Mechanisms of Ageing and Development, 66 (1992) 187-194 Elsevier ScientificPublishers Ireland Ltd.
187
PLASMIC MEMBRANES OF HEPATOCYTES AND ADRENOCORTICOCYTES IN RATS OF DIFFERENT AGES: EFFECT OF TESTOSTERONE
V.V. FROLKIS, A.L. KOBZAR and G.M. TJUKHTIN Institute of Gerontology, Kiev (Ukraine) (Received March 23rd, 1992) (Revision received May 19th, 1992)
SUMMARY
Experiments on adult (6-8-month-old and 26-28-month-old) Wistar rats revealed the hyperpolarization of plasmic membranes and activation of Na,K-ATPase of adrenocorticocytes in animals of both age groups and of hepatocytes of adult rats. No effect of testosterone was observed on the level of membrane potential and the activity of Na, K-ATPase of hepatoeytes of old rats. The effect of testosterone was prevented by inhibitors of protein biosynthesis (actinomycin D and cycloheximide) and a specific inhibitor of Na,K-ATPase (ouabain), but not by K+-channel blocker 2-aminopyridine. Testosterone was assumed to synthesize the specific factor, capable of activating Na, K-ATPase of plasmic membranes. The cytosole of hepatocytes and the blood serum of adult testosterone-treated rats activated the Na,K-ATPase of isolated plasmic membranes of hepatocytes of adult and old intact rats. During aging there was a decrease in the capacity of cells to synthesize the specific factor, which activated Na,K-ATPase of plasmic membranes.
Key words." Aging; Hepatocyte; Adrenocorticocyte; Testosterone; Hyperpolarization; Na,K-ATPase; Hyperpolarization factor INTRODUCTION
The process of aging is accompanied by changes in the secretory activity of sex glands, in the blood concentration of sex steroids and in the cell response to Correspondence to: Vladimir V. Froikis, Inst. of Gerontology, Vyshgorodskaya St. 67, 252655 KJev-114, Ukraine. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
188 hormones [3,5,10]. Previously, we have shown that sex steroids and some other hormones (estradiol propionate, testosterone, somatotropic hormone, insulin) induced the development of hyperpolarization of cell membranes [1,2]. This was assumed to be linked with the hormone-induced activation of synthesis of a special hyperpolarizing factor, which altered the status of plasmic membrane [1]. The purpose of the present study was to investigate the effect of testosterone on the level of polarization of cell membranes and of active ionic transport in the hepatocytes and adrenocorticocytes in animals of different age and to analyze the potential mechanism of the above effect. To solve this task the following have been undertaken: (1) The study of in vivo and in vitro effects of testosterone on the level of membrane potential of hepatocytes and adrenocorticocytes in animals of different ages. (2) The study of in vivo and in vitro effect of testosterone on the activity of Na,KATPase of membranes of hepatocytes and adrenocorticocytes. (3) The study of the effect of inhibitors of protein synthesis (actinomycin D, inhibitor of the transcription process; and cycloheximide, inhibitor of the translation process), inhibitors of K+-channels (2-aminopyridine) and Na,K-ATPase (ouabain) on the effect of a cell to testosterone. (4) The study of the effect of cytosole and blood serum of adult and old testosterone-treated rats on the activity of Na,K-ATPase of intact isolated plasmic membranes of hepatocytes, taken from animals of different age. MATERIALSAND METHODS Experiments were conducted on male Wistar rats aged 6-8 months. The level of membrane potential (MP) of the hepatocytes and adrenocorticocytes was measured according to [6] in the adrenocorticocytes and MP was measured following their in vivo and in vitro treatment with testosterone. Isolated adrenals were incubated in Krebs-Henselate solution [8] at 37°C [4]. Fractions of isolated plasmic membranes of hepatocytes were removed and purified according to Pietras and Szego [11]. The degree of purification was determined from the enrichment of the fraction with the marker enzyme of the plasmic membrane: 5'-nucleotidase [9]. Protein concentration in the samples was estimated using the method of Lowry [7]. Na, K-ATPase activity was determined according to Potapenko [12]. The fractions of isolated plasmic membranes of hepatocytes were incubated in vitro with the cytosole and blood serum of rats in ice for 40 rain at constant shaking. Adrenals were incubated in vitro in Krebs-Hanselate solution for 1 h at 37°C. The concentration of testosterone in the incubation medium was 20/~g/ml. The results obtained were processed statistically using bifactoral dispersion analysis ANOVA.
189 RESULTS
One hour after i.p. administration of testosterone (1 mg/kg) there was a significant hyperpolarization of the membranes of adrenocorticocytes of adult and old animals (Fig. 1). In response to testosterone administration the hyperpolarization of hepatocytes developed in adult rats only. In old animals, the MP level of hepatocytes remained unchanged. Pretreatment (30 min before testosterone) with inhibitors of protein synthesis actinomycin D (50/zg/kg) and cycloheximide (250 #g/kg) prevented the hyperpolarizing effect of the hormone upon both hepatocytes of adult animals and adrenocorticocytes of adult and old animals. A specific inhibitor of Na,KATPase, ouabain (73 #g/kg), also prevented the testosterone-induced hyperpolarization of plasmic membranes of hepatocytes and adrenocorticocytes. The blocker of K+-channels, 2-aminopyridine (10 mg/kg), produced no effect on the development of hyperpolarization of plasmic membranes (Fig. 1). The effect of testosterone on the MP level of adrenocorticocytes is thought to be linked with its direct effect upon the latter. This notion was supported by the results of experiments in which isolated adrenals of rats of different ages were in vitro incubated with testosterone. As is seen in Fig. 1, addition of testosterone to the medium in which isolated intact adrenals of animals removed from animals of both ages were incubated, produced significant hyperpolarization of the adrenocorticocytes membranes in both adult and old rats. A special series of experiments was undertaken to study the effect of testosterone upon the activity of Na,K-ATPase of rat hepatocytes and adrenocorticocytes. In the homogenate of adrenals and in the fraction of isolated plasmic membranes of hepatocytes, the basal Na,K-ATPase activity decreased from 8.0 and 11.1 nmol Pi/mg to 6.0 and 8.0 nmol Pi/mg in adult and old rats, respectively. Age-related differences have been established in the changes of Na,K-ATPase activity that occurred in cells of different type following testosterone administration. In adult rats, significant activation of Na,K-ATPase of hepatocytes (by 49%) and adrenocorticocytes (by 71%) was observed 1 h after i.p. testosterone (Fig. 2). In old rats, the activation of Na,K-ATPase was noted in adrenocorticocytes only, the degree being significantly higher in adult animals (Fig. 2). Testosterone in the dose used in this study did not activate Na,K-ATPase of old rat hepatocytes. Thus, the testosterone-induced hyperpolarization of plasmic membranes of adrenocorticocytes and hepatocytes was conditioned by the hormonal activation of Na,K-ATPase. Another series of experiments was carried out to study the effect of inhibitors of protein synthesis - - actinomycin D and cycloheximide - - on testosterone's effect upon the Na,K-ATPase activity. As is seen in Fig. 3, against the background Of pretreatment with one of the above inhibitors the testosterone produced no activation of Na,K-ATPase. The above effects are thought not to result from the direct
190
Adult mV
Adrenals
mV
1~
Liver
6O
59 #I
58 57 56 55 5@ 53 52
'
1 hour
36
0
1 hour
Old
60
mV
Adrenals
,1~
43
mV
Liver
42
58
2~ 41 40 39 38 37
52
1
0
1 hour
36 0
1 !
1 hour
Fig. 1. The size of membrane potential of hepatocytes and adrenocorticocytes from adult and old rats under testosterone action. 1, Under in vivo testosterone action (*, membrane potential significance changes in comparison with the control size, P < 0.05); 2, under in vitro testosterone action (*, membrane potential significance changes in comparison with the control size, P < 0.05); 3, under combined 2aminopiridine and testosterone action (*, membrane potential significance changes in comparison with the control size, P < 0.05); 4, under combined actinomycin D and testosterone action; 5, under combined cycloheximide and testosterone action; 6, under combined ouabaine and testosterone action.
effect u p o n the enzyme in view o f the fact t h a t neither testosterone, n o r a c t i n o m y c i n D n o r c y c l o h e x i m i d e p e r se d i d n o t affect the basal N a , K - A T P a s e activity when the fraction o f intact isolated m e m b r a n e s was i n c u b a t e d in vitro with each o f the inhibitors. The d a t a o b t a i n e d suggest t h a t the a c t i v a t i o n o f p r o t e i n synthesis in the cell cytosole resulted in the emergence o f a f a c t o r which a c t i v a t e d the N a , K - A T P a s e .
191
Adult nmol Pi/mg min
nmol Pi/mg min
25
20"
T T
2O
15.
15 10_
K
K
I0
5 5 0
0
Adrenals
Liver
Old nmol Pi/mg min
nmol Pi/mg min
20-
25 2O
1515 T
10-
IO
K
5-
T
K
5
0 Adenals
0
Liver
Fig. 2. The Na,K-ATPase activity hepatocytes and adrenocorticocytcs from adult and old rats under testosterone action. K, control rats; T, after testosterone injection (*, Na,K-ATPase activity significance changes in comparison with the control size, P < 0.05).
192 To check this assumption, we have performed a series of experiments, in which fractions of isolated plasmic membranes of hepatocytes of the intact rats were incubated in vitro with the cytosole of hepatocytes from testosterone-treated adult animals (Fig. 3). The cytosole of such animals is known to significantly activate the enzyme of isolated plasmic membranes of adult and old rats, whereas the cytosole of intact animals produces no effect upon the basal activity of Na,K-ATPase. Consequently, in old age the enzyme remains capable to adequately respond to the effect of activating factor. The cytosoles of hepatocytes of rats administered with one of
Adult membranes nmol Pi/mg min 25T
15-
i
T
m
20_
T
4~
TAd TAdTCg
K
II.F
lO_ 5
.I Membranes nmol Pi/mg min 25-
TCg
K
w
Adult cytosol
Adul' serum
Old membranes TS
2O TC 15
10
TC K
T,,,
K
5 O
Membranes
Incubation with old
TS
II
Incubation with adult
Fig. 3. Hepatocytes Na,K-ATPase activity under influence testosterone, protein biosynthesis inhibitors, hepatocyte cytosol and rat serum. K, control rats; T, testosterone-pretreatedrats (* Na,K-ATPase activity significance changes in comparison with the control size, P < 0.05); TAd, actimomycin D and testosterone-pretreated rats; TCg, cycloheheximide and testosterone-pretreated rats; TC, hepatocyte cytosole of testosterone-pretreatedrats (* Na,K-ATPase activity significancechanges in comparison with the control size, P < 0.05); TS, serum of testosterone-pretreated rats (* Na,K-ATPase activity changes in comparison with the control size, P < 0.05).
193
the above inhibitors 30 min prior to testosterone did not activate the Na,K-ATPase of intact plasmic membranes either (Fig. 3). Of substantial importance among testosterone's effects upon the cell are the membrane-genome mechanisms, which suffer during aging. Obviously, the old rats do not synthesize this factor in adequate quantities. This is evident from the fact that the cytosole of old rats did not activate the Na,K-ATPase of isolated plasmic membranes of old rats (Fig. 3). Apparently, lack of activation of Na,K-ATPase in the fraction of intact plasmic membranes of old rat hepatocytes at incubation with the hepatocyte cytosole and blood serum of testosterone-pretreated old animals was conditioned by the deficient synthesis of such a factor in aging. Na,K-ATPase-activating factor gets into the blood. Incubation of a fraction of isolated plasmic membranes of intact adult and old rats in the blood serum of adult testosterone-pretreated animals activated the enzyme compared with the incubation in the blood serum of intact adult rats (Fig. 3). Actinomycin D and cycloheximide, administered 30 min prior to testosterone, prevented the appearance of Na,KATPase-activating factor in the rat blood serum as well (Fig. 3). The blood serum of old rats did not alter the Na,K-ATPase activity of isolated plasmic membranes of intact old animals (Fig. 3). Thus, the administration of testosterone resulted in the synthesis in the cell of a factor, which activated Na,K-ATPase. This notion can be substantiated by the following. First, the hepatocyte cytosole and blood serum of insulin-treated adult animals activated Na,K-ATPase of isolated plasmic membranes of hepatocytes of intact rats. Second, inhibitors of protein synthesis prevented the hyperpolarization of membranes, the activation of Na,K-ATPase and the appearance of a factor in the hepatocyte cytosole and blood serum. The synthesis of testosterone-induced factor suffers with age and this can be substantiated by the following. First, there was no significant hyperpolarization of hepatocyte's membranes and activation of Na,K-ATPase in old rats against the background of testosterone effect. Second, the hepatocyte cytosole and blood serum of old rats lost their capacity to activate Na,K-ATPase of isolated plasmic membranes of the hepatocytes of intact old rats. At the same time, in old age the plasmic membrane per se retained its capacity to respond to hepatocyte cytosole and blood serum of adult rats. The activation of the ionic pump and the development of hyperpolarization are known to play a significant role in the cell reactivity, in the permeability of cell membranes and in the regulation of protein synthesis. The ageinduced weakening of synthesis of substances, which activate Na,K-ATPase and induce hyperpolarization, may be an important mechanism in the aging of a cell. REFERENCES 1 V.V. Frolkis, Is about hyperpolarization factor appearance under the activation of protein biosynthesis. Physiol. J., 26 (1980) 558-560.
194 V.V. Frolkis, Ageing Neurohumorai mechanisms, Kiev, 1981, 320 p. R. Ghanatian and K. Fortherby, Testosterone uptake by prostatic tissue from young and old rats. Gerontologia, 21 (1975) 211-215. 4 E.N. Gorban, The thyrotropin influence on membrane potential of thyrotrope gland: age peculiarities. Physiol. J., 24 (1979) 395-401. 5 S.M. Harman and P.D. Tsitouras, Reproductive hormones in ageing and men. 1. Measurement of sex steroids, basal luteinizing hormone and Leyding cell response to human horionic gonadotropin. J. Clin. Endocrinol. Metab., 51 (1980) 35-40. 6 P.G. Kostuk, Microelectrode tecniks, Kiev, 1960. 7 O.H. Lowry, N.J. Rosenbrouch, A.L. Farr and R.G. Randall, Protein measurement with the Pholin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 8 E.K. Mathews and M.S. Safran, Steroid production and membrane potential measurement in cells of the adrenal cortex. J. Physiol., 189 (1967) 149-161. 9 R.H. Michell and J.N. Hamthorn, The site of diphosphoinositide synthesis in rat liver. Biochem. Biophys. Res. Commun., 21 (1965) 333-338. 10 K. Nahoul and M. Roger, Age related decline of plasma bioactive testosterone in adult men. J. Steroid. Biochem. 35 (1990) 293-299. 11 R. Pietras and C. Szego, Membrane recognition and effector sites in steroid hormone action. Biochem. Actions Horm., 8 (1981) 307-363. 12 R.I. Potapenko, Acetyicholine and noradrenaline influence on synaptosomal membrane Na,KATPase activity from brain of adult and old rats. Neurochemistry, 5 (1986) 57-60. 2 3
187
PLASMIC MEMBRANES OF HEPATOCYTES AND ADRENOCORTICOCYTES IN RATS OF DIFFERENT AGES: EFFECT OF TESTOSTERONE
V.V. FROLKIS, A.L. KOBZAR and G.M. TJUKHTIN Institute of Gerontology, Kiev (Ukraine) (Received March 23rd, 1992) (Revision received May 19th, 1992)
SUMMARY
Experiments on adult (6-8-month-old and 26-28-month-old) Wistar rats revealed the hyperpolarization of plasmic membranes and activation of Na,K-ATPase of adrenocorticocytes in animals of both age groups and of hepatocytes of adult rats. No effect of testosterone was observed on the level of membrane potential and the activity of Na, K-ATPase of hepatoeytes of old rats. The effect of testosterone was prevented by inhibitors of protein biosynthesis (actinomycin D and cycloheximide) and a specific inhibitor of Na,K-ATPase (ouabain), but not by K+-channel blocker 2-aminopyridine. Testosterone was assumed to synthesize the specific factor, capable of activating Na, K-ATPase of plasmic membranes. The cytosole of hepatocytes and the blood serum of adult testosterone-treated rats activated the Na,K-ATPase of isolated plasmic membranes of hepatocytes of adult and old intact rats. During aging there was a decrease in the capacity of cells to synthesize the specific factor, which activated Na,K-ATPase of plasmic membranes.
Key words." Aging; Hepatocyte; Adrenocorticocyte; Testosterone; Hyperpolarization; Na,K-ATPase; Hyperpolarization factor INTRODUCTION
The process of aging is accompanied by changes in the secretory activity of sex glands, in the blood concentration of sex steroids and in the cell response to Correspondence to: Vladimir V. Froikis, Inst. of Gerontology, Vyshgorodskaya St. 67, 252655 KJev-114, Ukraine. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
188 hormones [3,5,10]. Previously, we have shown that sex steroids and some other hormones (estradiol propionate, testosterone, somatotropic hormone, insulin) induced the development of hyperpolarization of cell membranes [1,2]. This was assumed to be linked with the hormone-induced activation of synthesis of a special hyperpolarizing factor, which altered the status of plasmic membrane [1]. The purpose of the present study was to investigate the effect of testosterone on the level of polarization of cell membranes and of active ionic transport in the hepatocytes and adrenocorticocytes in animals of different age and to analyze the potential mechanism of the above effect. To solve this task the following have been undertaken: (1) The study of in vivo and in vitro effects of testosterone on the level of membrane potential of hepatocytes and adrenocorticocytes in animals of different ages. (2) The study of in vivo and in vitro effect of testosterone on the activity of Na,KATPase of membranes of hepatocytes and adrenocorticocytes. (3) The study of the effect of inhibitors of protein synthesis (actinomycin D, inhibitor of the transcription process; and cycloheximide, inhibitor of the translation process), inhibitors of K+-channels (2-aminopyridine) and Na,K-ATPase (ouabain) on the effect of a cell to testosterone. (4) The study of the effect of cytosole and blood serum of adult and old testosterone-treated rats on the activity of Na,K-ATPase of intact isolated plasmic membranes of hepatocytes, taken from animals of different age. MATERIALSAND METHODS Experiments were conducted on male Wistar rats aged 6-8 months. The level of membrane potential (MP) of the hepatocytes and adrenocorticocytes was measured according to [6] in the adrenocorticocytes and MP was measured following their in vivo and in vitro treatment with testosterone. Isolated adrenals were incubated in Krebs-Henselate solution [8] at 37°C [4]. Fractions of isolated plasmic membranes of hepatocytes were removed and purified according to Pietras and Szego [11]. The degree of purification was determined from the enrichment of the fraction with the marker enzyme of the plasmic membrane: 5'-nucleotidase [9]. Protein concentration in the samples was estimated using the method of Lowry [7]. Na, K-ATPase activity was determined according to Potapenko [12]. The fractions of isolated plasmic membranes of hepatocytes were incubated in vitro with the cytosole and blood serum of rats in ice for 40 rain at constant shaking. Adrenals were incubated in vitro in Krebs-Hanselate solution for 1 h at 37°C. The concentration of testosterone in the incubation medium was 20/~g/ml. The results obtained were processed statistically using bifactoral dispersion analysis ANOVA.
189 RESULTS
One hour after i.p. administration of testosterone (1 mg/kg) there was a significant hyperpolarization of the membranes of adrenocorticocytes of adult and old animals (Fig. 1). In response to testosterone administration the hyperpolarization of hepatocytes developed in adult rats only. In old animals, the MP level of hepatocytes remained unchanged. Pretreatment (30 min before testosterone) with inhibitors of protein synthesis actinomycin D (50/zg/kg) and cycloheximide (250 #g/kg) prevented the hyperpolarizing effect of the hormone upon both hepatocytes of adult animals and adrenocorticocytes of adult and old animals. A specific inhibitor of Na,KATPase, ouabain (73 #g/kg), also prevented the testosterone-induced hyperpolarization of plasmic membranes of hepatocytes and adrenocorticocytes. The blocker of K+-channels, 2-aminopyridine (10 mg/kg), produced no effect on the development of hyperpolarization of plasmic membranes (Fig. 1). The effect of testosterone on the MP level of adrenocorticocytes is thought to be linked with its direct effect upon the latter. This notion was supported by the results of experiments in which isolated adrenals of rats of different ages were in vitro incubated with testosterone. As is seen in Fig. 1, addition of testosterone to the medium in which isolated intact adrenals of animals removed from animals of both ages were incubated, produced significant hyperpolarization of the adrenocorticocytes membranes in both adult and old rats. A special series of experiments was undertaken to study the effect of testosterone upon the activity of Na,K-ATPase of rat hepatocytes and adrenocorticocytes. In the homogenate of adrenals and in the fraction of isolated plasmic membranes of hepatocytes, the basal Na,K-ATPase activity decreased from 8.0 and 11.1 nmol Pi/mg to 6.0 and 8.0 nmol Pi/mg in adult and old rats, respectively. Age-related differences have been established in the changes of Na,K-ATPase activity that occurred in cells of different type following testosterone administration. In adult rats, significant activation of Na,K-ATPase of hepatocytes (by 49%) and adrenocorticocytes (by 71%) was observed 1 h after i.p. testosterone (Fig. 2). In old rats, the activation of Na,K-ATPase was noted in adrenocorticocytes only, the degree being significantly higher in adult animals (Fig. 2). Testosterone in the dose used in this study did not activate Na,K-ATPase of old rat hepatocytes. Thus, the testosterone-induced hyperpolarization of plasmic membranes of adrenocorticocytes and hepatocytes was conditioned by the hormonal activation of Na,K-ATPase. Another series of experiments was carried out to study the effect of inhibitors of protein synthesis - - actinomycin D and cycloheximide - - on testosterone's effect upon the Na,K-ATPase activity. As is seen in Fig. 3, against the background Of pretreatment with one of the above inhibitors the testosterone produced no activation of Na,K-ATPase. The above effects are thought not to result from the direct
190
Adult mV
Adrenals
mV
1~
Liver
6O
59 #I
58 57 56 55 5@ 53 52
'
1 hour
36
0
1 hour
Old
60
mV
Adrenals
,1~
43
mV
Liver
42
58
2~ 41 40 39 38 37
52
1
0
1 hour
36 0
1 !
1 hour
Fig. 1. The size of membrane potential of hepatocytes and adrenocorticocytes from adult and old rats under testosterone action. 1, Under in vivo testosterone action (*, membrane potential significance changes in comparison with the control size, P < 0.05); 2, under in vitro testosterone action (*, membrane potential significance changes in comparison with the control size, P < 0.05); 3, under combined 2aminopiridine and testosterone action (*, membrane potential significance changes in comparison with the control size, P < 0.05); 4, under combined actinomycin D and testosterone action; 5, under combined cycloheximide and testosterone action; 6, under combined ouabaine and testosterone action.
effect u p o n the enzyme in view o f the fact t h a t neither testosterone, n o r a c t i n o m y c i n D n o r c y c l o h e x i m i d e p e r se d i d n o t affect the basal N a , K - A T P a s e activity when the fraction o f intact isolated m e m b r a n e s was i n c u b a t e d in vitro with each o f the inhibitors. The d a t a o b t a i n e d suggest t h a t the a c t i v a t i o n o f p r o t e i n synthesis in the cell cytosole resulted in the emergence o f a f a c t o r which a c t i v a t e d the N a , K - A T P a s e .
191
Adult nmol Pi/mg min
nmol Pi/mg min
25
20"
T T
2O
15.
15 10_
K
K
I0
5 5 0
0
Adrenals
Liver
Old nmol Pi/mg min
nmol Pi/mg min
20-
25 2O
1515 T
10-
IO
K
5-
T
K
5
0 Adenals
0
Liver
Fig. 2. The Na,K-ATPase activity hepatocytes and adrenocorticocytcs from adult and old rats under testosterone action. K, control rats; T, after testosterone injection (*, Na,K-ATPase activity significance changes in comparison with the control size, P < 0.05).
192 To check this assumption, we have performed a series of experiments, in which fractions of isolated plasmic membranes of hepatocytes of the intact rats were incubated in vitro with the cytosole of hepatocytes from testosterone-treated adult animals (Fig. 3). The cytosole of such animals is known to significantly activate the enzyme of isolated plasmic membranes of adult and old rats, whereas the cytosole of intact animals produces no effect upon the basal activity of Na,K-ATPase. Consequently, in old age the enzyme remains capable to adequately respond to the effect of activating factor. The cytosoles of hepatocytes of rats administered with one of
Adult membranes nmol Pi/mg min 25T
15-
i
T
m
20_
T
4~
TAd TAdTCg
K
II.F
lO_ 5
.I Membranes nmol Pi/mg min 25-
TCg
K
w
Adult cytosol
Adul' serum
Old membranes TS
2O TC 15
10
TC K
T,,,
K
5 O
Membranes
Incubation with old
TS
II
Incubation with adult
Fig. 3. Hepatocytes Na,K-ATPase activity under influence testosterone, protein biosynthesis inhibitors, hepatocyte cytosol and rat serum. K, control rats; T, testosterone-pretreatedrats (* Na,K-ATPase activity significance changes in comparison with the control size, P < 0.05); TAd, actimomycin D and testosterone-pretreated rats; TCg, cycloheheximide and testosterone-pretreated rats; TC, hepatocyte cytosole of testosterone-pretreatedrats (* Na,K-ATPase activity significancechanges in comparison with the control size, P < 0.05); TS, serum of testosterone-pretreated rats (* Na,K-ATPase activity changes in comparison with the control size, P < 0.05).
193
the above inhibitors 30 min prior to testosterone did not activate the Na,K-ATPase of intact plasmic membranes either (Fig. 3). Of substantial importance among testosterone's effects upon the cell are the membrane-genome mechanisms, which suffer during aging. Obviously, the old rats do not synthesize this factor in adequate quantities. This is evident from the fact that the cytosole of old rats did not activate the Na,K-ATPase of isolated plasmic membranes of old rats (Fig. 3). Apparently, lack of activation of Na,K-ATPase in the fraction of intact plasmic membranes of old rat hepatocytes at incubation with the hepatocyte cytosole and blood serum of testosterone-pretreated old animals was conditioned by the deficient synthesis of such a factor in aging. Na,K-ATPase-activating factor gets into the blood. Incubation of a fraction of isolated plasmic membranes of intact adult and old rats in the blood serum of adult testosterone-pretreated animals activated the enzyme compared with the incubation in the blood serum of intact adult rats (Fig. 3). Actinomycin D and cycloheximide, administered 30 min prior to testosterone, prevented the appearance of Na,KATPase-activating factor in the rat blood serum as well (Fig. 3). The blood serum of old rats did not alter the Na,K-ATPase activity of isolated plasmic membranes of intact old animals (Fig. 3). Thus, the administration of testosterone resulted in the synthesis in the cell of a factor, which activated Na,K-ATPase. This notion can be substantiated by the following. First, the hepatocyte cytosole and blood serum of insulin-treated adult animals activated Na,K-ATPase of isolated plasmic membranes of hepatocytes of intact rats. Second, inhibitors of protein synthesis prevented the hyperpolarization of membranes, the activation of Na,K-ATPase and the appearance of a factor in the hepatocyte cytosole and blood serum. The synthesis of testosterone-induced factor suffers with age and this can be substantiated by the following. First, there was no significant hyperpolarization of hepatocyte's membranes and activation of Na,K-ATPase in old rats against the background of testosterone effect. Second, the hepatocyte cytosole and blood serum of old rats lost their capacity to activate Na,K-ATPase of isolated plasmic membranes of the hepatocytes of intact old rats. At the same time, in old age the plasmic membrane per se retained its capacity to respond to hepatocyte cytosole and blood serum of adult rats. The activation of the ionic pump and the development of hyperpolarization are known to play a significant role in the cell reactivity, in the permeability of cell membranes and in the regulation of protein synthesis. The ageinduced weakening of synthesis of substances, which activate Na,K-ATPase and induce hyperpolarization, may be an important mechanism in the aging of a cell. REFERENCES 1 V.V. Frolkis, Is about hyperpolarization factor appearance under the activation of protein biosynthesis. Physiol. J., 26 (1980) 558-560.
194 V.V. Frolkis, Ageing Neurohumorai mechanisms, Kiev, 1981, 320 p. R. Ghanatian and K. Fortherby, Testosterone uptake by prostatic tissue from young and old rats. Gerontologia, 21 (1975) 211-215. 4 E.N. Gorban, The thyrotropin influence on membrane potential of thyrotrope gland: age peculiarities. Physiol. J., 24 (1979) 395-401. 5 S.M. Harman and P.D. Tsitouras, Reproductive hormones in ageing and men. 1. Measurement of sex steroids, basal luteinizing hormone and Leyding cell response to human horionic gonadotropin. J. Clin. Endocrinol. Metab., 51 (1980) 35-40. 6 P.G. Kostuk, Microelectrode tecniks, Kiev, 1960. 7 O.H. Lowry, N.J. Rosenbrouch, A.L. Farr and R.G. Randall, Protein measurement with the Pholin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 8 E.K. Mathews and M.S. Safran, Steroid production and membrane potential measurement in cells of the adrenal cortex. J. Physiol., 189 (1967) 149-161. 9 R.H. Michell and J.N. Hamthorn, The site of diphosphoinositide synthesis in rat liver. Biochem. Biophys. Res. Commun., 21 (1965) 333-338. 10 K. Nahoul and M. Roger, Age related decline of plasma bioactive testosterone in adult men. J. Steroid. Biochem. 35 (1990) 293-299. 11 R. Pietras and C. Szego, Membrane recognition and effector sites in steroid hormone action. Biochem. Actions Horm., 8 (1981) 307-363. 12 R.I. Potapenko, Acetyicholine and noradrenaline influence on synaptosomal membrane Na,KATPase activity from brain of adult and old rats. Neurochemistry, 5 (1986) 57-60. 2 3
