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Influence of the age on energy metabolism and purines turnover in watanabe hyperlipemic (whhl) rabbit aorta
115
Pharmacological Research, Vol. 21, Supplement 1, 1989
INFLUENCE OF THE AGE ON ENERGY METABOLISM AND PURINES TURNOVER IN WATANABE HYPERLIPEMIC (WHHL) RABBIT AORTA L.Pandolfo, A.Chinellato, M.Zambonin, M.De Biasi, E.Ragazzi & L.Caparrotta Department of Pharmacology, University of Padova, Padova (Italy) Key words: atherosclerosis, adenine nucleotides, rabbit aorta
Substancial evidence is present in the literature on the cellular composition of the atherosclerotic lesion as well as on the interactions among the cells involved in lesion formation during hypercolesterolemia (I), but despite the acquisition on the role of endothelium, smooth muscle, platelets, monocytes and macrophages, the genetic bases to atherosclerosis are still to be clarified. The Watanabe heritable hyperlipemic rabbit (WHHL) is considered the only animal model of human homozigous familial hypercolesterolemia (2), due to a similar LDL receptors deficiency, which causes the unability to bind LDL, with early increase of plasma lipids and aggressive atherosclerosis. In this model we studied energy metabolism of aortic wall related to purines turnover, considering the relevant role of ATP and adenosine in the regulation of vessel function and protection from injury and that of GTP in EDRF (endothelium dependent relaxing factor) transduction. Conflicting data are available on high energy phosphates in atherosclerotic aortic tissue (3) and there are no studies on bioenergetics in WHHL rabbits. Further, to investigate the evolution with age of atherosclerosis we measured purines turnover in aortas from rabbits of different age (young 3-10, aged 11-24 months). Segments of aortic arch (100-150 mg) from normal (New Zealand) and WHHL rabbits were rapidly frozen in liquid nitrogen and pulverized in 0.66 N PCA. The extracts were neutralized by 10% KOH 0.1 M Tris and analyzed for GTP, GDP, GMP, ATP, ADP, AMP, xanthine, adenosine, inosine and guanosine, NAD, NADP by high pressure liquid chromatographic procedure. The most interesting results about high energy 1043-6618/89/2110115--02/$03.00/0
© 1989 The Italian Pharmacological Society
116
Pharmacological Research. Vol. 21, Supplement 1.1989
adenine nucleotides content in aortic tissue are shown in Tab. 1. In Watanabe aorta the concentration of ATP and ADP, Total Adenylate Nucleotides (TAN) and adenylate energy charge were lower than in control rabbits. Ageing differently affected energy related metabolites in aortic tissue from" normal or WHHL rabbit. In control aortic arch the adenylate nucleotides variation induced by age did not cause significant unpairment of energy charge, by contrast in aged WHHL rabbits energy value was reduced by 14 % in respect to young animals (Tab.I), The results indicate considerable variations of energy parameters and metabolic differences in the susceptibility to the ageing between normal and Watanabe rabbits, suggesting that WHHL animals represent an interesting model for studying the relationship between metabolism and function of arterial wall in the early stage of atherosclerotic disease and in its evolution with the age. Tab . I Adenine nucleotides content in aortic arch from normal and WHHL rabbits of different age. CONTROL Young ATP ADP AMP TAN E
10.23+0.52 5.98+0.30 0.65+0.04 16.86+0.62 0.79+0.01
WHHL Aged
8.50+0.75 2.70+0.12 0 0 0.36+0.05 0 0 11.60+0.90 0 0 0.83+0.01 0
Young 5.16+0.93** 3.12+0.55** 0.63+0.05 8.90+1.46** 0.72+0.04
Aged 2.35+0.48 0 0** 2.65+0.57 1.07+0.49 6.07+1.44* 0.62+0.03**
TAN: Total Adenylate Nucleotides; E: Adenylate Energy Charge. Data expressed as prnoles/g proteins (except for E which is a ratio) are the mean ~ ES from 8 to 13 different animals. * p < 0.02; ** P < 0.001 difference vs control. 0 p < 0.02; 00 P < 0.001 difference due to age.
References 1. Ross R. The pathogenesis of atherosclerosis. An update. N. Eng. J. Med. 1986,314:
488-500. 2. Watanabe Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHLrabbit). Atherosclerosis 1980, 36: 261-268. 3. Heinle H. Metabolite concentration gradients in the arterial wall of experimental atherosclerosis. Exp, Mol. Path. 1987, 46: 312-320.
Pharmacological Research, Vol. 21, Supplement 1, 1989
INFLUENCE OF THE AGE ON ENERGY METABOLISM AND PURINES TURNOVER IN WATANABE HYPERLIPEMIC (WHHL) RABBIT AORTA L.Pandolfo, A.Chinellato, M.Zambonin, M.De Biasi, E.Ragazzi & L.Caparrotta Department of Pharmacology, University of Padova, Padova (Italy) Key words: atherosclerosis, adenine nucleotides, rabbit aorta
Substancial evidence is present in the literature on the cellular composition of the atherosclerotic lesion as well as on the interactions among the cells involved in lesion formation during hypercolesterolemia (I), but despite the acquisition on the role of endothelium, smooth muscle, platelets, monocytes and macrophages, the genetic bases to atherosclerosis are still to be clarified. The Watanabe heritable hyperlipemic rabbit (WHHL) is considered the only animal model of human homozigous familial hypercolesterolemia (2), due to a similar LDL receptors deficiency, which causes the unability to bind LDL, with early increase of plasma lipids and aggressive atherosclerosis. In this model we studied energy metabolism of aortic wall related to purines turnover, considering the relevant role of ATP and adenosine in the regulation of vessel function and protection from injury and that of GTP in EDRF (endothelium dependent relaxing factor) transduction. Conflicting data are available on high energy phosphates in atherosclerotic aortic tissue (3) and there are no studies on bioenergetics in WHHL rabbits. Further, to investigate the evolution with age of atherosclerosis we measured purines turnover in aortas from rabbits of different age (young 3-10, aged 11-24 months). Segments of aortic arch (100-150 mg) from normal (New Zealand) and WHHL rabbits were rapidly frozen in liquid nitrogen and pulverized in 0.66 N PCA. The extracts were neutralized by 10% KOH 0.1 M Tris and analyzed for GTP, GDP, GMP, ATP, ADP, AMP, xanthine, adenosine, inosine and guanosine, NAD, NADP by high pressure liquid chromatographic procedure. The most interesting results about high energy 1043-6618/89/2110115--02/$03.00/0
© 1989 The Italian Pharmacological Society
116
Pharmacological Research. Vol. 21, Supplement 1.1989
adenine nucleotides content in aortic tissue are shown in Tab. 1. In Watanabe aorta the concentration of ATP and ADP, Total Adenylate Nucleotides (TAN) and adenylate energy charge were lower than in control rabbits. Ageing differently affected energy related metabolites in aortic tissue from" normal or WHHL rabbit. In control aortic arch the adenylate nucleotides variation induced by age did not cause significant unpairment of energy charge, by contrast in aged WHHL rabbits energy value was reduced by 14 % in respect to young animals (Tab.I), The results indicate considerable variations of energy parameters and metabolic differences in the susceptibility to the ageing between normal and Watanabe rabbits, suggesting that WHHL animals represent an interesting model for studying the relationship between metabolism and function of arterial wall in the early stage of atherosclerotic disease and in its evolution with the age. Tab . I Adenine nucleotides content in aortic arch from normal and WHHL rabbits of different age. CONTROL Young ATP ADP AMP TAN E
10.23+0.52 5.98+0.30 0.65+0.04 16.86+0.62 0.79+0.01
WHHL Aged
8.50+0.75 2.70+0.12 0 0 0.36+0.05 0 0 11.60+0.90 0 0 0.83+0.01 0
Young 5.16+0.93** 3.12+0.55** 0.63+0.05 8.90+1.46** 0.72+0.04
Aged 2.35+0.48 0 0** 2.65+0.57 1.07+0.49 6.07+1.44* 0.62+0.03**
TAN: Total Adenylate Nucleotides; E: Adenylate Energy Charge. Data expressed as prnoles/g proteins (except for E which is a ratio) are the mean ~ ES from 8 to 13 different animals. * p < 0.02; ** P < 0.001 difference vs control. 0 p < 0.02; 00 P < 0.001 difference due to age.
References 1. Ross R. The pathogenesis of atherosclerosis. An update. N. Eng. J. Med. 1986,314:
488-500. 2. Watanabe Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHLrabbit). Atherosclerosis 1980, 36: 261-268. 3. Heinle H. Metabolite concentration gradients in the arterial wall of experimental atherosclerosis. Exp, Mol. Path. 1987, 46: 312-320.