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Aorta reactivity to acethylcholine in hypercholesterolemic rabbits: Comparison with energy metabolism and cholesterol content
Pharmacological Research Communications, Vo/. 20, Supplement I, 1988
103
AORTA REACTIVITY TO ACETHYLCHOLINE IN HYPERCHOLESTEROLEMIC RABBITS: COMPARISON WITH ENERGY METABOLISM AND CHOLESTEROL CONTENT E. Ragazzi, L. Pandolfo, G. Froldi, A. ChineHat% L. Caparrotta & G. Fassina Depvrtment of Pharmacology, University of Padova, Padova (Italy) Key words: cholesterol, adenine nucleotides, acethylcholine, aorta Attention has been focused on the function of endothelial ceZls in blood vesse]s, particularly on the phenomenon of endothelium dependent relaxation (Furchgott, 1984). The role of purines released from endothelial c~.lls is we]] known and it was also shown to be of pathophysiological significance in the control of vascular tone in hypertension and atherosclerosis (Burnstock, 1985). In aortas from normal and 196 cholesterol rich diet fed rabbits, we compared (i) the relaxing e f f e c t of acethytcholine, an arterial dilator stimulating the release of endothelium derived relaxing factor (EDRF), with (ii) adenine nucleotides and nucleosides related to energy metabolism and purine turnover, and with (iii) cholesterol content in serum and in aortic tissue extracts. The rationale of this protocol was to test the possible corre]ation of energy metabolism, with tipid infiltration and functional response to acethylFholine. Mate white New Zealand rabbits (2.5 - 3 kg) were fed either 196 cholesterol diet or standard diet for 10 weeks. After sacrifice, thoracic aorta was cut into 3 mm rings and two ring chains were suspended in tissue bath containing 20 ml of physiological solution gassed with 9596 02 - 596 CO 2. Isometric tension was recorded by high sensitivity isometric transducers. The arch segment and the descending thoracic distal segment of aorta were quicldy frozen in liquid nitrogen plus 696 PCA. Purines nucleotides and nucleosides were measured by HPLC procedures in neutralized tissue extracts. Cholesterol was determined at serum level and in abdominat aorta a f t e r ethanol extraction. Concentration-response curves of acethylcholine (30 n M - 0.1 raM) were obtained by cumulative addition of drug (Fig. 1) a f t e r pre-contraction by noradrenaline (0.3 0031-6989/88/20S10103-2/$03.00/0
© 1988 The Italian Pharmacological Society
104
Pharmacological Research Communications, Vol. 20, Supplement Io 1988
uM). Acethytcholine induced a significantly lower relaxation in cholesterol treated rabbit aortas than in aortas from control animals. A p,rogression in the loss of relaxing effect
was observed from
proximal
to distal aortic segments. The effect
of
acethytcholine was wel! correlated with the high cholesterol level (Table 1) both in serum and in aortic tissue of hypercholesterolemic animals. At metabolic level ATP, ADP, AMP and ADO, INO, XANT content in arch was 3 times higher than in thoracic descendent aorta. Hypercholesterolemic diet did not cause any significant alteration on energy metabolism and nucleosides at all aortic levels. These results suggest that in hypercholesterolemic rabbits the lower reactivity of aortic vessels to acethylcholine is not associated with alterations of adenine nucleotides and nuc]eosides in aortic tissue, but it is well corre]ated to the high cholesterol level in serum and in aortic tissue.
Figure ]
+,80"
Control Table 1.
+60.
Z 0
0 ....... 0
1% cholesterol diet
.y."~.
+40,
CHOLESTEROL
.~°o *°** o,.°
~[,.. o
AORTA mg/100mg f.t.
SERUM mg/100ml
o,26_+ o.18
4s + xo
3.2?+0.68
949+105
C
-20
b~ ~ O
--40 -60
Data are the mean + sere o f 11 animals, f.t. = f~'eah tissue. C = control HC = 1% c h o l e s t e r o l diet
-80 f'
10 "8
"
"
'
10 -7
~
I
10"6
¢
"
10-5
•
I
10 -4
I
10 -3
ACETHYLCHOLINE (M)
References Burnstock G. (1985) J. C a r d i o v a s c . PharmacoL 7: S 137 - S 146 F u r c h g o t t R.F. (1984) Ann. R a y . P h a r m a c o l . T o x i c o L 2 4 : 1 7 5 - 1 9 7
HC
103
AORTA REACTIVITY TO ACETHYLCHOLINE IN HYPERCHOLESTEROLEMIC RABBITS: COMPARISON WITH ENERGY METABOLISM AND CHOLESTEROL CONTENT E. Ragazzi, L. Pandolfo, G. Froldi, A. ChineHat% L. Caparrotta & G. Fassina Depvrtment of Pharmacology, University of Padova, Padova (Italy) Key words: cholesterol, adenine nucleotides, acethylcholine, aorta Attention has been focused on the function of endothelial ceZls in blood vesse]s, particularly on the phenomenon of endothelium dependent relaxation (Furchgott, 1984). The role of purines released from endothelial c~.lls is we]] known and it was also shown to be of pathophysiological significance in the control of vascular tone in hypertension and atherosclerosis (Burnstock, 1985). In aortas from normal and 196 cholesterol rich diet fed rabbits, we compared (i) the relaxing e f f e c t of acethytcholine, an arterial dilator stimulating the release of endothelium derived relaxing factor (EDRF), with (ii) adenine nucleotides and nucleosides related to energy metabolism and purine turnover, and with (iii) cholesterol content in serum and in aortic tissue extracts. The rationale of this protocol was to test the possible corre]ation of energy metabolism, with tipid infiltration and functional response to acethylFholine. Mate white New Zealand rabbits (2.5 - 3 kg) were fed either 196 cholesterol diet or standard diet for 10 weeks. After sacrifice, thoracic aorta was cut into 3 mm rings and two ring chains were suspended in tissue bath containing 20 ml of physiological solution gassed with 9596 02 - 596 CO 2. Isometric tension was recorded by high sensitivity isometric transducers. The arch segment and the descending thoracic distal segment of aorta were quicldy frozen in liquid nitrogen plus 696 PCA. Purines nucleotides and nucleosides were measured by HPLC procedures in neutralized tissue extracts. Cholesterol was determined at serum level and in abdominat aorta a f t e r ethanol extraction. Concentration-response curves of acethylcholine (30 n M - 0.1 raM) were obtained by cumulative addition of drug (Fig. 1) a f t e r pre-contraction by noradrenaline (0.3 0031-6989/88/20S10103-2/$03.00/0
© 1988 The Italian Pharmacological Society
104
Pharmacological Research Communications, Vol. 20, Supplement Io 1988
uM). Acethytcholine induced a significantly lower relaxation in cholesterol treated rabbit aortas than in aortas from control animals. A p,rogression in the loss of relaxing effect
was observed from
proximal
to distal aortic segments. The effect
of
acethytcholine was wel! correlated with the high cholesterol level (Table 1) both in serum and in aortic tissue of hypercholesterolemic animals. At metabolic level ATP, ADP, AMP and ADO, INO, XANT content in arch was 3 times higher than in thoracic descendent aorta. Hypercholesterolemic diet did not cause any significant alteration on energy metabolism and nucleosides at all aortic levels. These results suggest that in hypercholesterolemic rabbits the lower reactivity of aortic vessels to acethylcholine is not associated with alterations of adenine nucleotides and nuc]eosides in aortic tissue, but it is well corre]ated to the high cholesterol level in serum and in aortic tissue.
Figure ]
+,80"
Control Table 1.
+60.
Z 0
0 ....... 0
1% cholesterol diet
.y."~.
+40,
CHOLESTEROL
.~°o *°** o,.°
~[,.. o
AORTA mg/100mg f.t.
SERUM mg/100ml
o,26_+ o.18
4s + xo
3.2?+0.68
949+105
C
-20
b~ ~ O
--40 -60
Data are the mean + sere o f 11 animals, f.t. = f~'eah tissue. C = control HC = 1% c h o l e s t e r o l diet
-80 f'
10 "8
"
"
'
10 -7
~
I
10"6
¢
"
10-5
•
I
10 -4
I
10 -3
ACETHYLCHOLINE (M)
References Burnstock G. (1985) J. C a r d i o v a s c . PharmacoL 7: S 137 - S 146 F u r c h g o t t R.F. (1984) Ann. R a y . P h a r m a c o l . T o x i c o L 2 4 : 1 7 5 - 1 9 7
HC