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Cholesterol feeding attenuates endothelium-dependent relaxation response to acetylcholine in the main pulmonary artery of chickens
European Journal of Pharmacology, 129 (1986) 397-400 Elsevier
397
EJP 455SC Short communication
Cholesterol feeding attenuates endothelium-dependent relaxation response to acetylcholine in the main pulmonary artery of chickens 1 H a k k i E. A k s u l u , Selim Cellek a n d Rtistti K. T i i r k c r * Department of Pharmacology, Faculty of Medicine, University of Ankara, Ankara, Turkey Received 4 August 1986, accepted 19 August 1986
The endothelium-dependent relaxation in response to acetylcholine was significantly less in pulmonary artery strips from chickens fed 5% cholesterol for 4 weeks than in strips from animals fed an 'ordinary' diet. No relaxation was observed in the strips from either group when the endothelium was disrupted or the strips were pretreated with hydroquinone. These results indicate that the relaxing effect of acetylcholine is endothelium-dependent and that hypercholesterolemia leads to an impairement of the endothelium-mediated relaxation response of the pulmonary artery strips to acetylcholine. Endothelium-dependent relaxation; Acetylcholine; Hypercholesterolemia; (Chickens)
1. Introduction Endothelium derived relaxing factor (EDRF) has recently been described as a humoral substance(s) originating from vascular endothelium. This conclusion was based on experiments in which acetylcholine (ACh) and several other agents e.g. bradykinin, substance P, histamine, thrombin and Ca 2+ ionophore A23187 (for review see Furchgott, 1983) produced relaxation in precontracted vascular strips with intact endothelium but not in strips with disrupted endothelium. E D R F seems to be an endogenous endothelium-originating modulator for many vasoconstrictor and vasodilator agents (Cocks and Angus, 1983; Egleme et al., 1984). Apparent vascular morphological changes have been observed during experimental atheroscleroi This work was supported by grants from the Turkish Scientific and Technical Research Council (TAG-578) and Ankara University Research Foundation (No. 86-09-00-15). * To whom all correspondence should be addressed: A.0. Tip Fakiiltesi, Farmakoloji Enstitiisii, Sihhiye, Ankara, Turkey. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
sis. A high intake of cholesterol can produce atherosclerotic lesions in the vascular wall of rabbit and chickens. An increase in thickness and stiffness of the vascular wall and changes in permeability of the intimal surface to lipoproteins and albumin are well-known phenomena Occurring during the development of atherogenesis in chickens fed a high cholesterol diet (Siller, 1965). It is also known that all arteries are susceptible to cholesterol-induced atherogenesis, except the pulmonary artery in rabbits which remains free of vascular changes (Ibengwe and Suzuki, 1986). The effect of ACh has not yet been studied in the precontracted main pulmonary artery of chickens. The present data indicate that ACh produces endothelium-dependent relaxation of this artery from chickens and that this relaxation was significantly reduced in the same arterial segment from hypercholesterolemic animals. 2. Materials and methods Male chxckens aged 2 weeks and weighing 104.0 _+ 5.0 g (n = 26) were kept at 21°C in separate
398
cages and given sufficient water and ordinary food daily for a period of 1 week. Half of the animals were then fed ordinary chicken food while the others received 5% cholesterol added to the diet. The daily intake of additional cholesterol was kept between 800-1000 mg for each animal. This feeding program was adhered to for a period of 4 weeks. Body weight and serum cholesterol levels were measured at the end of this period. The main pulmonary artery was removed immediately after decapitation and transferred to a beaker containing oxygenated (5% CO 2 in 02) Krebs solution at room temperature. Surrounding tissues were dissected out and the arterial segments were cut spirally with 4 m m width and 2 cm length. The strips were suspended in 15 ml jacketed organ baths containing oxygenated and warmed (37 o C) Krebs solution. Isometric contractions were recorded on a Grass polygraph (Model 79 D) via force-displacement transducers (Grass FT.03) under 0.5 g initial tension. The strips were allowed to equilibrate for 1 h and the bathing medium was replaced every 15 min with Krebs solution. After this equilibration period noradrenaline was added to the bathing medium at the concentration of 10 7 M and submaximal contraction was elicited.
ACh was then added to the bathing medium and cumulative concentration-response curves were determined. The concentration-response studies were also repeated in rubbed and hydroquinonepreincubated unrubbed strips. Hydroquinone was added to the bathing medium at the concentration of 10 5 M 10 min before the test. The results were evaluated statistically with Student's t-test.
3. Results
Feeding the animals a high cholesterol diet for a period of 4 weeks did not produce a significant change in their body weights compared to the controls. The average body weight of the cholesterol-fed animals was 390.0 + 15.0 g while it was 360.0 + 16.0 g (n = 13) in animals fed the ordinary diet. The average serum cholesterol levels of control and cholesterol-fed chickens were 94.0 + 4.3 and 339.0 + 22.9 m g / d l (n = 13) respectively. ACh produced concentration-dependent relaxation in precontracted strips from normal and hypercholesterolemic chickens. The relaxing effect of ACh disappeared completely in endothelium-dis-
/
U MIN O'5gI
0,59
lj
n~-T
--'O
NA
,_
/
5=lO-m ~-r 5in0-" O'----'<'-----'O
40.SI~
ACh
HYD
/
I~ 7 O
NA
5=10"O 10.7 5=C ' O O O
(M)
ACh
Fig. 1. Tracings showing the effect of acetylcholine (ACh) on noradrenaline (NA)-precontracted main pulmonary artery strips from hypercholesterolemic (upper) and normal (lower) chickens before and after addition of hydroquinone (HYD) to the incubation bath. H Y D impaired the relaxing effect of ACh and converted it to a contractile one. The contractile effect of N A was slightly enhanced after HYD.
399
rupted strips. Pretreatment of the strips with hydroquinone (10 -5 M) also completely abolished the relaxing effect of ACh in strips with an intact endothelium (fig. 1). The relaxing effect of ACh was converted to a contractile one following hydroquinone pretreatment. Endothelium disruption or hydroquinone pretreatment also caused potentiation of the contractile response to noradrenaline. This enhancement was 30.0 _+ 5.0% (n = 4) for the hydroquinone pretreatment. There was no significant change or in some experiments, a slight decrease was observed in the contractile response to noradrenaline in the strips from hypercholesterolemic chickens. The concentration-response curve of ACh in hypercholesterolemic chicken strips was significantly reduced when compared with the curve from normal chickens (fig. 2). The maximum relaxing effect of ACh was also reduced by about 100% in the strips from hypercholesterolemic chickens as compared to the control.
I00
0 0 o
•~
50
ar
0
I
i0 -e
I
i0 -r
I
i 0 -6
1
i0 -s
EACh-~(M ) Fig. 2. The concentration-response curves of acetylcholine (ACh) in main pulmonary artery strips from normal (upper) and hypercholesterolemic (lower) chickens precontracted by noradrenaline (10-7 M). Each point represents the mean value from 13 experiments; vertical bars show S.E.M.
4. D i s c u s s i o n
The data presented indicate clearly that ACh produced an endothelium-dependent relaxation in the main pulmonary artery from chickens. The experiments in which ACh caused a relaxation in the noradrenaline-precontracted vascular strips with intact but not with disrupted endothelium made this evident. In addition, hydroquinone, a free-radical scavenger (Furchgott, 1983), when added to the incubation medium abolished the relaxing effect of ACh. The most interesting facet of this study is the close correlation between high serum cholesterol level and reduced relaxing effect of ACh in the main pulmonary artery strips. It is well known that increasing the dietary intake of cholesterol of rabbit and chickens produces atherosclerosis (Duff et al., 1957; Siller, 1965). Damage to endothelial cells is a consistent feature in cholesterol-induced atherosclerosis (Klimov et al., 1981) and these changes could interfere with endothelium-mediated vasodilation. It is also well-known that the pulmonary artery is a vasculature less susceptible to cholesterol-induced atherogenesis in rabbits (Ibengwe and Suzuki, 1986). Young chickens fed a high cholesterol diet for a short period do not develop the typical morphological changes of atherosclerogenesis in the pulmonary artery (Siller, 1965). However, a significant decrease in the relaxing effect of ACh was obtained in the precontracted pulmonary artery strips from hypercholesterolemic chickens in the present study. These results support the recent observation of Jayacody et al. (1985) who have shown that cholesterol feeding impaired endothelium-dependent relaxation of rabbit aorta. Moreover these authors also observed characteristic morphological features in the aorta from rabbits fed a high cholesterol diet. It was unlikely that such morphological changes could be expected in the main pulmonary artery of chickens following a short period on a high cholesterol diet. We therefore postulated that hypercholesterolemia depressed via some unknown mechanisms the AChinduced relaxation mediated by vascular endothelium and that this depression was not related to the production of atherosclerotic lesions. These findings were taken as an evidence that
400 b i o c h e m i c a l c h a n g e s in t h e v a s c u l a r e n d o t h e l i u m p r e c e d e the d e v e l o p m e n t of m o r p h o l o g i c a l changes in t h e v a s c u l a r wall o f c h i c k e n m a i n p u l m o n a r y artery.
References Cocks, T.M. and J.A. Angus, 1983, Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin, Nature (London) 305, 627. Duff, G.L., G.C. McMillan and A.C. Ritchie, 1957, The morphology of early atherosclerotic lesions of the aorta demonstrated by the surface techniques in rabbits fed cholesterol, Am. J. Pathol. 33, 845. Egleme, C., T. Godfraind and R.C. Miller, 1984, Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells, Br. J. Pharmacol. 81, 16.
Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Ibengwe, J.K. and H. Suzuki, 1986, Changes in mechanical responses of vascular smooth muscles to acetylcholine, noradrenaline and high-potassium solution in hypercholesterolemic rabbits, Br. J. Pharmacol. 87, 395. Jayakody, R.L., M.P.J. Senaratne, A.B.R. Thomson and C.T. Kappagoda, 1985, Cholesterol feeding impairs endothelium-dependent relaxation of rabbit aorta, Can. J. Physiol. Pharmacol. 63, 1206. Klimov, A.N., V.A. Nagornev and T.N. Lovagina, 1981, Functional characteristics of endothelium on the dynamics of experimental atherosclerosis, Pario Arterielle 7, 47. Siller, W.G., 1965, Spontaneous atherosclerosis in the fowl, in: Comparative Atherosclerosis, eds. J.C. Roberts, R. Straus and M.S. Cooper (Harper and Row Publish., New York) p. 66.
397
EJP 455SC Short communication
Cholesterol feeding attenuates endothelium-dependent relaxation response to acetylcholine in the main pulmonary artery of chickens 1 H a k k i E. A k s u l u , Selim Cellek a n d Rtistti K. T i i r k c r * Department of Pharmacology, Faculty of Medicine, University of Ankara, Ankara, Turkey Received 4 August 1986, accepted 19 August 1986
The endothelium-dependent relaxation in response to acetylcholine was significantly less in pulmonary artery strips from chickens fed 5% cholesterol for 4 weeks than in strips from animals fed an 'ordinary' diet. No relaxation was observed in the strips from either group when the endothelium was disrupted or the strips were pretreated with hydroquinone. These results indicate that the relaxing effect of acetylcholine is endothelium-dependent and that hypercholesterolemia leads to an impairement of the endothelium-mediated relaxation response of the pulmonary artery strips to acetylcholine. Endothelium-dependent relaxation; Acetylcholine; Hypercholesterolemia; (Chickens)
1. Introduction Endothelium derived relaxing factor (EDRF) has recently been described as a humoral substance(s) originating from vascular endothelium. This conclusion was based on experiments in which acetylcholine (ACh) and several other agents e.g. bradykinin, substance P, histamine, thrombin and Ca 2+ ionophore A23187 (for review see Furchgott, 1983) produced relaxation in precontracted vascular strips with intact endothelium but not in strips with disrupted endothelium. E D R F seems to be an endogenous endothelium-originating modulator for many vasoconstrictor and vasodilator agents (Cocks and Angus, 1983; Egleme et al., 1984). Apparent vascular morphological changes have been observed during experimental atheroscleroi This work was supported by grants from the Turkish Scientific and Technical Research Council (TAG-578) and Ankara University Research Foundation (No. 86-09-00-15). * To whom all correspondence should be addressed: A.0. Tip Fakiiltesi, Farmakoloji Enstitiisii, Sihhiye, Ankara, Turkey. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
sis. A high intake of cholesterol can produce atherosclerotic lesions in the vascular wall of rabbit and chickens. An increase in thickness and stiffness of the vascular wall and changes in permeability of the intimal surface to lipoproteins and albumin are well-known phenomena Occurring during the development of atherogenesis in chickens fed a high cholesterol diet (Siller, 1965). It is also known that all arteries are susceptible to cholesterol-induced atherogenesis, except the pulmonary artery in rabbits which remains free of vascular changes (Ibengwe and Suzuki, 1986). The effect of ACh has not yet been studied in the precontracted main pulmonary artery of chickens. The present data indicate that ACh produces endothelium-dependent relaxation of this artery from chickens and that this relaxation was significantly reduced in the same arterial segment from hypercholesterolemic animals. 2. Materials and methods Male chxckens aged 2 weeks and weighing 104.0 _+ 5.0 g (n = 26) were kept at 21°C in separate
398
cages and given sufficient water and ordinary food daily for a period of 1 week. Half of the animals were then fed ordinary chicken food while the others received 5% cholesterol added to the diet. The daily intake of additional cholesterol was kept between 800-1000 mg for each animal. This feeding program was adhered to for a period of 4 weeks. Body weight and serum cholesterol levels were measured at the end of this period. The main pulmonary artery was removed immediately after decapitation and transferred to a beaker containing oxygenated (5% CO 2 in 02) Krebs solution at room temperature. Surrounding tissues were dissected out and the arterial segments were cut spirally with 4 m m width and 2 cm length. The strips were suspended in 15 ml jacketed organ baths containing oxygenated and warmed (37 o C) Krebs solution. Isometric contractions were recorded on a Grass polygraph (Model 79 D) via force-displacement transducers (Grass FT.03) under 0.5 g initial tension. The strips were allowed to equilibrate for 1 h and the bathing medium was replaced every 15 min with Krebs solution. After this equilibration period noradrenaline was added to the bathing medium at the concentration of 10 7 M and submaximal contraction was elicited.
ACh was then added to the bathing medium and cumulative concentration-response curves were determined. The concentration-response studies were also repeated in rubbed and hydroquinonepreincubated unrubbed strips. Hydroquinone was added to the bathing medium at the concentration of 10 5 M 10 min before the test. The results were evaluated statistically with Student's t-test.
3. Results
Feeding the animals a high cholesterol diet for a period of 4 weeks did not produce a significant change in their body weights compared to the controls. The average body weight of the cholesterol-fed animals was 390.0 + 15.0 g while it was 360.0 + 16.0 g (n = 13) in animals fed the ordinary diet. The average serum cholesterol levels of control and cholesterol-fed chickens were 94.0 + 4.3 and 339.0 + 22.9 m g / d l (n = 13) respectively. ACh produced concentration-dependent relaxation in precontracted strips from normal and hypercholesterolemic chickens. The relaxing effect of ACh disappeared completely in endothelium-dis-
/
U MIN O'5gI
0,59
lj
n~-T
--'O
NA
,_
/
5=lO-m ~-r 5in0-" O'----'<'-----'O
40.SI~
ACh
HYD
/
I~ 7 O
NA
5=10"O 10.7 5=C ' O O O
(M)
ACh
Fig. 1. Tracings showing the effect of acetylcholine (ACh) on noradrenaline (NA)-precontracted main pulmonary artery strips from hypercholesterolemic (upper) and normal (lower) chickens before and after addition of hydroquinone (HYD) to the incubation bath. H Y D impaired the relaxing effect of ACh and converted it to a contractile one. The contractile effect of N A was slightly enhanced after HYD.
399
rupted strips. Pretreatment of the strips with hydroquinone (10 -5 M) also completely abolished the relaxing effect of ACh in strips with an intact endothelium (fig. 1). The relaxing effect of ACh was converted to a contractile one following hydroquinone pretreatment. Endothelium disruption or hydroquinone pretreatment also caused potentiation of the contractile response to noradrenaline. This enhancement was 30.0 _+ 5.0% (n = 4) for the hydroquinone pretreatment. There was no significant change or in some experiments, a slight decrease was observed in the contractile response to noradrenaline in the strips from hypercholesterolemic chickens. The concentration-response curve of ACh in hypercholesterolemic chicken strips was significantly reduced when compared with the curve from normal chickens (fig. 2). The maximum relaxing effect of ACh was also reduced by about 100% in the strips from hypercholesterolemic chickens as compared to the control.
I00
0 0 o
•~
50
ar
0
I
i0 -e
I
i0 -r
I
i 0 -6
1
i0 -s
EACh-~(M ) Fig. 2. The concentration-response curves of acetylcholine (ACh) in main pulmonary artery strips from normal (upper) and hypercholesterolemic (lower) chickens precontracted by noradrenaline (10-7 M). Each point represents the mean value from 13 experiments; vertical bars show S.E.M.
4. D i s c u s s i o n
The data presented indicate clearly that ACh produced an endothelium-dependent relaxation in the main pulmonary artery from chickens. The experiments in which ACh caused a relaxation in the noradrenaline-precontracted vascular strips with intact but not with disrupted endothelium made this evident. In addition, hydroquinone, a free-radical scavenger (Furchgott, 1983), when added to the incubation medium abolished the relaxing effect of ACh. The most interesting facet of this study is the close correlation between high serum cholesterol level and reduced relaxing effect of ACh in the main pulmonary artery strips. It is well known that increasing the dietary intake of cholesterol of rabbit and chickens produces atherosclerosis (Duff et al., 1957; Siller, 1965). Damage to endothelial cells is a consistent feature in cholesterol-induced atherosclerosis (Klimov et al., 1981) and these changes could interfere with endothelium-mediated vasodilation. It is also well-known that the pulmonary artery is a vasculature less susceptible to cholesterol-induced atherogenesis in rabbits (Ibengwe and Suzuki, 1986). Young chickens fed a high cholesterol diet for a short period do not develop the typical morphological changes of atherosclerogenesis in the pulmonary artery (Siller, 1965). However, a significant decrease in the relaxing effect of ACh was obtained in the precontracted pulmonary artery strips from hypercholesterolemic chickens in the present study. These results support the recent observation of Jayacody et al. (1985) who have shown that cholesterol feeding impaired endothelium-dependent relaxation of rabbit aorta. Moreover these authors also observed characteristic morphological features in the aorta from rabbits fed a high cholesterol diet. It was unlikely that such morphological changes could be expected in the main pulmonary artery of chickens following a short period on a high cholesterol diet. We therefore postulated that hypercholesterolemia depressed via some unknown mechanisms the AChinduced relaxation mediated by vascular endothelium and that this depression was not related to the production of atherosclerotic lesions. These findings were taken as an evidence that
400 b i o c h e m i c a l c h a n g e s in t h e v a s c u l a r e n d o t h e l i u m p r e c e d e the d e v e l o p m e n t of m o r p h o l o g i c a l changes in t h e v a s c u l a r wall o f c h i c k e n m a i n p u l m o n a r y artery.
References Cocks, T.M. and J.A. Angus, 1983, Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin, Nature (London) 305, 627. Duff, G.L., G.C. McMillan and A.C. Ritchie, 1957, The morphology of early atherosclerotic lesions of the aorta demonstrated by the surface techniques in rabbits fed cholesterol, Am. J. Pathol. 33, 845. Egleme, C., T. Godfraind and R.C. Miller, 1984, Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells, Br. J. Pharmacol. 81, 16.
Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Ibengwe, J.K. and H. Suzuki, 1986, Changes in mechanical responses of vascular smooth muscles to acetylcholine, noradrenaline and high-potassium solution in hypercholesterolemic rabbits, Br. J. Pharmacol. 87, 395. Jayakody, R.L., M.P.J. Senaratne, A.B.R. Thomson and C.T. Kappagoda, 1985, Cholesterol feeding impairs endothelium-dependent relaxation of rabbit aorta, Can. J. Physiol. Pharmacol. 63, 1206. Klimov, A.N., V.A. Nagornev and T.N. Lovagina, 1981, Functional characteristics of endothelium on the dynamics of experimental atherosclerosis, Pario Arterielle 7, 47. Siller, W.G., 1965, Spontaneous atherosclerosis in the fowl, in: Comparative Atherosclerosis, eds. J.C. Roberts, R. Straus and M.S. Cooper (Harper and Row Publish., New York) p. 66.