AGE DEPENDENT ALTERATIONS OF GLUCOCORTICOID RECEPTORS AND RELATIONSHIP TO ENZYME ACTIVITIES IN RAT LIVER

Aging has been defined as a general decline in body function associated with an impaired ability to maintain homeostasis (K i z a k i et at. 1998). Alterations in the adaptive responses of tissues and cells to a certain hormone and other biochemical stimuli are age-related as well as the changes in the induction of some enzymes (K a lim i et al. 1983). Glucocorticoid hormones (GH) regulate development and aging and are essential for the maintenance of basal and stress-related homeostasis. The effects of glucocorticoids are exerted by intracellular glucocorticoid receptor (GR) protein, ligand dependent transcriptional factor, that binds to specific DNA sequences, glucocorticoid response elements (GRE) thereby modulating the expression of target genes in a positive or negative manner (B eat

AGE DEPENDENT ALTERATIONS OF GLUCOCORTICOID RECEPTORS AND RELATIONSHIP TO ENZYME ACTIVITIES IN RAT LIVER.Mojca Vulovic, G. Koricanac, Esma Isenovic and Nevena Ribarac-Stepic, Department for Molecular Biology and Endocrinology,Vinca Institute of Nuclear Sciences, 11001 Belgrade, Serbia and Montenegro UDC 577.74: 574.15 Aging has been defined as a general decline in body function associated with an impaired ability to maintain homeostasis (K i z a k i et at.1998).Alterations in the adaptive responses of tissues and cells to a certain hormone and other biochemical stimuli are age-related as well as the changes in the induction of some enzymes (K a lim i et al. 1983).Glucocorticoid hormones (GH) regulate development and aging and are essential for the maintenance of basal and stress-related homeostasis.The effects of glucocorticoids are exerted by intracellular glucocorticoid receptor (GR) protein, ligand -dependent transcriptional factor, that binds to specific DNA sequences, glucocorticoid response elements (GRE) -thereby modulating the expression of target genes in a positive or negative manner (B eat 0 1989).Liver is a major target organ for glucocorticoid action.Functional modulation of hepatic GR during aging has been reported (R 0 t h 1974; K a lim i et al. 1983; K a lim i 1984; S h arm a and Tim ira s 1987; Dj 0 rd j e vi e-M ark 0 vic et at.1999; Ran hot r a and S h arm a 2000; Ran hot r a 2001).Most of the studies concerning the effects of aging on GR have focused either on the two ends of the lifespan, immature and senescent, or at two aduit age groups.
The effects of glucocorticoids on intermediary metabolism by modulation of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) gene expression in the liver are well known (S c hub art 1986).The regulation is exerted by binding of GRs to the GREs in promoter region of these genes (D an esc h et at.1987; Jan t zen et at.1987).However, data analyzing age-related effects on TAT and TO enzymes activity is scanty.
The main goal of the presented study was to examine the concentration and ligand affinity (Kd) of rat liver GR during the aging.In parallel, the activity of TO and TAT enzymes that represent biological indicators of the GR function, were also studied in the liver of all aged groups.
The experiments were performed on male Mill Hill hooded rats aged 3, 6, 12, 18,21 and 24 months.Three months old rats were chosen as the appropriate control group.The animals were killed by cervical dislocation, the livers were minced and homogenized using Poter-Elvejhem homogenizer in buffer pH 7.0 (20 mM Tris-HCI, 1 mM EDTA, 10% glycerol, 50 mM NaCl, 2 mM DTT, 20 mM Na-phosphate buffer) 1:2 (w/v).The homogenate was centrifuged at 105 000 x g, for 90 min, at 4°C and the obtained cytosolic fractions were incubated for 18 h at 4°C, with increasing concentrations of 3H_ triamcinolone acetonide (TA) (Amersham, SA 32 Ci/mmol) IP ranging from 0.495 to 30 nM, in the absence or in the presence of 1000-fold excess of unlabeled steroid.The unbound steroid was removed by charcol-dextran absorption binding technique.The number and affinity of specific glucocorticoid binding sites was calculated using S cat c h a r d plot analyses (1949).TO enzyme activity was determined in rat liver homogenates according to the method of K n 0 x and A u e r b a c h (1955) and the activity of TAT was assayed in rat liver cytosol fraction by the method of D i a m 0 n d s ton e (1966).Protein concentration was measured by method of Low r y et at.(1951).Statistical significance of differences was estimated by One-way ANOVA test, and the differences was considered statistically significant at p<0.05.
The obtained results indicate the alterations in the affinity of GR during the aging (Fig. 1).Decrease in the receptor dissociation constant value (Kd) was observed in all age groups with significant changes in 6 (p<O.OOl), 12 (p<O.Ol) and 18month-old rats (p<O.Ol), as compared to three-month-old control group.The concentration of GR in rat liver cytosol was reducing from 6 up to 21 months of age (Fig. 2) and the pattern of changes was similar to those of Kd value.A significant decrease was recorded in 6 (p<O.OOl), 12 (p<O.OOl) and 18-month-old animals (p<O.Ol) comparing to 3-month-old -ones, while at 24 months of age a significant increase in GR level (p<O.Ol) was observed.However, published studies indicate that the number of hepatic GR could be decreased, increased or unchanged, during the aging.The presented results are in agreement with published evidence showing that the concentration of the GR is reduced in the liver of 6-month-old rats as compared to the corresponding control (S h arm a and Tim ira s 1987), as well as with the data indicating that the concentration of GR varies depending on the age group (Dj 0 r d j e v i e-M ark 0 vic et at.1999).Although Rot h (1974) reported an increase in the number of glucocorticoid binding sites in the liver of 12-month-old Sprague-Dawley rats comparing to 3-month-old animals, K a lim i et at.(1983) observed no changes in the receptor concentration of adult (2-3 months) and senescent (24-28 months) rats of the same strain.
Age-related changes in the GR are followed by the changes in TO and TAT activities.The activities of TO (Fig. 3) and TAT (Fig. 4) in rat liver were decreasing during the aging.A significant decrease in the activity of TO was noticed in 6 (p<O.OOl), 12 (p<O.OOl) and 18-month-old rats (p<O.Ol).TAT enzyme activity was significantly decreased in 6 (p<0.05),  12 (p<O.01),18 (p<O.01)and 21-month-old animals (p<O.OI).However, increased activities of TO and TAT were observed in 24 months old animals comparing to the corresponding controls.According to Rat h a and K a nun g 0 (1974) TAT synthesis occurs to the same extent in both, young and old rats, while some authors observed age-related alterations in TAT mRNA and activity (D j 0 r d j e v i e-M ark 0 vic et al. 1999).
Besides, a reduced level of TAT mRNA and activity has been noticed in old rats, without age-dependent differences for the same parameters of TO (Wei lin g e rand G u i g 0 Z 1986).The observed changes in GR, as well as TO and TAT activities can be related to altered cell responsiveness to glucocorticoid action during the aging.

Fig. 1 .Fig. 4 .Fig. 2 .
Fig.1.The dissociation constant (Kd) for glucocorticoid receptor in rat liver during the aging.The results represent the mean ( SD of 4 independent experiments and are expressed as percentage of control value obtained for 3-month-old animals.**p<O.OI; ***p<O.OOI.

Fig. 3 .
Fig.3.The effects of aging on TO enzyme activity.TO enzyme activity was determined in rat liver homogenate of 3, 6, 12, 18, 21 and 24-month-old rats.The results represent the mean (SD of 4 independent experiments and are expressed as percentage of control value obtained for 3-month-old animals.**p<O.OI; ***p<O.OOI.