Changes in the concentration of nucleic acids in the aorta of rats in relation to body weight

JOURNAL OF ATHEROSCLEROSISRESEARCH

397

CHANGES IN T H E C O N C E N T R A T I O N OF NUCLEIC ACIDS IN T H E A O R T A OF RATS IN R E L A T I O N TO BODY W E I G H T

E. A. SAVINO*, M. R. MALINOW**, L. GERSCHENSON, H. C. MOGUILEVSKY*, C. A. MARUFFO, J. R. DEPAOLI AND A. BALDASSARRE Institute of Physiology, Buenos A ires Medical School and Hospital Instituto de Cardiologla, FundaciSn Pombo de Rodrlguez, National Academy of Medicine, Buenos Aires (Argentina)

(Received September 8, 1964)

INTRODUCTION Changes in the concentration of pentose nucleic acid (PNA) in relation to ageing have been reported in several tissues. WULFF AND FRESHMAN1 indicated that the P N A content decreases in the myocardium and cerebellum of rats, while HYDI~N2 described a progressive increase in the average amount of P N A in the anterior horn cells of man up to the age of 40. Results reported by MANDEL in cattle indicate t h a t older animals have a decreased concentration of nucleic acids in the aorta 3. In the present study, nucleic acids have been determined in the aorta of rats and an increase has been found with advancing age. MATERIALAND METHODS Male rats weighing between 60 and 290 g, from the Instituto Bacteriol6gico Malbrdn, reared on a balanced laboratory diet ("Forramex", Molinos, Rio de la Plata)

were used. These weights corresponded to rats of 30 and 170 days, respectively. The animals were sacrificed b y head concussion; the whole aorta was rapidly excised and freed from adventitia. A thin portion of diaphragmatic muscle was also removed and blotted in filter paper. Organs were weighed without delay on a torsion balance to the nearest mg. The m e t h o d for determination of pentose nucleic acid phosphorus (PNA-P) and deoxypentose nucleic acid phosphorus (DNA-P) has already been described 4. Tissues were homogenized at 50,000 d.p.m, in 10 ml of distilled water at 4°C for 10 min in a Buhler Apparatus (Karl Kolb Technical Supplies, Frankfurt/Main, Germany), and then for 3 min with a Teflon pestler. D r y weight and nucleic acids were determined on aliquots. Deoxypentose nucleic acid (DNA) and pentose nucleic acid (PNA) were studied b y the method of OGUR AND ROSEN5 modified as follows. * Fellow from the Consejo Nacional de Investigaciones Cientificas y Tdcnicas de la Repfiblica Argentina. ** Presently at the Oregon Regional Primate Research Center, Beaverton, Oregon, U.S.A. j . Atheroscler. Res., 5 (1965) 397-402

E.A. SAVINO et al.

398

The homogenate, m a i n t a i n e d in a cold bath, was p r e c i p i t a t e d twice with cold (4-6°C) 5 °/o (v/v) trichloro-acetic acid and then centrifuged. (All centrifugations were performed at 5000 r.p.m, for 30 rain). The residue was e x t r a c t e d with e t h a n o l - e t h e r (3 : 1 v/v) at 40°C for 15 rain and then centrifuged. The residue was washed again with e t h a n o l - e t h e r at room t e m p e r a t u r e and centrifuged as before. Extraction of P N A . Tile residue was e x t r a c t e d with 3 ml 1 N perchloric acid (PCA) at 37°C for 30 min and then centrifuged. The residue was r e - e x t r a c t e d with 2 ml PCA a n d centrifuged. The s u p e r n a t a n t s were c o m b i n e d and tile volume was a d j u s t e d to 5 ml with PCA. Extraction of D N A . The residue from P N A e x t r a c t i o n was e x t r a c t e d twice with PCA in e x a c t l y similar fashion except t h a t the t e m p e r a t u r e of extraction was 80°C. Measurement of the extinction at 261 m # (the m a x i m u m for liver a n d a o r t a supernatants) was made as described by OGUR AND ROSENS; the results have been expressed as phosphorus of nucleic acids ( P N A - P a n d DNA-P). In order to prevent minor variations in tile m e t h o d from interfering with the results, 12 or 13 r a t s were used each d a y with weights e q u a l l y d i s t r i b u t e d t h r o u g h o u t the studied range*; the experiment was c o n d u c t e d for four consecutive days. The percentage w a t e r content was determined in the a o r t a a n d d i a p h r a g m a t i c muscle of simultaneously sacrificed similar rats. Results were a n a l y z e d with the r coefficient of regression using the tables of FISHER AND YATES 6. In a n o t h e r group of male rats weighing 60 or 290 g the a o r t a and the d i a p h r a g m were s t u d i e d histologically. In order to learn w h e t h e r the findings were confined to one strain of rats, animals of the Instituto de .Fisiologla were also included. Since results were similar, b o t h groups are described together. After fixation in 4 °/o formaldehyde a n d e m b e d d i n g in paraffin, the following staining techniques were p e r f o r m e d v: h e m a t o x y l i n - e o s i n ; VAN GIESON'S trichromic acid; reticulin (Rio HORTEGA); PAS (MACMANuS); f u c h s i n - p a r a l d e h y d e (GOMORI); t o l u i d i n blue. In aortic and d i a p h r a g matic sections s t a i n e d with h e m a t o x y l i n - e o s i n , t h e nuclei within squares of 100 × 100 # were c o u n t e d a n d the results of 10 such m e a s u r e m e n t s in each organ averaged. Care was t a k e n to include the whole arterial wall in this observation. In most of the older rats this was not possible because the vessel was t h i c k e r than 100 #; in such a case counting was s t a r t e d at the i n t i m a a n d e x t e n d e d up to the inner t h i r d of the adventitia. In six other animals weighing e i t h e r 60 or 290 g the a d v e n t i t i a was s t r i p p e d from the aorta; because findings were similar t h e y have been pooled together with those from i n t a c t arteries. Countings were c o m p a r e d with STUDENT'S t test 6. RESULTS

Chemical determinations Figs. 1-3 show t h a t P N A - P and D N A - P increase in the aorta of r a t s with * The age of the present rats can be calculated from the weight curve previously determined in 100 animals of this strain on the same diet. The weight increases linearly up to 270 g (140 days of age) and then there is a gradual change of slope up to 320 g (200 days of age). (Data kindly provided by DR. A. BALDASSARRE,Instituto Bacterioldgico Malbrdn, Buenos Aires, Argentina).

j. Atheroscler. Res., 5 (1965) 397-402

RELATION OF NUCLEIC ACID CONCENTRATION IN RAT AORTA TO BODY" WEIGHT

399

b o d y weight a n d t h a t the P N A - P / D N A - P ratio decreases. The same figures also i n d i c a t e t h a t n o changes are o b s e r v e d i n the d i a p h r a g m . TABLE I COEFFICIENT

OF

CORRELATION

Variable against body weight

OF

AORTA

AND

versus

DIAPHRAGM

Aorta

BODY

WEIGHT

IN

MALE

RATS

Diaphragm

number

r

t

number

of

of

values

values

PNA 48 DNA 48 P/D ratio 48 Percentage dry weight of organ 47

0.94 0.86 --0.87

18.8 49 11.4 49 12.4 49

--0.02

0.13

r

t

0.09 --0.06 --0.10

0.62 0.41 0.69

0.10

0.67

47

PNA-P j.lg/100 rn cj

Zl ;o!

t

19 •

.o°

17 15

iii

o

50

I

.

.

e

t

o

o o

oo

t

I

70

k

t •

o

14

.

:

° o

°

°G o •

•1 "

.

c0m

o

o

o I

90

I

o

.

I

.

I

110

.I

.

130

I

o

o

o

o

.

I

.

150 BOO¥

.I

I

o

o

o

co

..

I

.I

.

I

i

170 190 210 WEIGHT ( 9 )

oo

oo o

I

.

70 I 230

I

250

t

7 270

I

I

290

I

I

310

Fig, 1. PNA-P in the aorta (black dots) and the diaphragm (white circles) of male rats against body weight. Values expressed in/~g/100 mg fresh tissue.

I n T a b l e I it can be seen t h a t the coefficient of correlation r is n e a r one i n the c o m p a r i s o n s of the aorta, a n d a p p r o a c h e s zero in those of the d i a p h r a g m , b e i n g t h u s c o m p a t i b l e w i t h the results r e p o r t e d i n the figures. F u r t h e r m o r e , the s a m e t a b l e i n d i c a t e s t h a t n o c h a n g e s were o b s e r v e d in the w a t e r c o n t e n t of the a o r t a n o r of the d i a p h r a g m a t i c muscle. H istologic o b s e r v a t i o n s A o r t a . No changes were d e t e c t e d i n the i n t i m a of the older rats; differences were n o t e d in the m e d i a : the m y o c e l l u l a r nuclei b e c a m e t h r o u g h a n increase in the t r a n s v e r s e d i a m e t e r , a n d lipofuchsin p i g m e n t a r o u n d the nuclei of the myocells. Collagen fibers a p p e a r e d b e t w e e n the

the following less fusiform was o b s e r v e d cells, a n d the

j . Atheroscler. Res., 5 (1965) 397-402

E . A . SAVZ~O et al.

400

r e t i c u l i n fibers h a d a m o r e c o m p l i c a t e d p a t h w a y . T h e P A S - p o s i t i v e m a t e r i a l , a l t h o u g h u n i f o r m l y d i s t r i b u t e d , s h o w e d a slight t e n d e n c y to be c o n c e n t r a t e d a r o u n d t h e e l a s t i c lamellae. The g r e a t e s t c h a n g e s o c c u r r e d in t h e a d v e n t i t i a : a m a r k e d increase of f i b r o a d i p o s e tissue a n d of t h e n u m b e r of l y m p h o c y t e s w h i c h were c o n c e n t r a t e d u p o n t h e e x t e r n a l elastic l a m e l l a e f o r m i n g n o d e - l i k e s t r u c t u r e s . ONA-P yo/lOOmg d,,:

45

35

! " ..b

P

"'.:''':i 25

T• I •

16 i

50

o o

" i

i

70

i

i

90

*

i

110

i

i

130

i

i

i

i

i

i

7 o ?.':i I

I

150 170 190 210 BOOY WEIGHT ( g )

230

I

Z50

I

270

290

t

t

310

Fig. 2. DNA-P in the aorta (black dots) and the diaphragm (white circles) of male rats against body weight. Values expressed in /,g/100 mg fresh tissue. P/D Patio .68 .64 .60 .56

r.

•

.'|

: -.

•

.52

.48 .44 .40 o

.86 '90 I .82 78

o oO=

o o o

o

o

o

~

o o

o

o

o

o o

o

o

s'o

7'0

o

o

:o =

o

co

,

oo

r

oo o

o

o

o

9'0

110

130 150 170 190 BOGY WEIGHT ( g )

210

230

250

270

290

Fig. 3. P / D ratio in the aorta (black dots) and the diaphragm (white circles) of male rats against body weight. T h e increase in l y m p h o c y t e s is i n d i c a t e d on T a b l e I I . I t will b e seen t h a t older r a t s h a d m a n y m o r e nuclei p e r s u r f a c e u n i t t h a n y o u n g e r ones a n d t h a t t h i s difference d i s a p p e a r e d if l y m p h o c y t e s were n o t i n c l u d e d in t h e c o u n t i n g s ( y o u n g e r r a t s h a d p r a c t i c a l l y no infiltration).

j . Atheroscler. Res., 5 (1965) 397-402

RELATION OF NUCLEIC ACID CONCENTRATION IN RAT AORTA TO BODY WEIGHT

401

D i a p h r a g m . N o h i s t o l o g i c a l differences were n o t e d in t h e d i a p h r a g m . T h e o l d e r

rats, t h o u g h , h a d a s l i g h t l y lower n u m b e r of nuclei t h a n t h e y o u n g e r ones. TABLE II NUMBER

OF NUCLEI

IN AORTA

AND

DIAPHRAGM

60-g Rats

Aorta Diaphragm

OF RATS

290-g Rats

number of animals

total nuclei ( A )

number of animals

total nuclei ( B )

without lymphocytes (C)

9 6

42.20 ~_ 0.27 25.90 ± 0.23

9 6

71.27 ~ 0.82 24.13 ± 0.24

41.98 ± 0.54

P (aorta): A vs. B < 0.001; B vs. C < 0.001; A vs. C > 0.9 P (diaphragm): A vs. B < 0.001 Nuclei/1000 /,2. Ten measurements per organ in each animal; average ± standard error of the mean. DISCUSSION

T h e c o n c e n t r a t i o n of nucleic acids o b s e r v e d in t h e p r e s e n t r a t s is s i m i l a r t o those a l r e a d y d e s c r i b e d 4 in s p i t e of s t r a i n diffelences. Values, t h o u g h , are h i g h e r t h a n t h o s e i n d i c a t e d b y MANDEL 3 w h o r e p o r t e d t h e following c o n c e n h a t i o n s in t h e a o r t a of r a t s : P N A - P , 10.5 :]: 0.8; D N A - P , 9.0 4- 0.7 in # g / 1 0 0 m g of fresh tissue; a n d P / D ratio, 1.19 ± 0.10. I t is n o t possible to a c c o u n t for these differences b e c a u s e MANDEL s t u d i e d the w h o l e a o r t a a n d we s t r i p p e d t h e a d v e n t i t i a . I t is l i k e l y t h e n t h a t t h e lower results m a y i n d i c a t e t h e presence of a f a t - r i c h tissue such as t h e a d v e n t i t i a . Also, t h e h i g h e r v a l u e s (especially of D N A - P ) o b t a i n e d b y us c o u l d i n d i c a t e a m o r e t h o r o u g h e x t r a c t i o n , since t h e y were seen even in y o u n g e r r a t s where no l y m p h o c y t i c infiltration was observed. T h e p r e s e n t r e s u l t s s h o w t h a t aging in rats, as j u d g e d b y i n c r e a s i n g w e i g h t of t h e animals, b r i n g s a b o u t a h i g h e r c o n c e n t r a t i o n of nucleic acids in t h e a o r t a . T h e histologic findings i n d i c a t e t h a t these differences can p r o b a b l y be a s c r i b e d to t h e a c c u m u l a t i o n of l y m p h o c y t e s o b s e r v e d in o u r o l d e r a n i m a l s a n d this of course m a y obscure a n y d e c r e a s e in t h e tissue i n d u c e d b y age 3. W h e t h e r t h e l y m p h o c y t e s s h a r e w i t h t h e fixed a r t e r i a l cells m e t a b o l i c r e a c t i o n s of t h e v a s c u l a r wall c a n n o t be s t a t e d a t the p r e s e n t t i m e . N o m e n t i o n of an increase in t h e n u m b e r of l y m p h o c y t e s in t h e a d v e n t i t i a of r a t s is m a d e in t h e c o m p r e h e n s i v e p a p e r on s e n e s c e n t a n i m a l s b y WILENS AND SPROUL 8. W h e n t h e r e s u l t s o b t a i n e d in a n o t h e r tissue s t u d i e d for c o m p a r i s o n are a n a l y zed, it is seen t h a t n o c h a n g e s in t h e c o n c e n t r a t i o n of nucleic a c i d s were a p p a r e n t in t h e d i a p h r a g m a l t h o u g h a s l i g h t decrease in t h e n u m b e r of nuclei was observed. T h e fact t h a t no s t r i c t c o r r e l a t i o n was p r e s e n t here b e t w e e n c h e m i c a l a n d h i s t o l o g i c m e t h o d s Call b e e x p l a i n e d e i t h e r b e c a u s e of s c a t t e r e d v a l u e s of D N A levels, t o c h a n g i n g d e g r e e s of c o n t r a c t i o n of t h e d i a p h r a g m a t i c muscles, or t o o t h e r u n d e j . Atheroscler. Res., 5 (1965) 397-402

402

E . A . SAVINO et al.

t e r m i n e d factors. S i m i l a r reasonings m a y p r o b a b l y e x p l a i n t h e h i g h e r n u m b e r of nuclei f o u n d in t h e c o n t r a c t e d a r t e r i a l walls as c o m p a r e d w i t h t h e r e l a x e d d i a p h r a g m . I t is f r e q u e n t l y s t a t e d t h a t m e t a b o l i c r a t e s a r e m o r e m e a n i n g f u l if results are e x p r e s s e d p e r u n i t weight of D N A . O u r findings s h o w t h a t in t h e p a r t i c u l a r case of t h e a o r t a in r a t s , this nucleic a c i d m a y n o t r e p r e s e n t a r t e r i a l cells b u t i n f i l t r a t i v e e l e m e n t s w h i c h m a y b e u n r e l a t e d to t h e i n t r i n s i c v a s c u l a r m e t a b o l i s m . F u r t h e r m o r e , in t h e light of t h e p r e s e n t findings it is clear t h a t c h a n g e s r e p o r t e d in t h e a o r t i c c o n c e n t r a t i o n of D N A a n d P N A in r a t s in r e l a t i o n t o r e m o v a l of e n d o c r i n e g l a n d s , for i n s t a n c O , 9, m a y t h u s h a v e a d u a l m e c h a n i s m : m o d i f i c a t i o n of t h e nucleic a c i d c o n t e n t of fixed a r t e r i a l cells a n d / o r v a r i a t i o n s in t h e n u m b e r of mobile c o n n e c t i v e tissue elements. SUMMARY

P e n t o s e nucleic a c i d p h o s p h o r u s ( P N A - P ) a n d d e o x y p e n t o s e nucleic a c i d p h o s p h o r u s ( D N A - P ) h a v e b e e n d e t e r m i n e d in t h e a o r t a a n d t h e d i a p h r a g m a t i c m u s c l e of m a l e rats. A n i m a l s w e i g h i n g b e t w e e n 60 a n d 290 g were s t u d i e d . T h e a o r t i c conc e n t r a t i o n of b o t h nucleic acids i n c r e a s e d w i t h b o d y w e i g h t while t h e P / D r a t i o decreased. N o m o d i f i c a t i o n s were o b s e r v e d in t h e d i a p h r a g m . T h e w a t e r c o n t e n t w a s s i m i l a r in t h e a o r t a as well as in t h e d i a p h r a g m t h r o u g h o u t t h i s r a n g e of b o d y weight. H i s t o l o g i c findings suggest t h a t t h e c h a n g e s o b s e r v e d in t h e a o r t a m a y be t o t a l l y or p a r t i a l l y e x p l a i n e d t h r o u g h l y m p h o c y t i c i n f i l t r a t i o n of t h e i n n e r a v d e n t i t i a . T h e possible significance of these o b s e r v a t i o n s is d i s c u s s e d . REFERENCES

1 V. J. WULFF AND M. FRESHMAN,Arch. Biochem. Biophys., 95 (1961) 181. H. HYD~N, In: J. BRACHET AND A. E. MIRSKY (EDs.), The Cell, Academic Press, New York, 1961, Vol. 4, p. 301. 3 p. MANDEL, In: ]3. PRUSIK, Z. REINIS, AND O. RIEDL (EDs.), Metabolismus Parietis Vasorum, Prague, 1962, p. 25. 4 L. GERSCHENSON, M. R. MALINOW, J. L. LACUARAAND H. C. ]VIOGUILI~VSKY,J. Atheroscler. Res., 2 (1962) 365. 5 M. OGUR AND G. ROSEN, Arch. Biochem., 25 (1950) 262. s R. H. FISHER AND F. YATES, Statistical Tables for Agricultural, Biological and Medical Research, Oliver and Boyd, Edinburgh, 1953. 7 G. GOMORI, Microscopic Histochemistry, Univ. Chicago Press, Chicago, 1958. 8 S. L. WILENS AND E . E . SPROUL, Am. J. Pathol., 14 (1938) 201. o L. GERSCHENSON, V. G. FOGLIA, M. R. MALINOW, H. C. MOGUILEVSKY, J. R. DEPAOLI, J. L. LACUARAAND R. YABO, Ang~iologie (Paris), in the press. J. Atheroscler. Res., 5 (1965) 397-402