2 synthase is expressed in major quantities in large blood vessels and it seems that in PHA, that characteristic is reversed, probably due to a more pronounced endothelial alterations there. This downregulated alteration of PGI2 function, induces greater vasoconstriction and tendency to intravascular coagulation (Wideman et al., 2005). In contrast, it has been proposed that in broiler chickens PGI2 and prostaglandin E2 (PGE2) probably lower pulmonary artery pressure indirectly by altering cardiac output and not through a direct action on blood vessels (Robbins et al., 2001; Lopes et al., 2002).
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It is well established that acetylcholine (Ach), and some of its agonists, could stimulate the synthesis of relaxing factors in the endothelium, and thus induce hyperpolarization of vascular smooth muscle cells. This hyperpolarizing effect is resistant to the opposing effects of NOS and cyclooxygenase inhibitors (Stebel and Wideman, 2008; Chen et al., 1988). Hence, it is likely that a third endothelial hyperpolarizing agent could have a role in the pathogenesis of PAH. Release of endothelial hyperpolarizing factor (EDHF) is initiated through activation of cholinergic muscarinic receptors, which are pharmacologically different from those responsible for NO release. The expression of M1 Ach receptors mediates EDHF liberation, whereas M2 and M3 receptors should do so for NO (Yanagisawa et al., 1998). Relaxation of vascular smooth muscle induced by EDHF is associated with K+ channels activation (Komori and Suzuki, 1987). Thus, EDHF could induce hyperpolarization by promoting the opening of various K+ channels, after Ca2+ or ATP activation. Distribution of these type of K+ channels could change within animal species, and also, according to the correspondent arterial system. For instance, in the dog´s pulmonary artery, EDHF action is associated with ATP dependent K+ channels, whereas in the rat´s mesenteric artery, the correspondent action is Ca2+ dependent (Garland et al., 1995; Doughty et al., 1999). The abovementioned characteristics, were demonstrated by EDHF inactivation with depolarizing agents (Gambone et al., 1997). The objective of the present study was to evaluate the possible participation of EDHF on endothelial dependent vasodilatation in pulmonary arterial rings and its possible intervention in the inherent mechanisms of PAH in broilers. It should be noted that the role of EDHF in pathogenesis of PAH in broiler chickens has not been previously reported.
2. Materials and methods 2.1. Vascular rings preparation Commercial male broiler chickens with ages ranging from 28 to 42 days, were obtained from a local commercial farm in Bogotá (Colombia; 2638 m above sea level). They were classified according to the so called cardiac index (CI = right ventricular weight/total ventricular mass weight (McCulloch and Randall, 1998; Alexander et al., 1960). That value in non-pulmonary hypertensive broilers (NPHBs) were under 25%, (average mean ± standard deviation values: 21.3 ± 1.8). PHB, correspondent values were 43.1 ± 6.6 (Hernández, 1987; Julian, 1993). Moreover, clinical and necropsy findings were taken into account as to discard subjects which had lesions and clinical signs of illnesses different from PAH. All broilers chickens were subjected to anesthesia with inhaled 8% isoflurane. Under deep anesthesia maintained with isoflurane (6%), death was provoked by incision in the aorta. The heart and lungs were immediately separated, the pulmonary artery devoid of surrounding fat, was placed in a Petri dish with a Krebs– Henseleit solution (119 mM NaCl, 4.7 mM KCl, 2.5 mM, CaCl21.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3 and 5.6 mM glucose), maintained at 42 °C and continuously bubbled with a gas mixture (5% CO2 and 95% O2). Three to 5 mm rings were taken at 2 mm distance from the pulmonary artery bifurcation (Alexander and Jensen, 1959). Each ring was fixed at opposing points from each other, using two stainless steel wires. One of them was secured within the rings bath and the other, to an isometric transducer (MLT0202, Panlab, Spain) connected to a dispositive for data acquisition (Power Lab, USA) which provided information directly to a computer. Every ring´s bath was maintained at 42 °C and contained 20 ml of Krebs-Henseleit solution with the abovementioned bubbling mixture. The pulmonary arterial rings were stabilized
for 30 min by Krebs-Henseleit solution changes every 5 min and 2 g basal tension. Ach, phenylephrine y SNAP were obtained from Sigma Aldrich (St. Louis, MO). NaHCO3, C6H12O6, KCl, CaCl2, NaCl, KH2PO4, MgSO4, and C6H8O6, from Merck Laboratories (USA). All concentrations of used solutions were expressed as final molar concentration in the organs bath. Employed reagents were diluted in bi-distilled water (MiliQ), except for SNAP which was diluted in DMSO. None of the abovementioned vehicles modified the level of contraction or dilatation of the arterial rings used. 3. Experimental protocols 3.1. Assay 1: Vascular response to phenylephrine and Ach with intact endothelium Following a period of stabilization, the first vascular contraction was induced by KCl (40 mM) to obtain maximum response for each vascular ring, which was taken as 100% for comparative purposes. After sequential washings with Krebs-Henseleit solution to obtain a basal tension of 0 g, the second contraction was obtained with phenylephrine (10 4 M) and no differences were found in the force of contraction between the two experimental groups. Following a maximal induced contraction with a 10 4 M phenylephrine, the vascular rings were exposed to increasing concentrations of Ach (10 7–10 2 M). Contractions induced by phenylephrine were expressed as a percentage of the maximum contraction quantified with the use of KCl. Ach induced relaxation was quantified as a percentage of the level of contraction acquired by the use of 10 4 M phenylephrine. 3.2. Assay 2: Vascular response to Ach in endothelium deprived vascular rings The endothelium was removed by rubbing (three times) the lining of the vascular rings with a cotton swab. A 100% contractile response was induced with 10 4 M phenylephrine. Following phenylephrine response stabilization, the vascular rings were challenged with increasing Ach concentrations (10 7–10 2 M). Results were expressed as percentage of relaxation induced by different Ach concentrations, in relation to the contraction induced by 10 4 M phenylephrine. 3.3. Assay 3: Vascular response to S-Nitroso-N-acetyl-D,Lpenicillamine (SNAP), a NO donor A contractile response was induced with 10 4 M phenylephrine. SNAP was then added to the bath (50 nM). Results were expressed in relation to phenylephrine induced contraction. 3.4. Assay 4: Vascular relaxation response to Ach, following constriction with KCl Vascular contraction was induced with KCl (20 mM). From there on, increasing concentrations of Ach were employed (10 7– 10 2 M) and the relaxation responses were expressed as a percentage of the abovementioned contraction. 3.5. Assay 5: Vascular response to Ach in presence of apamin and Charybdotoxin (K channel blockers) Apamin (50 nM) and Charybdotoxin (50 nM) were used 30 min before obtaining a contractile response induced with 10 4 M phenylephrine. Increasing Ach concentrations (10 7–10 2 M) were used
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to induce relaxation. Results were expressed in relation to the maximal contraction induced by 10 4 M phenylephrine. 3.6. Data analysis Data obtained for muscular contraction and the effect of each treatment were based on real time observations using Labchart V6 software for Windows XP and values were expressed as mean ± standard deviations and stored in digital format. Statistical analysis (t student) was doing using the Statview software. The differences were considered significant at P = 0.05. 4. Results 4.1. Vascular response to phenylphrine and Ach with intact endothelium 4.1.1. Contractile response of pulmonary arterial rings from PHB and NPHB to phenylephrine In order to determine possible differences in the contractile response of vascular rings, a concentration curve was prepared. Lower concentrations to 1 10 6 phenylephrine did not induce an increment in vascular tone in rings of PHB and NPHB. Only phenylephrine concentrations of 1 10 6,1 10 5,1 10 4 could induce contraction of vascular rings of both, non-hypertensive and hypertensive broilers, but no statistical differences were detected. Lower phenylephrine concentrations did not have any effect. Fifty percent was the highest level of contractile response in the studied broiler chickens, regardless of their health condition (Fig. 1). 4.1.2. Response of pre-contracted pulmonary artery rings from PHB and NPHB to Ach To determine possible differences in vasodilator responses due to a cholinergic stimulus, a correspondent response concentration curve was generated. The major vasodilator effect in NPHB was observed using a 1 10 7 M Ach concentration, obtained after precontraction with 1 10 4 M phenylephrine. More concentrated treatments did not enhance that response. In contrast, in vascular rings taken from PHB, vasodilatations occurred with concentrations ranging from 1 10 5 M to 1 10 2 M. However differences in relaxation (%) between PHB and NPHB were observed only at 10 4–10 2 M Ach concentration. (Fig. 2). 4.1.3. Response of arterial rings from chickens with varying CI value Given that Ach generates responses with statistical differences in rings from both, NPHB and PHB, we tried to establish if there was, in vascular rings from PHB animals, a possible relationship between the exaggerated response to cholinergic stimuli and the CI. To do that, PHB were divided in two subgroups: in the first one, chickens with CI values between 25 and 32, with no apparent le-
Fig. 2. Dose–response curve to Ach in phenylephrine pre-contracted rings (1 10– 4 M). Values are expressed as mean ± SD of relaxation induced by Ach; aaa, bbb p < 0.001. (a) Comparison of Ach-induced response in vascular rings of NPHB and PHB. (b) Comparison of Ach-induced responses in vascular rings of of NPHB and PHB, with or without endothelium.
Fig. 3. Response to Ach in arterial rings pre-contracted with phenylephrine 1 10– 4 M. Values are expressed as mean ± SD. cc p < 0.01 and aaa, bbb, ccc p < 0.001. (a) Comparison of Ach-induced responses in vascular rings from chickens with CI < 25 and chickens with CI between 25 and 32. (b) Comparison of Ach-induced response in vascular rings from chickens with CI < 25 and chickens with IC between 44 and 52. (c) Comparison of Ach-induced responses in vascular rings of chickens with IC between 25 and 32 and chicken with IC between 44 and 52.
sions different from augmentation of the right ventricle mass. In subgroup 2, animals with CI ranging from 44 to 52 and overt lesions: hydropericardium, ascites and fibrin deposits in the corporal cavity. The relaxing effect of increased concentrations of Ach on vascular rings of NPHB and PHB are shown in Fig. 3. Statistical differences were found between subgroups 1 and 2 and NPHB broilers. For subgroup 1, the difference was detected at 10 4 M concentration, whereas that was true for subgroup 2, from concentrations of 10 5 M. Furthermore, there was a greater degree of vasodilatation in vascular rings prepared from subgroup 2, than in those obtained from broilers in subgroup 1.
n=15
n=24
4.2. Vascular response to Ach in endothelium deprived vascular rings The same methodology described in the preceding paragraph, was employed in denuded endothelium arterial rings taken from NPHB and PHB broilers. The vasodilator response to Ach was abolished when the endothelium was denuded in vascular rings from all broilers used (Fig. 2). 4.3. Vascular response to SNAP, a NO donor
Fig. 1. Contraction level of pulmonary artery rings from hypertensive and nonhypertensive chickens in response to phenylephrine.
After verifying the involvement of vascular endothelium in the response to Ach, we intended to study the involvement of endothe-
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Fig. 4. Comparative response to NO donor, SNAP, in pulmonary arterial rings from hypertensives and non-hypertensives broilers. Values are expressed as mean ± SD of relaxation induced by NO donor (SNAP) in phenylephrine pre-contracted rings.
Fig. 6. Charybdotoxin and Apamin effect on Ach-induced relaxation in phenylephrine pre-contracted rings from hypertensives and non-hypertensive broilers. Values are expressed as mean ± SD. a p < 0.05. aaa p < 0.001.
phenylephrine. Thereafter, the rings were exposed to different Ach concentrations. Vascular rings from NPHB showed greater vasodilatations than those from PHB, at all Ach concentrations employed, as seen in Fig. 6. 5. Discussion
Fig. 5. Ach-induced relaxation in K pre-contracted (20 mM) pulmonary arterial rings from chicken broilers with or without signs of HPA. Values are expressed as mean ± SD. ⁄p < 0.05.
lium-derived vasodilator factors in this response. Fig. 4 shows the effect of different concentrations of NO donor in pulmonary vascular rings pre contracted with phenylephrine 1 10 4 M. The figure shows that there are no statistically significant differences between rings from PHB and NPHB, after exposure to different concentrations of NO donor SNAP. 4.4. Vascular relaxation response to Ach following constriction with KCl Having noted that arterial rings from hypertensive and nonhypertensive broiler chickens, respond similarly to the NO donor, and after considering that PGI2 and PGE2 did not induce decreased vascular resistance in broilers, we studied the possible involvement of the third vasodilator agent, EDHF in the vasodilatation initiated by Ach. To do this, we compared the ability of Ach to relax KCl (20 mM) pre contracted arterial rings, obtained from NPHB and PHB. Fig. 5 shows how under condition of high extracellular K concentration, the Ach-induced vasodilating responses were reversed, in contrast to results obtained when contraction was induced with phenylephrine. In this case, NPHB showed the highest vasodilatation induced by Ach. Furthermore, differences in the relaxation induced by all concentrations of Ach are statistically significant. 4.5. Vascular response to Ach in presence of apamin and charymbdotoxin (K channel blockers) To determine a possible participation of Ca2+ activated K+ channels in the process of vasodilatation, through a cell hyperpolarizing effect, apamin (50 nM) and Charybdotoxine (50 nM) were added to the baths using rings previously contracted with 1 10 4 M
It appears that this is the first study to compare the vascular rings response of PHB and NPHB to phenylephrine and Ach. No differences were found in the effect of phenylephrine between PHB and NPHB. Pulmonary hypertensive broilers showed greater vasodilatation, dependent on the presence of the endothelium, and activated by Ach in vascular rings previously contracted with the alpha-1 agonist phenylephrine. Most likely, the observed response is not due to greater numbers of muscarinic receptors in the endothelial cells in PHB, since low concentrations of Ach did not elicit different vascular responses as seen in the correspondent assays with vascular rings obtained from NPHB. Several researchers have proposed that the endothelial dependent pulmonary vasodilatation is controlled by NO, PGI2 and EDHF (Campbell and Harder, 2001; Dora, 2001; Feletou and Vanhoutte, 2000). Vasodilatation evoked by the hyperpolarizing factor, which is resistant to NOS and prostaglandins pharmacological inhibitors, represents a fraction of the total vascular relaxation induced by muscarine cholinergic receptors in the endothelium (Chen et al., 1988). EDHF action is mediated by K+ channels opening, which leads to cellular hyperpolarization by K+ leaking (Garland et al., 1995). Under conditions of high potassium concentration dependent vasocontraction, cell hyperpolarization is not allowed. Therefore, EDHF relaxing action is thus impeded (Fukao et al., 1995; Gerber et al., 1998). From there, it can be suggested that previously observed disappearance of vasodilatation in vascular rings of PHB broilers contracted with phenylephrine (in this case potassium induced contraction), is associated with inactivation of the EDHF mechanism. It can be also suggested that the relaxing effect of EDHF should play a major role in vasodilatation in the pulmonary arteries of PHB. Possibly, the augmented vasorelaxation observed in vascular rings from healthy chickens, could be explained from a previous report, that NPHB express more eNOS in the endothelium of pulmonary arterioles of NPHB, than PHB (Moreno De Sandino and Hernández, 2003). The abovementioned results, together with others obtained in different experimental animal models (Morio et al., 2007), could suggest that PHB develop adaptive responses to overcome pathogenic effects. The present findings could reinforce this statement, since broiler chickens with higher CI developed a more pronounced vasodilating response, possibly dependent on EDHF. EDHF func-
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tion, through K+ channels activation (either by Ca2+ or ATP), is modified in various manners, according to the animal species and specific vascular tree (Doughty et al., 1999; Gambone et al., 1997). Apamin and Charybdotoxine K+ channel blockers were presently employed, as to find the type of K+ channels involved in EDHF relaxation in the pulmonary artery rings from broiler chickens. The use of apamin and Charybdotoxine disrupted the previously observed differences in vascular rings of PHB. It means that those differences could be associated with the EDHF action, when there was a potassium induced contraction. Also, that in broiler chickens the hyperpolarizing activity is mediated through Ca2+ activated K+ channels. Likewise, the action of apamin and Charybdotoxine on vascular rings response, clearly showed NO vasodilating activity, this due to inability of prostacyclin to relax the arterial rings from broiler and the inctivación of EDHF. In vascular rings from NPHB there was greater dilatation than in those rings from PHB. This could imply that there was a major production of NO in healthy broilers, as in an earlier study (Moreno De Sandino and Hernández, 2003). In acute hypoxia, there is inhibition of opening of KCa and KATP channels, which elevates intracytoplasmic Ca2+ concentration and subsequent vasoconstriction, due to alterations in membrane action potential. During chronic hypoxia there is a reduction of genetic expression of K+ channels in arterial smooth muscle cells (Coppock et al., 2001; López-Barneo et al., 1988; Nelson and Quayle, 1995; Wang et al., 1997), which leads to enhanced contractility. However, contrasting present findings with abovementioned observations, it might be suggested that in chickens with PHB, an augmented EDHF function, accompanied by K+ channels enhanced activity, could represent a compensatory alternative to hypoxic deleterious effects on expression and activity of K+ channels. EDHF over expression could be facilitated by chronic exposure to NO synthases inhibitors (Brandes et al., 2000; Scotland et al., 2001). Moreover, it has been proposed that PHB have diminished quantities of the NO synthases (Brandes et al., 2000). If we take together the abovementioned findings, it can be explained why in the present study there was excess activity of EDHF, and therefore a possible compensatory role of this molecule in the pathogenesis of PAH. Given that ET-1 levels are elevated in PAH broilers (Gomez et al., 2007; Gomez et al., 2008), it can be suggested that the EDHF vasodilating effect can be cancelled by ET-1 excess. It is proposed EDHF as an important vasodilator in the pulmonary arteries of PHB, which may play a compensatory role against the deleterious effects of hypoxia or various vasoconstrictor factors like ET-1. The greater response to Ach in PHB with CI values ranging from 44 and 55, appears to be unexpected if data from previous studies are taken into account. In those works (Gomez et al., 2007; Gomez et al., 2008; Moreno De Sandino and Hernández, 2003), there was lesser amounts of eNOS in the endothelium of PHB, as compared to healthy ones. That would imply a lost of the principal endothelium derived factor, the NO, in PHB. It is considered that observed differences in vascular rings in NHPB and HPB are not necessary related to NOS activity. However, we studied NO participation on the observed response. Therefore present study was conducted to examine if the endothelium of PHB and NPHB produced vasodilation of similar magnitude and also to see if the observed response was not due to the excessive reaction of vascular rings of PHB to NO. Pulmonary artery rings of both, healthy and PHB, responded in a similar manner when subjected to SNAP, a NO donor. Perhaps the observed greater response to Ach in artery rings of PHB is not related to an exaggerated vasodilation to NO. Besides, in order to specifically see NO involvement in the Ach induced vasodilation in rings from PHB or NPHB, we decided to discard or inactivate the vasodilating action of endothelial derived prostacyclin and EDHF. It should be
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emphasized that prostacyclin and prostaglandin E2, do not modify vascular resistance in pulmonary blood vessels and arterial pulmonary rings (Wideman et al., 2005; Stebel and Wideman, 2008; Odom et al., 2004). Given that EDHF action was presently abolished by 2 different procedures, it is feasible to propose that data in Figs. 5 and 6 clearly show specifically the involvement of NO in the vasodilation of arterial rings from NPHB and PHB. Furthermore, we could discard NO participation in the presently observed differences in Ach induced. Hence, that inhibition of NOS should not probably deny or confirm the obtained. Hence, the possibility that, under the in vitro conditions of the present work, NO was probably not responsible for the observed difference. It is desirable to undertake future studies, as to widen our knowledge on the effect of nitric oxide arterial preparations. Financial support This work was made possible by funds given to C.O. by Dirección de Investigación, Sede Bogotá. Universidad Nacional de Colombia. Acknowledgements Doctors Nestor Guerrero, Alex Garcia (Pollo Fiesta) and Martha Pulido gave important support to bring about the present work, financially supported by División de Investigaciones, Sede Bogota, National University of Colombia. References Adnot, S., Raffestin, B., Eddhahibi, S., Braquet, P., Chabrier, P., 1991. Lost of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. Journal of Clinical Investigation 87, 155–162. Alexander, A.F., Jensen, R.G., 1959. Cardiac changes in cattle with high mountain (brisket) disease and in experimental cattle maintained at high altitudes. American Journal of Veterinary Research 20, 680–689. Alexander, A.F., Will, D.H., Grover, J.F., Reeves, J.T., 1960. Pulmonary hypertension and right ventricular hypertrophy in cattle at high altitude. American Journal of Veterinary Research 21, 199–204. Baghbanzadeh, A., Decuypere, E., 2008. Ascites syndrome in broilers: physiological and nutritional perspectives. Avian Pathology 37, 117–126. Balog, J.M., 2003. Ascites syndrome (pulmonary hypertension syndrome) in broiler chickens: Are we seeing the light at the end of the tunnel? Avian and Poultry Biology Reviews 14, 99–126. Brandes, R.P., Schmitz-Winnenthal, F.H., Feletou, M., Godecke, A., Huang, P.L., Vanhoutte, P.M., Busse, R., 2000. An endothelium-derived hyperpolarizing factor distinct from no and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial no synthase knockout mice. Proceedings of the National Academy of Sciences of the United States of America 97, 9747–9752. Budhiraja, R., Tuder, R.M., Hassoun, P.M., 2004. Endothelial dysfunction in pulmonary hypertension. Circulation 109, 159–165. Campbell, W.B., Harder, D.R., 2001. Prologue: EDHF–What is it? American Journal of Physiology. Heart and Circulatory Physiology 280, H2413–H2416. Chen, G., Suzuki, H., Weston, A.H., 1988. Acetylcholine releases endotheliumderived hyperpolarizing factor and EDRF from rat blood vessels. British Journal of Pharmacology 95, 1165–1174. Coppock, E.A., Martens, J.R., Tamkun, M.M., 2001. Molecular basis of hypoxiainduced pulmonary vasoconstriction: role of voltage-gated K+ channels. American Journal of Physiology Lung Cell Molecular Physiology 281, 1–12. Cueva, S., Sillau, H., Valenzuela, A., Ploog, H.P., 1974. High altitude induced pulmonary hypertension and right heart failure in chickens. Research in Veterinary Science 16, 370–374. Dora K.A., 2001 Cell–Cell communication in the vessel wall. Vascular Methods 6 43– 50. Doughty, J.M., Plane, F., Langton, P.D., 1999. Charybdotoxin and apamin block edhf in rat mesenteric artery if selectively applied to the endothelium. American Journal of Physiology Heart Circulatory Physiology 276, H1107–H1112. Feletou, M., Vanhoutte, P.M., 2000. Endothelium dependent hyperpolarization of vascular smooth muscle cells. Acta Pharmacologica Sinica 21, 1–18. Fukao, M., Hattori, Y., Kanno, M., Sakuma, I., Kitabatake, A., 1995. Evidence for selective inhibition by lysophosphatidylcholine of acetylcholine-induced endothelium-dependent hyperpolarization and relaxation in rat mesenteric artery. British Journal of Pharmacology 116, 1541–1543. Gambone, L.M., Murray, P.A., Flavahan, N.A., 1997. Synergistic interaction between endothelium-derived no and prostacyclin in pulmonary artery: Potential role for K1 ATP channels. British Journal of Pharmacology 121, 271–279.
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