Effect of steroid hormones on age dependent changes in rat arginase isoenzymes

Experimental Gerontology, Vol. 19, pp. 191-198,1984 Printed in the USA.All rightsreserved.

0531-5565/84$3.00 + .00 Copyright©1984PergamonPress Ltd

EFFECT OF STEROID HORMONES ON AGE DEPENDENT CHANGES IN RAT ARGINASE ISOENZYMES

A. NIRMALKUMAR and G.D. KALYANKAR* Department of Biochemistry, Minto Regional Institute of Ophthalmology, Bangalore *Department of Biochemistry and Biophysics, St. John's Medical College, Bangalore-560034, India Abstract-Age dependent changes in rat liver and kidney arginase isoenzymesfrom day 1 to 73 weeks have been studied. It has been observed that total liver arginase activity (activity/liver) increases with age, but there is no appreciable change in the proportions of isoenzymes, their pH optima, electrophoretic mobilities and inhibition characteristics, although Kmincreases with age. Corticosterone induces liver arginase activity and the degree of induction decreases with age. The hormone has no effect on kidney arginase. Testosterone treatment, on the other hand, induces kidney arginase without any effect on liver enzyme.

LIGHTBODY(1938) WAS the first to study age-dependent changes in rat liver arginase, (E.C. 3.5.3.1), who observed an oscillating pattern of enzyme activity during development and a decrease in old age. Charbonneau et al. (1967) reported that the rat liver arginase activity was maximum when body weight reached about 100 g and then began to decrease. Highest activity both in liver and kidney cortex has been reported in six week-old rats (Shukla and Kanungo, 1969). Isozymic forms of arginase was demonstrated by the use of DEAE cellulose columns (Gasiorowska et al. 1970). In liver they showed the presence of A~ and A3 forms and A~ and A4 in kidneys, A~ being the main form of the enzyme in liver and A4 in kidneys. The above observations were confirmed (Porembska et al., 1971) using starch gel electrophoresis. All the above forms have similar mol. wts. (120,000) and almost similar affinities for the substrate. However, Rao and Kanungo (1974) have reported the same specific activity for liver arginase in 10 and 70 weeks old rats. They also found a single molecular form of the enzyme in these age groups. The urea cycle enzymes are known to be inducible by nutrients like proteins, and hormones (Schimke, 1963; Ashida and Harper, 1961). The present studies were undertaken to see whether the age dependent changes in arginase could be related to the hormonal changes in the animals. Influence of two hormones, namely, corticosterone and testosterone has been studied.

*All correspondence should be addressed to Dr. G.D. Kalyankar. 191

192

A.N. K U M A R AND (;.I). KAI YANKAR

MATERIALS

AND

METHODS

The chemicals used were from the following sources: L-Arginine (BDH), DEAE-Cellulose (Bio Rad Laboratories, USA), corticosterone, testosterone propionate, acrylamide, biscrylamide and N.N.N'.N'-tetramethylene ethylene diamine (Sigma, USA). Other chemicals were of analytical grade made locally.

Experimental animals The animals used for the study were male albino rats (Wistar strain). They were maintained o,1 Hind Lever rat feed. Rats from day 1 to 73 weeks of age were used.

Tissues for enz)'me study Animals were killed by cervical dislocation, livers and kidneys were separated, washed thoroughly with cold 5 m M Tris-HCl buffer containing 5 m M MnCI~ (pH 7.5) to remove blood, blotted and kept frozen till they were used.

Separation o f arginase isoenzymes The frozen tissues were weighed and homogenized with 5 volumes of solution containing 5 m M MnCI~-I00 m M KCI in 10 m M Tris-HCl buffer (pH 7.5) in a Teflon glass Potter type holnogenizer for 1 minute. The homogenate is left for an hour at 4°C with gentle stirring and then kept at - 10°C for 24 hours. It is then thawed, centrifuged at 4°C and the sediment extracted once more as described above. The combined extracts were dialysed overnight against Tris-HCl buffer (pH 8.3) containing 5 m M MnCl2. The precipitate, if any, was centrifuged and the clear supernatant was subjected to DEAE-cellulose chromatography. The above procedure extracted almosl complete arginase activity from tissues.

DEAE-cellulose column chromatography This was done using a slightly modified procedure of Gasiorowska et al. (1970). The dialysed tissue extract was applied on to a DEAE-cellulose column (1 × 10 cms) previously equiliberated with 5 m M Tris-HCl buffer (pH 8.3). The elution was carried out with 50 ml of equiliberating buffer and 5 ml fractions were collected. Elution was continued with 50 ml of the buffer containing 0.15 M KCI in case of liver and with 0.25 M KCI in case of kidney, Each fraction was analysed for protein and arginase activity.

A rginase as~av Arginase activity was determined by incubating 0.1 ml of the respective tissue homogenate (0.5 percem in case of liver and 5 percent in case of kidney) with 250/zmoles of glycine-NaOH buffer pH 10.00 and 1 ?.mole of manganese chloride in a volume of 2.0 ml, for 15 minutes at 37°C, at the end of which 50 #moles of arginine (0.5 m l p H 10.0) was added and incubation continued for 15 minutes more. The reaction was arrested by addition of 0.5 ml of 10% T C A and centrifuged at 3000 rpm for 10 minutes. Urea liberated was estimated, taking an appropriate aliquot of tim supernatant, by diacetyl-monoxilne method. One unit of arginase activity is equal to 1 /,mole of urea formed.

Protein estimation Proteins in tissue homogenates and column fractions were estimated according to I..o,ary et al. (1951).

H o r m o n e trealnlen[

Ailimals of various ages were injected (i.p.) with saline suspension o1"3 rag/100 g body v, eight of corticosterone or 1 rag/100 g body weight of testosterone propionate fur 7 days. The controls ~ere treated appropriately.

Polyacrylamide gel electrophoresi~ Gel etectrophoresis was carried according to the procedure of Davis (1964) using 5070 acrylamide gel at pH 8.3.

STEROID HORMONES AND RAT AR(ilNa, SE ISOENZYMES

193

RESULTS AND DISCUSSION

Age dependent changes Table 1 shows that rat liver arginase activity increased slowly from day 1 to day 14, then a sudden increase was observed on day 21. From day 28 onwards, there were marginal changes in the enzyme activity, except at the eighth week. Though total liver arginase activity (units/liver) increased with age, when expressed per 100 g body weight, there was an increase up to eight weeks and a gradual decrease thereafter. The data closely agrees with earlier results (Greengard et al., 1970; Porembska, 1973; and Lamers and Mooren, 1980). Table 2 gives the kidney arginase activity at various ages. The activity/g tissue shows fluctuating pattern with age, but the total arginase activity (activity/pair of kidney) gradually increases and decreases at the 73rd week. Figure 1 gives the pattern of chromatographic separation of liver arginase isoenzymes. The proportions of isoenzymes (Table 3) from day 1 to 73 weeks show marginal variations. This shows that both isoenzymes undergo simultaneous changes in their concentrations during the lifespan studied. The pH profiles of liver isoenzymes, A, and A3, isolated from rats of ages 7, 13, 25 and 73 weeks show similar pH optima which lies between 10.0-10.5, which is also the pH optima of the original homogenate. The KI, values of A, and A3 arginase isoenzymes (Table 4 and Figure 2) determined by TABLE 1. LIVER ARGINASE ACTIVITY AT VARIOUS AGES

Age 1 7 14 21 28 7 8 13 25 73

Number o f animals

day days days days days weeks weeks weeks weeks weeks

A rginase activity (× 10~) units/g/hr.

10 6 6 5 5 6 5 5 5 3

0.81 0.70 0.91 3.63 3.56 3.40 5.55 3.40 4.10 3.54

44± + ± 44444-

A rginase activity (× 10~) units~liver~hr,

0.07 0.10 0.08 0.08 0.31 0.30 0,05 0,12 0,38 0.29

0.17 0.29 0.70 5.40 7.43 17.42 30.60 32.60 53.70 53.20

± + ± 44+ + ± 44-

Arginase activity ( × 10~) units~hr./1 O0 g body wt.

0.06 0.04 0.04 0.26 0.45 1.10 3.00 1.60 3.70 2.40

2.98 2.10 2.70 14.20 15.20 18.30 24.20 19.20 16.90 14.20

TABLE 2. KIDNEY ARG1NASE AT VARIOUS AGES

Age (weeks)

Number o f animals

7 8 13 25 73

6 5 5 5 3

Arginase activity ( × 104) units/g/hr. 0.13 0.15 0.11 0.14 0.11

44444-

0.003 0.005 0.003 0.009 0.004

Total arginase activity ( x 10~) units~hr. 0.12 0.17 0.21 0.29 0.23

194

A,N. KUMAR AND G,D. KAI.YANKAR

TABLE 4. K,n

TABLE 3. DISTRIBUTION OF ARGINASE ISOENZYMES

A,

AND A3 IN LIVER AT VARIOUS AGES

Age 1 7 14 21 28 7 8 13 25 73

day days days days days weeks weeks weeks weeks weeks

A, 80 80 90 85 85 90 90 80 80 80

VALUES FOR

Aj

AND

A3

LIVER ARGINASE 1SOENZYMES AT VARIOUS AGES

A

Age

20 20 10 15 15 10 10 20 20 20

(weeks)

A~ (raM)

A3 (raM)

7 25 73

6.6 12.5 20.00

7.7 12,5 16.6

The values are average of three experiments in each age group.

Lineweaver-Burk plots show that the affinity of the substrate to the enzyme decreases with age. The diamino acids, lysine and ornithine are competitive inhibitors of arginase (Gasioroskawa et al., 1970). The inhibitory action of the above amino acids showed no age dependent changes. Liver arginase isoenzymes AI and A3 isolated from 7, 25 and 73 weeks rats on polyacrylamide disc electrophoresis at pH 8.3 showed that A1 from all three ages studied remained near the origin and A3 moved towards the anode. The mobility of AI and A3 in all the ages studied showed that isoenzymes exist in the same molecular forms. Figure 3 gives a pattern of chromatographic separation of kidney arginase isoenzymes AI and A4. Like liver arginase, the kidney isoenzymes A~ and A4 do not show any appreciable age dependent variations (Table 5). The pH optima for AI are between 9.5-10.0, at all ages studied. The above data clearly show that concentrations of arginase isoenzymes in liver and kidney remain almost constant and they may exist in the same molecular forms. As the synthesis of isoenzymes are under the control of different genes, from the above data, it is clear that the genes for A, and A3 of liver and A~ and A4 of kidney remain unchanged throughout.

TABLE 5. DISTRIBUTION OF ARGINASE ISOENZYMES AND

A4

A,

IN KIDNEYS AT VARIOUS AGES

Age (weeks)

A,

A

7 13 25 73

9 5 5 5

91 95 95 95

The values are average of three experiments in each age group.

STEROID HORMONES

195

AN[) RAT AR(ilNASE ISOENZYMES

~80--¢-- A R G I N A S E --o--

PROTEIN

160"

ARGINASE ---,0- - PROTEIN

//~ /

A~

I/,0.

120 ' Z

ox oZ 500"

• 12

I,

Iv.

-10

100-

8 ~

~

z

60.

z

/.0

<

20

li'i

250"

I00-

/d-"°,, \

/,-

Kcl ,'

',

...... L,

G z ILl tf

4 o_

,.°-.. ~,~ ~/'

....

5- 9 10 12 FRACTION NO

......

15 16

1

2

L,

5"" 9

12

I/.

16

FRACTION NO

FIG. 1. Separation of Liver arginase isoenzymes on DEAE-Cellulose CoLumn. Each fraction is 5 ml.

Flo. 3. Separation of Kidney arginase isoenzymes on DEAE-Cellulose Column. Each fraction is 5 ml.

25 WEEKS

7 WEEKS

At

73 WEEKS

AI

AI

o~

f 05'

±

0.8

O~

06

03

04

02" ~J

Oi

V

i' 01

0 /.

f

ol

08

o~

08

01

OL.

08

A3 25

2.5

0

Z0 iS

01

0/,,

08

Lt.

0.l

I

0/,

08

01

0/.,

08

[$] FIG. 2. Line Weaver-Burke Plots of A1 and A3 arginase isoenzymes from rat liver of various ages.

196

A.N, KUMAR AND G.D. KAI.YANKAR

Effect of steroid hormones Glucocorticoids elevate the urea cycle enzymes, secondary to protein catabolism. Greengard et al. (1970) studied the effect of hydrocortisone on the developmental formation of arginase in rat liver. They observed that after the age of 28 days a single dose of hydrocortisone did not cause any increase in the enzyme activity. They also concluded that induction of arginase in liver of adult rats requires high daily doses of hydrocortisone for a week. Berlin and Schimke (1965) observed that arginase responds very slowly either to the treatment with cortisone or to increase in dietary protein. They suggested that the enzymes which are in constant usage or whose physiological activity is controlled by availability of substrate would not be required to have the ability to fluctuate rapidly to any environmental stimulus. Rao and Kanungo (1974) did not observe any increase in the specific activity of liver arginase at 10 and 70 weeks after treating with cortisone (3 rag/100 g body weight) for three days. Probably this might be due to the limited time of hormone treatment. In view of the above variations, rats of ages 6, 12, 24 and 72 weeks were treated with corticosterone (3 rag/100 g body weight) for seven days. Corticosterone treatment increases liver arginase activity (Table 6) in all the four age groups studied. But the degree of induction decreases from 98% at 7 weeks to 24°70 at 73 weeks, exhibiting an age dependent decrease in degree of induction. Our above data are in agreement with the observations of Rahman and Peraino (1973) on the induction of ornithine aminotransferase in response to high protein diet alone or in combination with glucocorticoids. Isoenzymes from liver of corticosterone treated rat were separated on DEAE-cellulose column. The proportions of the isoenzymes do not (Table 7) show any change except in case of seven week rats where the proportions change from 90:10 to 80:20. The elution characteristics, pH optima and electrophoretic mobil±ties of these isoenzymes are similar to those of respective controls. The interesting observation in the present study is that TABLE 6. EFFECT OF CORTICOSTERONE AND TESTOSTERONE ON LIVER AND KIDNEY ARGINASE AT VARIOUS AGES ( X l0 4 UNITS/G/TISSUE/HR.)

Liver

Kidney

A g e (weeks)

Normal

Corticosterone a

Testosterone b

Normal

Corticosterone a

Testosterone b

7 (n = 5)

3.40 + 0.30

6.73 + 0.48 (+98%)

3.97 + 0.08

0.13 + 0.003

0.14 + 0.007

0.25 + 0.010 (+95%)

13 (n = 5)

3.40 4- 0.12

P = NS 3.41 + 0.13

0.11 + 0.003

P = NS 0.11 + 0.006

25 (n = 5)

4.10 4- 0.38

P = NS 4.64 + 0.44

0.14 4- 0.009

P = NS 0.15 + 0.010

73 (n = 5)

3.54 + 0.29

P < 0.001 5.00 + 0.14 (+47%) P < 0.001 5.60 + 0.32 (+36%) P < 0.01 4.40 + 0.19 (+24%) P < 0.05

P = NS 3.65 + 0.17

0.11 + 0.004

P = NS 0.12 + 0.005

P < 0.001 0.21 + 0.008 (+94%) P < 0.001 0.27 + 0.024 (+97%) P < 0.001 0.22 + 0.010 (+100%) P < 0.001

P = NS

a C o r t i c o s t e r o n e : 3 r a g / 1 0 0 g b o d y ~ e i g h t for 7 days. b T e s t o s t e r o n e p r o p i o n a t e : 1 r a g / 1 0 0 g b o d y weight for 7 days. NS = N o t significant.

P = NS

STEROID HORMONES AND RAT ARGINASE ISOENZYMES

197

TABLE 7. VARIATIONS IN ARGINASE ISOENZYMES IN LIVER AFTER CORTICOSTERONE TREATMENT AND IN KIDNEY AFTER TESTOSTERONE TREATMENT

Liver

Kidney

Age (weeks)

Normal A~:A3

Corticosterone AI:A3

Normal A~:A4

Testosterone A,:A4

7 13

90:10 80:20

80:20 80:20

9:91 5:95

5:95 2:98

25 73

80:20 80:20

80:20 80:20

5:95 5:95

2:98 2:98

T h e values are a v e r a g e o f t h r e e e x p e r i m e n t s in each age g r o u p .

though there is a decrease in degree of induction of enzyme activity with age, the proportions of isoenzymes do not show any change, indicating that both the isoenzymes are induced simultaneously with age. Corticosterone at the given dose has no effect on kidney arginase. In a variety of tissues, increase in arginase occur in association with growth. Such changes include the increase in kidney arginase with testosterone treatment, in liver of females with thyroxine, the increase in testes at puberty and the increase in liver and mammary gland during lactation. Kochakian (1945) and Frieden and Fischel (1968) have observed that administration of testosterone increases kidney arginase activity in rat and mice. Based on the above data, rats of 6, 12, 24 and 72 weeks were injected with 1 rag/100 g body weight of testosterone propionate to study the effect of hormone on kidney arginase with age. Testosterone treatment doubles the kidney arginase activity (Table 6) in all the four ages studied, showing that age has no effect on the induction of enzyme activity in response to the hormone. Further, the proportions of kidney arginase isoenzymes (Table 7) show marginal changes in the major fraction. Thus, the kidney isoenzymes change simultaneously in all ages studied, as was observed with liver. The elution characteristics, pH optima of the above isoenzymes are similar to those from their respective controls. Testosterone at the dose given has no effect on liver arginase activity in any of the four age groups studied. Adelman (1975) classified the altered adaptive increase in the activities of a number of enzymes in response to a broad spectrum of stimuli into four categories. Some enzyme adaptations are altered in time course a n d / o r magnitude of response whereas others for reasons not known are not altered at all. Our data on the induction of liver arginase by corticosterone treatment resembles the second category of the above classification wherein the magnitude of response decreases with age. The data on the increase in kidney arginase in response to testosterone treatment is similar to the fourth category of the above classification, where the magnitude of response did not show any change with age. SUMMARY Age dependent changes in rat liver arginase activity from day 1 to 73 weeks show variations when the enzyme activity expressed per g of liver, but the total arginase activity (ac-

198

A.N. KUMARAND G.D. KALYANKAR

tivity/liver) increases with age. The proportions of arginase isoenzymes A, and A~ in liver do not show any age dependent change. The pH optima, inhibition characteristics by diamino acids, the electrophoretic mobilities of the isoenzymes are same in all the ages studied, whereas Km increases with age. Corticosterone induces liver arginase, but the degree of induction decreases with age from 98°7o at 7 weeks to 24°7o at 73 weeks. However, no change in the proportions of the isoenzymes or their physio-chemical characteristics is observed. Kidney arginase activity from 7 weeks to 73 weeks shows variations with age, but the proportions of isoenzymes do not show any change. Testosterone induces kidney arginase to the same extent in all the ages studied. The proportions of kidney arginase isoenzymes AI and A4 do not show any appreciable change with age after hormone treatment. REFERENCES ADELMAN,R.C. (1975) In Enzyme Induction, Basic Life Sciences, Vol. 6, 303, Ed. Parke, D.V., Plenum Press, London. ASHIDA, K. and HARPER, A.E. (1961) Proc. Soc. Exp. Biol. Med. 107, 151. BERLIN, C.M. and SCrlIMKE, R.T. (1965) MOI. Pharmacol. l, 149. CHARBONNEAU, R., ROBERG, A. and BERLINGUET, L. (1967) Can. J. Biochem. 45, 1427. DAvis, B.J., Ann. New York Acad. Sci. (1964) 121,404. FRIEDEN, E.M. and FISCHEL, S.S. (1968) Biochem. Biophys. Res. Commun. 31, 515. GASIOROWSKA, I., POREMBSKA, Z., JACHIMOWICZ, J. and MOCHNAIKA, 1. (1970) Acta Biochim. Pol. 7, 19. GREENGARD, O., SgHIa, M.K. and KNOX, W.E. (1970) Arch. Biochem. Biophys. 137, 477. KOCrIAKtAN, C.D. (1945) J. BioL Chem 155, 579. LAMERS, W.H. and MOOREN, P.G. (1980) BioL Neonate 37, 113. LIGrtTBODY, H.D. (1938) J. Biol. Chem. 124, 196. LOWRY, O.A. ROSEBROUGH, N.J., PARR, A.L. and RANDALL, R.J. (1951) J. Biol. Chem. 193, 265. POREMBSKA, Z (1973) Enzyme, 15, 198. POREMBSKA,Z., JACHIMOWICZ, J. and GASIOROWSKA,I. (1971) Bull. Acad. Pol. Ser. Sci. Biol. 19, 27. RAHMAN, Y.E. and PERAINO, C (1973) Exp. Geront. 8, 93. RAO, S.S. and KANUNGO, M.S. (1974) Indian J. Biochem. Biophys. l l , 208. SCHIMKE, R.T. (1963) J. Biol. Chem. 238, 1012. SHUKLA,S.P. and KANUNGO,M.S. (1969) Exp. Geront. 4, 57.