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Turnover of sulfated glycosaminoglycans in the rat aortic wall
Comp. Biochem. Physiol., 1974, Vol. 49B,pp. 113 to 118. Pergamon Press. Printed in Great Britain
TURNOVER OF SULFATED GLYCOSAMINOGLYCANS IN THE RAT AORTIC WALL B. H E R M E L I N , J. PICARD, A. PAUL-GARDAIS and P. LEVY Laboratory of Biochemistry, Faculty of Medicine St.-Antoine, 27 rue Chaligny, 75571 Paris, Cedex 12, France (Received 25 ffuly 1973)
Abstract--1. The turnover of sulfated glycosaminoglycanswas studied in the
rat aortic wall. Carrier-free radioactive sulfate was administered by intraperitoneal injection to young and old rats. The specific activity and half-time of sulfated glycosaminoglycanswere determined. 2. In dermatan sulfate, the specific activity and half-time were respectively higher and shorter than in the other glycosaminoglycans. 3. Incorporation of radioactive sulfate in every glycosaminoglycan was decreased in old rats compared to young rats. The half-time was increased in old rats. 4. These results suggest a diminution of glycosaminoglycan metabolism in the aortic wall with aging. INTRODUCTION AN INCREASEof glycosaminoglycan content was noted in the arterial wall of different animals receiving atherogenic diets. Similar variations have been observed in human species with aging(Kirk & Dyrbye, 1957; Kaplan & Meyer, 1960; Buddecke, 1962; Kumar et al., 1967). All these works were essentially based upon histochemical observations and they did not allow us to state if increased metachromasia was an explanation of the modification of the relative proportions of the different glycosaminoglycans. Biochemical determinations of the arterial wall glycosaminoglycan content in different animals (rat, rabbit) have not shown a significant increase; a decrease has only been observed in complicated and calcified lesions from human aortas. The well-known hypothesis of glycosaminoglycan-increased synthesis in animals or in humans with maturation and atherosclerotic involvement was not based upon reliable biochemical data. An eventual glycosaminoglycan increase with maturation could be explained either by an excessive synthesis or by a catabolic decrease. Previously, we have shown that it was possible to determine, by an electrophoretic micromethod, the relative concentration of the different glycosaminoglycans in the arterial wall of the rat. Using this method, we studied the variations of the glycosaminoglycan distribution in the rat aorta with maturation. We had already observed that the radioactive sulfate injected intraperitoneally was rapidly incorporated in the glycosaminoglycans of the rat arterial wall and could 113
114
B. HERMELIN,J. PICARD, A. PAuL-GARDAIS AND P. LEVY
also be d e t e c t e d in t h e n u c l e o t i d e sulfate p r e c u r s o r s ( P i c a r d et al., 1967). F o r t h i s r e a s o n w e s t u d i e d t h e t u r n o v e r o f g l y c o s a m i n o g l y c a n s in t h e aortic wall o f t h e rat in o r d e r to d e t e r m i n e in vivo t h e flux a n d t u r n o v e r o f t h e s e m a c r o m o l e c u l e s . T h e p r e s e n t p a p e r r e c o r d s t h e r e s u l t s o f t h e t u r n o v e r rates of d e r m a t a n sulfate, h e p a r a n sulfate a n d c h o n d r o i t i n sulfate in t h e m e d i a a n d i n t i m a o f t h e t h o r a c i c a o r t a in rats w i t h a g i n g u s i n g t h e p r o c e d u r e set u p in o u r l a b o r a t o r y ( G a r d a i s et al. 1969). MATERIALS AND METHODS The experiment was performed with two groups of rats: (1) forty rats three months old (300 g), and (2) thirty rats 18 months old (900 g). T h e y were injected intraperitoneally with 500/~1 of sodium sulfate (~5S) containing 0"5 mCi. T h e animals were killed at various times: 24, 48, 72 and 98 hr after the isotope injection.
Analytical methods 1. Extraction of glycosaminoglycans. T h e thoracic aortas were removed, and washed in an isotonic solution. T h e different layers (media intima and adventicia) were separated, and then treated with acetone at 4°C. T h e acetone powders of these different layers were treated with 2% crystalline papain (Sigma) in an aqueous solution of 0"05 M sodium E D T A and 0"005 M cysteine at p H 7"3 for 6 hr at 65°C (Antonopoulos et al., 1964). T h e enzyme was inactivated by heating for 2 min at 100°C. T h e proteolysis digest was centrifuged and the supernatant was dialyzed against running water then distilled water and the glycosaminoglycans were precipitated in the previously dialyzed fraction by 1% of cetyl pyridinium chloride (CPC) in 0"03 M aqueous sodium chloride. After precipitation for 18 hr at 30°C, the CPC glycosaminoglycan complexes were collected by centrifugation, and were dissolved in a 1"25 M NaC1 solution and purified by alternate precipitations with ethanol (3 vol.) and CPC. The ethanol precipitated glycosaminoglycans were stored in a vacuum at 4°C. 2. Separation ofglycosaminoglycan peptides. T h e glycosaminoglycans were separated by electrophoresis on cellulose acetate strips (Cellogel) according to the procedure previously described (Gardais et al., 1969). Electrophoresis was performed in a 0"1 M pyridine formate buffer, p H 3, 150 V, for 1 hr. After running, the strips were stained with 0"5% alcian blue. T h e use of this electrophoretic separation for this work has been previously described (Gardais et al., 1973). 3. Liquid scintillation counting. After electrophoretic migration, the stained areas corresponding to the different glycosaminoglycans were cut out, put in liquid scintillation vials, dried, dipped in a scintillation fluid (5 g of PPO in 1000 ml of toluene) and then counted with a Beckman L S 150 scintillator. Previous work has established that radioactive counting does not depend on the surface and staining intensity of our samples in the experimental conditions (Schwartz, 1970). These results agree with previous observations (Nunez & Jacquemin, 1961). We have also verified that the damping in the scintillation fluid did not affect the counting (Schwartz, 1970). 4. Determination of glycosaminoglycan content. After counting, the stained areas were taken out of the scintillation fluid, dried in order to eliminate toluene and dissolved in 1 ml of 80% acetic acid. T h e fractions were measured by colorimetry at 675 nm with a standard reference (Gardais et al., 1969). Specific radioactivity was expressed in counts/rain/per bemole of hexosamine. T h e hexosamine content was determined by the colorimetric method of Elson & Morgan (1933). Glucosamine HC1 was used as a standard. Samples were hydrolyzed with 4 N HC1 for 4 hr at 100°C.
SULFATED GLYCOSAMINOGLYCANS IN THE RAT AORTIC WALL
115
RESULTS For the different groups of rats, the distribution of sulfated glycosarninoglycans in the arterial wall is indicated in Table I. These results did not show any significant change with aging. In the arterial wall of these same rats the concentration of total glycosaminoglycans remained stable and the determination of the molar ratio of sulfate to hexosamine in the sulfated glycosaminoglycans did not show a significant change. These results did not provide evidence for a marked variation in glycosaminoglycan sulfation with aging. The radioactive sulfate was rapidly incorporated in the aortic glycosaminoglycans. Maximal labelling occurred 24 hr after injection in younger rats. This delay increased with age and maximal labelling occurred 4 8 b r after injection in older rats. TABLE 1--GLYCOSAMINOGLYCAN
(GAG) DISTRIBUTION FROM
GAG
Young rats
Old rats
HS DS CS
54 25 21
52.5 29"5 18
AORTIC MEDIA INTIMA
Values are expressed as a percentage of the total GAG. Z: normality test (0.12
T A B L E 2 - - S P E C I F I C RADIOACTIVITIES OF SULFATED GLYCOSAMINOGLYCANS IN THE ARTERIAL WALL (MEDIA I N T I M A ) OF OLD RATS (18 MONTHS OLD)
T i m e (hr) 24 GAG HS DS CS
Specific radioactivity 38,400 ± 9000 129,000 ± 22,500 76,800 _+18,600
48 Z 0.48 0-15 0.23
Specific radioactivity 56,400 + 8400 148,500 + 17,400 95,400 ± 9000
72
96
Z
Specific radioactivity
Z
0.46 0.35 0.43
44,400 + 18,000 85,800 _+18,000 73,200 ± 9600
0.44 0.76 0.23
Specific radioactivity 39,600 + 8400 69,600 + 9000 69,600 ± 9000
Z 0.21 0.44 0.21
Specific radioactivities are expressed in counts/rain per /xmole hexosamine. Mean values (seven rats) are followed by standard deviations and the normality test (Z).
t~
Z
>
.> > T A B L E 3 - - S P E C I F I C RADIOACTIVITIES OF SULFATED GLYCOSAMINOGLYCANS IN THE ARTERIAL WALL (MEDIA INTIMA) OF YOUNG RATS (3 M O N T H S OLD)
7 >O
T i m e (hr) 24 GAG HS DS CS
Specific radioactivity 160,000 + 43,000 290,000 _+62,000 215,000 +_66,000
48 Z 0.26 0.60 0.30
Specific radioactivity 108,000 + 24,000 180,000 + 36,000 152,000 + 30,000
72 Z 0.30 0.55 0-66
Specific radioactivity 81,000 + 12,000 120,000 + 18,000 101,000 ± 16,000
>
96 Z
Specific radioactivity
Z
0.57 0.82 0.63
55,800 + 7800 72,000 + 13,800 68,000 ± 8400
0.57 0.38 0.79
Specific radioactivities are expressed in counts/min per /~mole hexosamine. Mean values (ten rats) are followed by standard deviations and the normality test (Z).
SULFATED GLYCOSAMINOGLYCANS IN THE RAT AORTIC WALL
117
TABLE 4---HALF-TIMES OF SULFATED GLYCOSAMINOGLYCANS IN THE RAT ARTERIAL WALL (MEDIA INTIMA)
GAG
Young rats
Old rats
HS DS CS
2 1"6 2-3
4 2 4
Values are expressed in days and were calculated with specific radioactivities (Tables 2 and 3). sulfation. Various results have been observed in humans and other animals (Kirk & Dyrbye, 1957; Buck & Heagy, 1958; Kaplan & Meyer, 1960; Buddecke, 1962; Kumar et al., 1967; Ichida & Kalant, 1968). On aging the glycosaminoglycan content is affected differently in the arterial wall of the human compared with that of the rat. These observations can be related to a different chemical composition as was previously suggested. The relative resistance of the rat to an atherogenic diet should be associated with the high rate of heparan sulfate in the arterial wall of the rat. The concentration of glycosaminoglycans in the arterial wall of the rat can be modified in some experimental conditions (Picard et al., 1967). It has been observed that the turnover of sulfated glycosaminoglycans in the arterial wall of the rat changes with aging. These variations occur without any change in the level of glycosaminoglycan sulfation. With aging glycosaminoglycans appear to turn over less rapidly; these findings are related to alterations in the enzymatic activities involved in the metabolism of these compounds. This slower metabolism may be associated with alterations in the degradation and synthesis process. In this case, it is impossible to determine whether the alteration is related to sulfate activation or to sulfate transfer on the glycosaminoglycans; to obtain such findings it would be necessary to determine the intraarterial pool of PAPS. In the arterial wall of young rats, dermatan sulfate has a turnover faster than the other arterial wall sulfated glycosaminoglycans. It is also faster than dermatan from the skin of the same animals. These results agree with other works (Schiller & Dorfman, 1955, 1957); the turnover of dermatan sulfate is also faster in old rats. However, the results show that this slower metabolism may be applied to the three sulfated glycosaminoglycans. Although our findings are concerned with the turnover of sulfate, identical determinations of glycosaminoglycan turnover have been obtained with radioactive sulfate or glucidic precursors (14C) (Katsura & Davidson, 1966; Buddecke et al., 1971). These results are in agreement with an active metabolism of the glycosaminoglycans in the arterial wall which is the localization of an active biosynthesis of these compounds involving sugar nucleotide and sulfate nucleotide precursors (Picard & Gardais, 1967; Picard et al., 1969). This metabolic activity of the arterial wall slows down with aging. Acknowledgements--We are grateful to the D.G.R.S.T. for supporting this work.
118
B. HERMELIN,J. PICARD,A. PAUL-GARDAISAND P. LEVY
REFERENCES ANTONOPOULOS C. A., GARDELL S., SZlRMAI J. & DE TYSSONSK E. R. (1964) Determination of glycosaminoglycans from tissues on the microgram scale. Biochem. biophys. Acta 83 1-19. BUCK R. C. & HEAGY F. C. (1958) Uptake of radioactive sulfate by various tissues of normal and cholesterol fed rabbits. Can.ft. Biochem. Physiol. 36, 63-69. BUDDECKE E. (1952) Chemical changes in the ground substance of the vessel wall in atherosclerosis, o7. Atheroscler. Res. 2, 32--46. BUDDECKE E., KRESSE H. & SEGETH G. (1971) Chemical and metabolic heterogenicity of x4C and 35S sulfate labelled glycosaminoglycans of bovine arterial tissue. Z. Klin. Chem. Klin. Biochem. 9, 74. ELSON L. A. & MORGAN W. T. J. (1933) A colorimetric method for the determination of glucosamine and chondrosamine. Biochem.ft. 27, 1824-1828. GARDAI$ A., PICARD J. & HERMELIN B. (1973) Distribution des glycosaminoglycanes dans la paroi artdrielle de cinq esp6ces. Comp. Biochem. Physiol. 44B, 507-515. GARDAIS A., PICARD J. & TARASSE C. (1969) Microfractionnement et microdosage des glycosaminoglycanes par dlectrophordse sur bandes d'acdtate de cellulose gdlatinisdes. ft. Chromatog. 42, 396-407. ICHIDA T. & KALANTN. (1968) Aortic glycosaminoglycans in atheroma and alloxan diabetes. Can. ft. Bioehem. 46, 249-260. KAPLAN n . & MEYER K. (1960) Mucopolysaccharides of aorta at various ages. Proc. Soc. exp. Biol. Med. 105, 78-81. KATSURA N. & DAVIDSON E. A. (1966) Metabolism of connective tissue polysaccharides in vivo--IV. T h e sulphate group. Biochim. biophys. Aeta 121, 135-143. KIRK J. E. & DYRBYE M. D. (1957) Mucopolysaccharides of human arterial t i s s u e - - I I . Analysis of total isolated mucopolysaccharide material, ft. Gerontol. 12, 23-31. KUMAR V., BERENSON G. S., RuIz H., DALFERES E. R. & STRONG J. P. (1967) Acid mucopolysaccharides of human a o r t a - - I . Variation with maturation..7. Atheroscler. Res. 7, 573-581. NUNEZ J. & JACQUEMIN C. (1961) Comptage de radiochromatogrammes par scintillation liquide..7. Chromatog. 5, 271-273. PICARD J. & GARDAIS A. (1967) Nucleotides sulfates du tissu conjonctif. Bull. Soc. chim. Biol. 49, 1689-1705. PICARD J., GARDAIS A., LACORD-BONNEAUM. & HERMELIN B. (1969) Le mdtabolisme des glyeosaminoglycanes dans la paroi artdrielle. 7thInt. CongressClin. chem., p. 83, Geneva. Karger, Basle. PICARD J., LACORD-BONNEAUM. & GARDAISA. (1967) Incorporation du radiosulfate (35 S) dans les mucopolysaccharides de l'aorte de rat. Colloque Int., C N R S , Paris, Vol. II, pp. 769-787. SCHILLER R. S. & DORFMAN A. (1955) T h e biosynthesis of mucopolysaccharides in the skin of alloxan diabetic rats. Biochem. biophys. Acta 16, 304-310. SCHILLER S. & DORFMANA. (1957) T h e metabolism of mucopolysaccharides in animals--IV. T h e influence of insulin, ft. biol. Chem. 227, 625-632. SCHWARTZ C. (1970) Application de la m6thode de fractionnement electrophor6tique a l'6tude du m6tabolisme des mucopolysaccharides. D E A de Biochimie Fac. Med., St Antoine, Paris.
Key Word Index--Sulfated glycosaminoglycans; rat; aorta wall; glycosaminoglycan; dermatan sulfate.
TURNOVER OF SULFATED GLYCOSAMINOGLYCANS IN THE RAT AORTIC WALL B. H E R M E L I N , J. PICARD, A. PAUL-GARDAIS and P. LEVY Laboratory of Biochemistry, Faculty of Medicine St.-Antoine, 27 rue Chaligny, 75571 Paris, Cedex 12, France (Received 25 ffuly 1973)
Abstract--1. The turnover of sulfated glycosaminoglycanswas studied in the
rat aortic wall. Carrier-free radioactive sulfate was administered by intraperitoneal injection to young and old rats. The specific activity and half-time of sulfated glycosaminoglycanswere determined. 2. In dermatan sulfate, the specific activity and half-time were respectively higher and shorter than in the other glycosaminoglycans. 3. Incorporation of radioactive sulfate in every glycosaminoglycan was decreased in old rats compared to young rats. The half-time was increased in old rats. 4. These results suggest a diminution of glycosaminoglycan metabolism in the aortic wall with aging. INTRODUCTION AN INCREASEof glycosaminoglycan content was noted in the arterial wall of different animals receiving atherogenic diets. Similar variations have been observed in human species with aging(Kirk & Dyrbye, 1957; Kaplan & Meyer, 1960; Buddecke, 1962; Kumar et al., 1967). All these works were essentially based upon histochemical observations and they did not allow us to state if increased metachromasia was an explanation of the modification of the relative proportions of the different glycosaminoglycans. Biochemical determinations of the arterial wall glycosaminoglycan content in different animals (rat, rabbit) have not shown a significant increase; a decrease has only been observed in complicated and calcified lesions from human aortas. The well-known hypothesis of glycosaminoglycan-increased synthesis in animals or in humans with maturation and atherosclerotic involvement was not based upon reliable biochemical data. An eventual glycosaminoglycan increase with maturation could be explained either by an excessive synthesis or by a catabolic decrease. Previously, we have shown that it was possible to determine, by an electrophoretic micromethod, the relative concentration of the different glycosaminoglycans in the arterial wall of the rat. Using this method, we studied the variations of the glycosaminoglycan distribution in the rat aorta with maturation. We had already observed that the radioactive sulfate injected intraperitoneally was rapidly incorporated in the glycosaminoglycans of the rat arterial wall and could 113
114
B. HERMELIN,J. PICARD, A. PAuL-GARDAIS AND P. LEVY
also be d e t e c t e d in t h e n u c l e o t i d e sulfate p r e c u r s o r s ( P i c a r d et al., 1967). F o r t h i s r e a s o n w e s t u d i e d t h e t u r n o v e r o f g l y c o s a m i n o g l y c a n s in t h e aortic wall o f t h e rat in o r d e r to d e t e r m i n e in vivo t h e flux a n d t u r n o v e r o f t h e s e m a c r o m o l e c u l e s . T h e p r e s e n t p a p e r r e c o r d s t h e r e s u l t s o f t h e t u r n o v e r rates of d e r m a t a n sulfate, h e p a r a n sulfate a n d c h o n d r o i t i n sulfate in t h e m e d i a a n d i n t i m a o f t h e t h o r a c i c a o r t a in rats w i t h a g i n g u s i n g t h e p r o c e d u r e set u p in o u r l a b o r a t o r y ( G a r d a i s et al. 1969). MATERIALS AND METHODS The experiment was performed with two groups of rats: (1) forty rats three months old (300 g), and (2) thirty rats 18 months old (900 g). T h e y were injected intraperitoneally with 500/~1 of sodium sulfate (~5S) containing 0"5 mCi. T h e animals were killed at various times: 24, 48, 72 and 98 hr after the isotope injection.
Analytical methods 1. Extraction of glycosaminoglycans. T h e thoracic aortas were removed, and washed in an isotonic solution. T h e different layers (media intima and adventicia) were separated, and then treated with acetone at 4°C. T h e acetone powders of these different layers were treated with 2% crystalline papain (Sigma) in an aqueous solution of 0"05 M sodium E D T A and 0"005 M cysteine at p H 7"3 for 6 hr at 65°C (Antonopoulos et al., 1964). T h e enzyme was inactivated by heating for 2 min at 100°C. T h e proteolysis digest was centrifuged and the supernatant was dialyzed against running water then distilled water and the glycosaminoglycans were precipitated in the previously dialyzed fraction by 1% of cetyl pyridinium chloride (CPC) in 0"03 M aqueous sodium chloride. After precipitation for 18 hr at 30°C, the CPC glycosaminoglycan complexes were collected by centrifugation, and were dissolved in a 1"25 M NaC1 solution and purified by alternate precipitations with ethanol (3 vol.) and CPC. The ethanol precipitated glycosaminoglycans were stored in a vacuum at 4°C. 2. Separation ofglycosaminoglycan peptides. T h e glycosaminoglycans were separated by electrophoresis on cellulose acetate strips (Cellogel) according to the procedure previously described (Gardais et al., 1969). Electrophoresis was performed in a 0"1 M pyridine formate buffer, p H 3, 150 V, for 1 hr. After running, the strips were stained with 0"5% alcian blue. T h e use of this electrophoretic separation for this work has been previously described (Gardais et al., 1973). 3. Liquid scintillation counting. After electrophoretic migration, the stained areas corresponding to the different glycosaminoglycans were cut out, put in liquid scintillation vials, dried, dipped in a scintillation fluid (5 g of PPO in 1000 ml of toluene) and then counted with a Beckman L S 150 scintillator. Previous work has established that radioactive counting does not depend on the surface and staining intensity of our samples in the experimental conditions (Schwartz, 1970). These results agree with previous observations (Nunez & Jacquemin, 1961). We have also verified that the damping in the scintillation fluid did not affect the counting (Schwartz, 1970). 4. Determination of glycosaminoglycan content. After counting, the stained areas were taken out of the scintillation fluid, dried in order to eliminate toluene and dissolved in 1 ml of 80% acetic acid. T h e fractions were measured by colorimetry at 675 nm with a standard reference (Gardais et al., 1969). Specific radioactivity was expressed in counts/rain/per bemole of hexosamine. T h e hexosamine content was determined by the colorimetric method of Elson & Morgan (1933). Glucosamine HC1 was used as a standard. Samples were hydrolyzed with 4 N HC1 for 4 hr at 100°C.
SULFATED GLYCOSAMINOGLYCANS IN THE RAT AORTIC WALL
115
RESULTS For the different groups of rats, the distribution of sulfated glycosarninoglycans in the arterial wall is indicated in Table I. These results did not show any significant change with aging. In the arterial wall of these same rats the concentration of total glycosaminoglycans remained stable and the determination of the molar ratio of sulfate to hexosamine in the sulfated glycosaminoglycans did not show a significant change. These results did not provide evidence for a marked variation in glycosaminoglycan sulfation with aging. The radioactive sulfate was rapidly incorporated in the aortic glycosaminoglycans. Maximal labelling occurred 24 hr after injection in younger rats. This delay increased with age and maximal labelling occurred 4 8 b r after injection in older rats. TABLE 1--GLYCOSAMINOGLYCAN
(GAG) DISTRIBUTION FROM
GAG
Young rats
Old rats
HS DS CS
54 25 21
52.5 29"5 18
AORTIC MEDIA INTIMA
Values are expressed as a percentage of the total GAG. Z: normality test (0.12
T A B L E 2 - - S P E C I F I C RADIOACTIVITIES OF SULFATED GLYCOSAMINOGLYCANS IN THE ARTERIAL WALL (MEDIA I N T I M A ) OF OLD RATS (18 MONTHS OLD)
T i m e (hr) 24 GAG HS DS CS
Specific radioactivity 38,400 ± 9000 129,000 ± 22,500 76,800 _+18,600
48 Z 0.48 0-15 0.23
Specific radioactivity 56,400 + 8400 148,500 + 17,400 95,400 ± 9000
72
96
Z
Specific radioactivity
Z
0.46 0.35 0.43
44,400 + 18,000 85,800 _+18,000 73,200 ± 9600
0.44 0.76 0.23
Specific radioactivity 39,600 + 8400 69,600 + 9000 69,600 ± 9000
Z 0.21 0.44 0.21
Specific radioactivities are expressed in counts/rain per /xmole hexosamine. Mean values (seven rats) are followed by standard deviations and the normality test (Z).
t~
Z
>
.> > T A B L E 3 - - S P E C I F I C RADIOACTIVITIES OF SULFATED GLYCOSAMINOGLYCANS IN THE ARTERIAL WALL (MEDIA INTIMA) OF YOUNG RATS (3 M O N T H S OLD)
7 >O
T i m e (hr) 24 GAG HS DS CS
Specific radioactivity 160,000 + 43,000 290,000 _+62,000 215,000 +_66,000
48 Z 0.26 0.60 0.30
Specific radioactivity 108,000 + 24,000 180,000 + 36,000 152,000 + 30,000
72 Z 0.30 0.55 0-66
Specific radioactivity 81,000 + 12,000 120,000 + 18,000 101,000 ± 16,000
>
96 Z
Specific radioactivity
Z
0.57 0.82 0.63
55,800 + 7800 72,000 + 13,800 68,000 ± 8400
0.57 0.38 0.79
Specific radioactivities are expressed in counts/min per /~mole hexosamine. Mean values (ten rats) are followed by standard deviations and the normality test (Z).
SULFATED GLYCOSAMINOGLYCANS IN THE RAT AORTIC WALL
117
TABLE 4---HALF-TIMES OF SULFATED GLYCOSAMINOGLYCANS IN THE RAT ARTERIAL WALL (MEDIA INTIMA)
GAG
Young rats
Old rats
HS DS CS
2 1"6 2-3
4 2 4
Values are expressed in days and were calculated with specific radioactivities (Tables 2 and 3). sulfation. Various results have been observed in humans and other animals (Kirk & Dyrbye, 1957; Buck & Heagy, 1958; Kaplan & Meyer, 1960; Buddecke, 1962; Kumar et al., 1967; Ichida & Kalant, 1968). On aging the glycosaminoglycan content is affected differently in the arterial wall of the human compared with that of the rat. These observations can be related to a different chemical composition as was previously suggested. The relative resistance of the rat to an atherogenic diet should be associated with the high rate of heparan sulfate in the arterial wall of the rat. The concentration of glycosaminoglycans in the arterial wall of the rat can be modified in some experimental conditions (Picard et al., 1967). It has been observed that the turnover of sulfated glycosaminoglycans in the arterial wall of the rat changes with aging. These variations occur without any change in the level of glycosaminoglycan sulfation. With aging glycosaminoglycans appear to turn over less rapidly; these findings are related to alterations in the enzymatic activities involved in the metabolism of these compounds. This slower metabolism may be associated with alterations in the degradation and synthesis process. In this case, it is impossible to determine whether the alteration is related to sulfate activation or to sulfate transfer on the glycosaminoglycans; to obtain such findings it would be necessary to determine the intraarterial pool of PAPS. In the arterial wall of young rats, dermatan sulfate has a turnover faster than the other arterial wall sulfated glycosaminoglycans. It is also faster than dermatan from the skin of the same animals. These results agree with other works (Schiller & Dorfman, 1955, 1957); the turnover of dermatan sulfate is also faster in old rats. However, the results show that this slower metabolism may be applied to the three sulfated glycosaminoglycans. Although our findings are concerned with the turnover of sulfate, identical determinations of glycosaminoglycan turnover have been obtained with radioactive sulfate or glucidic precursors (14C) (Katsura & Davidson, 1966; Buddecke et al., 1971). These results are in agreement with an active metabolism of the glycosaminoglycans in the arterial wall which is the localization of an active biosynthesis of these compounds involving sugar nucleotide and sulfate nucleotide precursors (Picard & Gardais, 1967; Picard et al., 1969). This metabolic activity of the arterial wall slows down with aging. Acknowledgements--We are grateful to the D.G.R.S.T. for supporting this work.
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B. HERMELIN,J. PICARD,A. PAUL-GARDAISAND P. LEVY
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Key Word Index--Sulfated glycosaminoglycans; rat; aorta wall; glycosaminoglycan; dermatan sulfate.