Enzymatic sulfation of steroids—VII. Hepatic cortisol sulfation and glucocorticoid sulfotransferases in old and young male rats

Exp. Gevont.Vol.13,pp. 425-429.

0531-5565/78/1201-0425502.0010

O Pergamon Press Ltd. 1978. Printed in Great Britain.

ENZYMATIC SULFATION OF STEROIDS--VII. HEPATIC CORTISOL SULFATION A N D GLUCOCORTICOID SULFOTRANSFERASES IN OLD AND Y O U N G MALE RATS* SANFORD S. SINGERt and LAWRENCEBRUNS Department of Chemistry, University of Dayton, Dayton, OH 45469, U.S.A. (Received 12 June 1978)

INTRODUCTION AGING modifies the glucocorticoid-mediated tyrosine aminotransferase (TAT, EC 2.6.1.5) response to cold stress in mice; diminishes their basal TAT activity; and decreases the TAT response to fasting and near-physiologic doses of glucocorticoid (Finch et aL, 1969). Similar effects have been reported with other rodents. This suggests that a factor essential for physiologic glucocorticoid action is depleted in old animals. Its extensive production appears to occur, after a lag, when they are stressed. But it may not be replaced completely or maintained as long as in young animals. On the other hand, the TAT response to pharmacologic doses of glucocorticoid (Finch et al., 1969; Gregerman, 1959; Rapaport et al., 1964) is unaltered in aged rodents. This suggests that their induction mechanism is fully operational when challenged with adequate glucocorticoid. Thus the lesion due to aging may involve glucocorticoid secretion, activation, or metabolism. The first alternative is unlikely, since basal glucocorticoid levels in several species (Finch et al., 1969; Rapaport et al., 1964; Grad et al., 1967) and corticosterone secretion in coldstressed mice (Finch et al., 1969) are similar regardless of age. In addition, decreased glucocorticoid activation due to glucocorticoid receptor--Cortisol Binder II (Litwack et al., 1973)--depletion is not consistent with reported differences between pharmacologic and physiologic glucocorticoid effects. Receptor diminution should affect both similarly. Indeed Roth (1974) reports no change in receptor concentration on old rats. Aging effects may thus be due to a presently unstudied glucocorticoid metabolite. For example, much of administered glucocorticoid is rapidly converted to sulfated metabolites by rat liver (Fiala and Litwack, 1966; Singer et aL, 1970; Tsong and Koide, 1973; Carlstedt-Duke and Gustafsson, 1975). We have already reported data (Singer and Litwack, 1971 ; Singer et al., 1975) suggesting that these compounds and the 3 cytoplasmic proteins--Cortisol Binders I, III and IV (Litwack and Singer, 1973)--they associate with may be involved in control of TAT induction .~n rats and guinea pigs. By analogy they could also be involved in aging effects on glucocorticoid actions. Consequently, study of STI, STII and STIlI--the 3 rat liver glucocorticoid sulfotransferases we described earlier (Singer et al., 1976; Singer, 1978a)--could provide a useful tool for understanding aging effects on glucocorticoid actions. The adrenals (Singer, 1978b) and gonads (Singer and Sylvester, 1976) appear to regulate tb_e 3 enzymes in a complex individualized fashion. Since they possess very different substrate preferences (Singer et al., 1976) change of their relative concentrations as rats age, could lead to very different pro*Supported by a grant from the Universityof Dayton Research Council. tTo whom to address correspondence. 425

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SANWORD S. gINGER AND LAWRENCE BRUNS

d u c t i o n o f g l u c o c o r t i c o i d sulfates due to physiologic a n d p h a r m a c o l o g i c g l u c o c o r t i c o i d levels. T h i s m a n u s c r i p t describes o u r initial c o m p a r i s o n o f hepatic g l u c o c o r t i c o i d (cortisol) sulfation in y o u n g a n d o l d male rats. W e r e p o r t changes o f relative a m o u n t s o f STI, S T I I a n d S T I I I t h a t could be involved in r e p o r t e d aging effects on glucocorticoid actions. METHODS Male and female CDR Fisher rats (Charles River Labs., Wilmington, MA) were purchased; or bred from selected animals and raised in this laboratory. Males were used for aging studies, females for PAPS-3'-phosphoadenosine-5'-phosphosulfate--preparation.* Young males (9-19 week)were bred here and maintained until use. The old rats were purchased at 6 weeks of age and maintained here until use at 20-27 months old. Animals were fed Purina chow and tap water a d libitum. [1,2:sI-I]Cortisol(45 Ci/mmol) and other [gH] steroids (Singer et al., 1976) were from New England Nuclear (Boston, MA). Nonradioactive steroids were purchased from Sigma (Saint Louis, MO). Steroid purity was checked periodically as described earlier (Singer et al., 1976). Cytosols were prepared after decapitation of individual rats. Livers were removed rapidly, trimmed, chilled and homogenized immediately in 1 vol of ice-cold TSM-0.050 M Tris-0.25 M sucrose-3.0 mM mercaptoethanol (pH 7.5). This and all other preparative steps were carried out at 0-4°C. The 50~ homogenates were centrifuged at 35,000 x g (Sorval RC-2B, 30 min). Pellets were discarded and the supernatants were then recentrifuged at 105,000 x g (Beckman L5-65B, 1 h). The resultant supernatant, 50Yo cytosol, contains most of the cortisol sulfotransferase activity (Singer et al., 1976). Suitably diluted cytosol samples were assayed for cortisol sulfotransferase activity at pH 6"8 as described earlier (Singer et al., 1976). Then reaction mixtures were extracted with CH2C1.2 and the aqueous residues which contain the steroid sulfates produced--were counted (Singer et al., 1976; Singer, 1978a). The enzyme activity, determined from the radioactivity measurements (Singer et al., 1976) is given as nmol steroid sulfated h -x per ml of undiluted enzyme sample. Kinetic properties of enzyme samples from old and young rats were as reported earlier for young animals (Singer et al., 1976). To fractionate the individual glucocorticoid sulfotransferases, 3 ml aliquots of cytosol from young or old rats were chromatographed on 2 x 50 cm columns of DEAE Sephadex A-50. Columns were eluted at 30 ml h -x with linear gradients consisting of 300 ml each of TSM and TSM-0.30 M KCI. Three ml fractions were collected. Enzyme assays were carried out as described above. Protein was estimated from the 280 nm absorbance. Salt gradients were determined from conductivity measurements (Singer, 1978b). The fractionation recovered 70-80 ~o of the enzyme activity loaded. Paired columns were always eluted together. One contained cytosol from a young rat. The other contained cytosol from an old animal. The presence or absence of the individual sulfotransferases was determined by comparison (Singer et al., 1976). RESULTS AND DISCUSSION Y o u n g (9-19 week) a n d o l d (20-27 m o n t h ) males were c o m p a r e d . T h e y o u n g g r o u p possessed typical a d u l t cortisol sulfotransferase activity a n d g l u c o c o r t i c o i d sulfotransferases (Singer e t aL, 1976; Singer, 1978a). T h e first g r o u p o f o l d rats tested were 20-23.5 m o n t h s old. Six experiments (Table 1A) gave m e a n cortisol sulfotransferase activity o f 29.2 4- 20 n m o l 11-1 per ml o f cytosol, c o m p a r e d to 15.8 ± 2.8 for the y o u n g rats used for controls. The difference between the means o f the 2 groups was n o t significant due to the large s p r e a d o f values in the o l d group. This s p r e a d o f d a t a suggested that the 20-23.5 m o n t h time p o i n t might represent a changeover p o i n t for the hepatic glucococ o r t i c o i d sulfotransferase activity. To test this a second g r o u p o f rats was aged further. E x a m i n a t i o n o f the survivors (n = 3) o f this g r o u p a t 26.5-27 m o n t h s o l d (Table 1B) s h o w e d t h a t the s p r e a d o f enzyme activities in this g r o u p was less extensive t h a n t h a t in the 20-23.5 m o n t h group. P o o l e d d a t a f r o m all the experiments (Table 1C) show n o m a j o r difference o f the m e a n cortisol sulfotransferase activity per ml cytosol between o l d a n d *PAPS(3'-phosphoadenosine-5'-phosphosulfate) was prepared as described in manuscript VI of this series. This has been submitted to Analyt. Biochem.

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CORTISOL 15ULFATION IN OLD AND YOUNG MALE-RATS

young groups. Enzyme activity values expressed per 100 g body weight are 80.5 q- 48 and 65.9 -t- 10 for old and young groups, respectively. It appears that if a changeover of enzyme activity occurs, it may precede 20 months. TABLE 1. HEPATIC CORTISOL SULFOTRANSFERASE ACTIVITY IN OLD AND YOUNG MALE RATS

Experiment (A) 1 2 3 4 5 6 average of 1-6

Hepatic CS activity per ml cytosol Old rats Young rats 47.1 19"9 16"1 18.6 23"6 15'1 61.1 18"5 12'1 15.3 15'4 17.2 29.2 ± 20 15.8 ± 2.8

(B)

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8 9 average of 7-9

26.7 25.0 20.9 4- 8.5

13.5 11.8 20.0 15.1 + 4.5

398 5:65 13.9 5:2.6 26.5 4- 17 80.5 ± 48

296 5:28 10.5 4- 1-2 17.6 ± 3.6 65.9 4- 19

(C) Summary of 1-9 Body weights (g) Liver weights (g) Activity per ral cytosol* Activity per 100 g body weight

(A) and (B) show 9 experiments in which an old (20-27 month) and a young (9-19 week) male rat were sacrificed by decapitation. Liver cytosols were prepared and assayed for cortisol sulfotransferase (CS) activity as in Methods. CS activity is nmol cortisol sulfated h -1 4- SEM. (C) summarizes the data from all experiments. *Each g of liver yields 1 ml of cytosol. Comparison of the substrate preference of the cytoplasmic steroid sulfotransferase activities of the old and young groups indicate d (not shown) that old rats had much higher abilities to sulfate dehydroepiandrosterone relative to cortisol than young rats. This suggested that livers from old and young animals contained different sulfotransferases. Increased STII production was deemed probable, since this enzyme sulfates dehydroepiandrosterone preferentially (Singer et aL, 1976). This prediction was supported by sulfotransferase fractionation on DEAE Sephadex A-50 (as in Fig. 1). All animal pairs tested showed decreased relative amounts of STIII in old rats. In all cases, STI was present in significant amounts in livers from old rats and STII was elevated extensively. Figure 1 compares the old and young rats of experiment 3, tb.e median experiment of the first series. Enzyme profiles from all old rats tested were qualitatively similar. Enzyme profiles from young rats always contained large amounts of STIII (80-9070 of recovered activity) and small amounts of STII (10-207o of recovered activity). No STI was observed in any young rats (Fig. lb; Singer et al., 1976; Singer, 1978a; Singer and Sylvester, 1976; Singer, 1978b). The data suggest that hepatic sulfation of physiologic amounts of glucocorticoid in old rats is likely to be diminished due to the presence of large amounts of STII supplying the ability to sulfate steroids like dehydroepiandrosterone at the expense of glucocorticoid sulfation. If glucocorticoid sulfates are involved in control of glucocorticoid metabolism and glucocorticoid effects on TAT and other glucocorticoid parameters (IngelmanSundberg, 1976; Singer and Litwack, 1971 ; Singer et al., 1975) this could lead to decreased

428

S A N r O g D S. SINGER A N D L A W R E N C E BRUN$

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FIG. 1. Fractionation of the glucocorticoid sulfotransferases of cytosol samples from livers of old and young rats. Three ml aliquots of 50 % cytosol from the old (A) and young (B) rats of experiment 3 were loaded on 2 × 50 cm DEAE Sephadex A-50 columns and eluted at 30 ml h -1, overnight. Elution of the 2 columns was carried out together, Each was eluted with a linear gradient consisting of 300ml of TSM and 300 ml of TSM-0.30 M KCI. Three ml fractions were collected. Enzyme activities, protein content and salt concentrations of indicated fractions were determined as in the Methods section. Roman numerals represent the individual sulfotransferases, STI, STII and STIII (Singer et al., 1976). The molar quantities are the salt concentrations measured at the peaks of eluted sulfotransferase activity. basal TAT levels and other reported effects of age on physiologic glucocorticoid parameters. However, large pharmacologic doses of glueocorticoid should compete successfully for sulfation. Thus they would be expected to cause age-independent glucocorticoid effects as reported (Finch et al., 1969; Gregerman, 1959; Rapaport et al., 1964; Grad et aL, 1967). Presently we are examining the temporal relationship between the sulfotransferase changeover and known glucocorticoid effects and ascertaining whether changes of sulfotransferase populations on aging are accompanied by changes of in vivo glucocorticoid metabolism. Once these goals have been attained, the basis for the age-mediated change of the enzymes can be explored and related to the failure of physiologic actions of glucocorticoids in old age. SUMMARY Old (20°27 month) and young (9-19 week) male CDR Fisher rats exhibited hepatic cortisol sulfotransferase activities of 80.5 4- 48 and 65.9 4- 19 nmol It -1 per 100 g body weight. The substrate preferences of the hepatic steroid sulfotransferase activity from the 2 groups were very different; old animals sulfating much more dehydroepiandrosterone than young ones. This was due to major differences of the relative amounts of STI, STII and STIII--the 3 rat liver steroid sulfotransferases that sulfate glucocorticoids--in the 2 groups. STIII predominates in young rats, comprising 80-90% of the hepatic cortisol sulfotransferase activity. In old animals the relative amount of STIII decreases markedly; STI is elevated greatly; and significant amounts of S T I - - n o t found in young males--are observed. These differences of the individual enzyme activities may be of use for understanding the basis for dissimilarities between physiologic and pharmacologic glucocorticoid effects in old rodents.

CORTISOLSULFATIONIN OLD AND YOUNGMALERATS

429

REFERENCES

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