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Ecdysterone induces acetylcholinesterase in mammalian brain
0306-4492184$3.00+ 0.00 ,’ 1984 Pergamon Press Ltd
Camp. Biochem. f’h~siol. Vol. 78C, No. 1. pp. 193-195, 1984 Printed in Great Britain
ECDYSTERONE
R. E.
CATALAN’,
INDUCES ACETYLCHOLINESTERASE IN MAMMALIAN BRAIN M.
D. ARAGONES~,
J. E. GODLY*
and A. M.
MARTINEZ’
‘Departamento de Bioquimica y Biologia Molecular, Centro de Biologia Molecular (CSIC-UAM), Universidad Autbnoma de Madrid, Madrid-34, Spain and 2Departamento de Bioquimica, Colegio Universitario Arcos de Jal6n, Madrid-17, Spain
(Received 4 August 1983) Abstract-l. The effects of ecdysterone on brain acetylcholinesterase of both sexes have been studied in in citro conditions.
(AChE)
in immature
and adult rats
2. Ecdysterone produced an increase of AChE in rat brain slices. The most remarkable effect was found in immature male rats. In vitro assay using a purified AChE from electric eel showed no effect. 3. Pretreatment with cycloheximide or actinomycin D abolished the ecdysterone action on brain AChE. 4. These results suunort the idea that induction of AChE may be involved in the heterophilic actlon .. of ecdysterone.
INTRODUCTION
MATERIALS
The concept of hormonal heterophily, that invertebrate hormones have some effects on metabolic and molecular events in vertebrates, has been reviewed before (Burdette, 1974). Thus, the heterophilic action of some insect-metamorphosing steroids has been described and a certain degree of regulation by these steroids on the protein, lipid and carbohydrate metabolism has been suggested. More recently, we have demonstrated that a close relationship exists between ecdysterone and the cyclic AMP-protein kinase system in some mammalian tissues (Catalin et al., 1979a; Catalan et al., 1979b; Catalin et al., 1980; Catalan et al., 1982a; Catalin et al., 1982b). On the other hand, it has been suggested that cholinergic neuronal functions can be regulated by steroid hormones. Thus, 17-P-estradiol increases the activity of acetylcholinesterase (AChE; EC 3.1.1.7) in vivo, in several female rat brain areas (Iramain et al., 1980), as well as in rats of various ages (Moudgil and Kanungo, 1973). In addition, testosterone and estradiol increase the AChE and choline acetyltransferase of cerebral hemisphere of male rats of various ages (James and Kanungo, 1980). With the knowledge that steroid hormones are effective in regulating AChE, we became interested in investigating the possibility that ecdysterone also can influence this enzyme in the mammalian brain. This assumption can be further supported by the fact that ecdysterone induces AChE in Drosophila melanogaster cell-line (Cherbas et al., 1977; Best-Belpomme and Courgeon, 1977) and in Aedes aegypti cell-line (Cohen, 198 1). The present study was undertaken to investigate the heterophilic action of ecdysterone on AChE mammalian brain. The possibility that the effect of ecdysterone on AChE activity may be age- or sexdependent was tested by using immature and adult rats of both sexes. We have also investigated the possible resemblances between ecdysterone and sex hormones, in an attempt to extend the understanding of ecdysterone heterophilic action. c BP 7*11c M
AND METHODS
Animals Immature (5-week-old) and adult (20-week-old) albino rats of Wistar strain were used throughout these experiments. Animals were maintained on a standard laboratory diet with water ad libitum and housed in temperature and humidity controlled quarters with 14 hr light and 10 hr dark.
Incubation conditions Rat cerebral cortex slices were prepared and incubated in Krebs-Ringer-bicarbonate buffer (pH 7.4) as previously described (CatalBn et al., 1979~) with the tested steroids at the appropriate concentrations. The ecdysterone was dissolved in 0.9% NaCl and the other steroids in a methanolethylene glycol (1: 3, v/v) mixture. Controls were handled in the same way except that saline or ethanol-ethylene glycol was added instead of ecdysterone or sex hormones. The incubations were terminated by the collection of slices and their immediate homogenization in 155 mM uhosohate buffer @H 8) in a proportion of 1% (w/v). Aliiuots’were taken for AChE assay and protein determination. Experiments for &dying the involvement of protein synthesis in the ecdysterone action were carried out by preincubating the slices in the presence of actinomycin D (8 x 1O-6 M) or cycloheximide (10-j M) at 37°C for 30 min prior to the steroid treatment. In vitro experiments were also done by using a purified AChE from electric eel (Electrophorus electricus). A 0.05 pg sample of the enzyme was preincubated in 155 mM phosphate buffer (pH 8) with ecdysterone at the aourooriate concentrations to make a total volume of 1ml for 30 min. The substrate was added and the AChE assay was performed. I.
.
Analytical determinations AChE activity was measured essentially by the method of Ellman et al. (1961) as described (Catalin et al., 1981). Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard.
Statistical analysis Statistical significance of the experimental data was carried out with the Students t-test. A probability of 0.05 was used as the significant level. All results are expressed as the mean *SD for G3 determinations. 193
R. E. CATALAN et al.
194 Table
1. Effect of ecdysterone
on acetylcholinesterase
AChE 5 week Ecdysterone
Female
0 3 x IO-‘M 3 x lo-‘M 3 x lo-‘M
62.2 15.9 16.5 19.6
Assay conditions as described *P < 0.01, **P < 0.05.
k + + k
(AChE)
(nmol/mg
4.4 17.0 9.3’* 5.9**
estradiol
in brain slices
protein/min) 20 week
Male 83.5 100.2 114.4 125.3
in the Materials
Table 2. Effect of testosterone,
activity
f f + f
Female
13.9 -7.0** 14.8’ 23.8*
and Methods
Male
f 3.2 f 6.2 f 9.1 f7.8
on acetylcholinesterase (nmol/mg
Control Testosterone 3 x lo-‘M 3 x 10m6 M 3 x IO-‘M Estradiol 3 x lo-‘M 3 x 1O-6 M 3 x IO-‘M Progesterone 3 x lo-‘M 3 x 10-6M 3 x 1O-5 M
f f f f
3.2 5.8” 7.8** 11.7*
(AChE)
in brain slices
protein/min) 20 week
5 week Treatment
59.3 68.2 70.6 77.8
section. The data are given as mea” f SD (N = 5).
and progesterone AChE activity
42.3 40.0 45.9 51.1
Female
Male
Female
Male
62.2 + 4.4
83.5 f 13.9
4i.3 f 3.2
59.3 f 3.2
59.4 f 13.5 65.1 + 9.8 70.2+ II.4
93.5 f IO.8 99.4 f 5.1** 113.5 f 13.2’
35.0 f 13.5 34.5 f 9.2 58.2 ?r 3.7*
64.3 f 6.7 60.6 + 9.1 67.9 f 10.7
65.9 + 12.3 63.8 i 5.9 70.1 * 11.1
90.7 i 8.7 103.0 * 10.5 121.1 i 12.5’
51.0 + 13.3 49.8 + 7.8 56.7 + 13.0*’
72.3 + 8.8*’ 17.1 + 7.5* 89.0 + 12.9’
73.4 i 9.3 69.7 & 6.5 75.9 f 12.0”
93.5 i 7.9 88.5 + 8.4 99.1* 7.3”
48.8 + 10.1 45.9 ? 4.3 53.3 f 10.1”
65.2 + 4.7’* 70.6 7 6.7’ 72.9f8.1’
Assav conditions as described ;P < 0.01, **p < 0.05.
in the Materials and Methods section. The data are given as mean f SD (N = 5).
Reagents
Table
Ecdysterone, testosterone, 17-b-estradiol acetate, progesterone, 5,5’-dithio(bis)dinitrobenzoic acid, cycloheximide, actinomycin D and AChE from electric organ of Electrophorus electricus were obtained from Sigma Chemical Co., St. Louis, U.S.A. Acetylthiocholine iodide was obtained from Merck Co., Darmstadt, F.R.G. All other reagents were of the highest analytical grade available.
RESULTS
The variations in the AChE activity in cerebral cortex slices from immature and adult rats of both sexes after administration of ecdysterone are given in Table I. Ecdysterone was able to stimulate significantly AChE activity in immature as well as adult male rats in a dose-dependent manner in a range from 3 x lo-’ to 3 x 10m5M. Immature female rats were less sensitive to the ecdysterone action while adult females were totally unresponsive. To compare the ecdysterone action with that of other steroid hormones, experiments were performed with testosterone, estradiol and progesterone, in the same experimental conditons. The results obtained are summarized in Table 2. AChE from immature male rats was sensitive to the action of the three hormones tested, especially to the testosterone action. On the other hand, in female rats of the same age, only progesterone caused a significant effect. Estradiol as well as progesterone evoked an increase of AChE in adult rats of both sexes, whereas testosterone caused a similar effect only in female rats. To acquire a greater knowledge of the mechanism by which ecdysterone affects the brain AChE, two kinds of experiments were also carried out. In the first, a purified enzyme from electric eel was assayed in the presence of ecdysterone at the concentrations
3. Effect of ecdysterone on a purified acetylcholinesterase fAChEj from electric eel AChE activity (pmol/mg protein/min)
Treatment Control Ecdysterone
3 x lo-‘M 3 x 10-6M 3 x IO-sM
50.3 46.8 51.4 55.1
f f + *
5.4 6.1 8.3 3.4
Assay conditions as described in the Materials and Methods The data are given as mea” f SD (N = 5).
section.
described above. The results are shown in Table 3. The enzyme was found unaffected by ecdysterone in the range of concentrations tested. To learn whether the ecdysterone action on brain AChE represents enhanced enzyme synthesis, we performed a second experiment in the presence of cycloheximide or actinomycin D. As can be seen in Table 4, either cycloheximide or actinomycin D abolished the ecdysterone effect. DISCUSSION
In previous reports (Catalan et al., 1979a; Catalan et al., 1979b; Catalan et al., 1980; Catalan et al., 1982a; Catalan et al., 1982b) we have indicated that ecdysterone can exert some effects on mammalian metabolic systems. Granting that the steroid hor-
Table 4. Effect of ecdysterone on acetylcholinesterase (AChE) in brain slices in the presence of cycloheximide or actinomycin D Treatment Control Ecdysterone Ecdysterone Ecdysterone
3 x lo-’ M + Cycloheximide + Actinomycin D
AChE activity (nmol/mg protein/min) 72.5 106.6 76.0 74.3
f f + *
8.2 13.0’ 6.2 10.1
Assay conditions as described in the Materials and Methods section. The data are given as mean k SD (N = 5). *P < 0.01.
Ecdysterone induces acetylcholinesterase manes are known as regulators of brain AChE (Iramain et al., 1980; James and Kanungo, 1980; Moudgil and Kanungo, 1973) we sought to find out whether ecdysterone has a similar effect on AChE in mammalian brain. The results reported here indicate that ecdysterone is capable of increasing brain AChE, in the same way that the increase occurs in insects (Cherbas et al., 1977; Best-Belpomme and Courgeon, 1977; Cohen, 1981). This fact is not surprising since it has been described that the molecular structure of invertebrate AChE is quite analogous to the vertebrate type (Morton and Singh, 1980). We must point out that the effect of ecdysterone is greater in immature than in adult rats. This fact would seem to suggest that the steroid hormone receptors are susceptible to ecdysterone in the immature rats. These results are in agreement with those reported by Csaba et al. (1978) in newborn rats and with those of Ashburner (1980) who indicated that ecdysteroid receptors were similar to those for oestrogen or progesterone in vertebrates. Comparison of ecdysterone with sex hormones suggests that the invertebrate hormone behaves in a manner similar to that of progesterone in immature rats, due to the fact that this is the only sex hormone that affects the AChE either in immature male or female rats. Furthermore, the lack of effect showed by ecdysterone in adult rats suggests that in adult animals, the receptors are completely formed and they are unable to recognize alien steroids. Among the mechanisms which may explain the ecdysterone action on AChE, the following may be considered: a direct effect on AChE catalytic properties or an effect through stimulation of enzyme synthesis. The first possibility is a rather unlikely mechanism since no effect was observed on a purified enzyme. However, this consideration deserves further studies due to the source of the purified enzyme. In pursuing the second approach outlined above, we have presented here evidence that the ecdysterone action is due to an increase of the enzyme synthesis at the transcription and translation levels. These findings agree with the general fact that the effect of steroid hormones is mediated through a specific cytoplasmatic receptor protein which binds the hormone to form an activated steroid-receptor complex. It is then translocated to the nucleus where it binds to specific chromosomal sites, causing specific changes in gene expression. Our studies indicate that the changes in the AChE induced by estradiol, progesterone and testosterone generally agree with those of other researchers (Iramain et al., 1980; James and Kanungo, 1980; Moudgil and Kanungo, 1973) who have stated that the intraperitoneal administration of steroid hormones stimulates the brain AChE. Discrepancies found may be due to different experimental conditions. Thus, in in vivo conditions the effect of these hormones might be due to their metabolites, as suggested previously (James and Kanungo, 1978). On the other hand, our results confirm previous data (James and Kanungo, 1978; Moudgil and Kanungo, 1973) which demonstrate that AChE activity is higher in immature than in adult rats, probably due to higher metabolism of acetylcholine associated with learning.
195
The outcomes of these and related investigations accomplished in our laboratory may represent an important advance in understanding the heterophilic action of ecdysterone. Acknowledgements-The skilful technical assistance of MS B. G. Miguel, Mr A. Robles, Mr J. E. Godoy and Mr J. Blizquez is gratefully appreciated. This work was supported in part by grants from the Fondo de Investigaciones Sanitarias. REFERENCES Ashburner M. (1980) Chromosomal action of ecdysterone. Nuturc, Lond. 285, 435-436. Best-Belpomme M. S. and Courgeon A. M. (1977) Ecdysterone and acetylcholinesterase activity in cultured Drosophila cells. FEBS Lett. 82, 345-347. Burdette W. J. (1974) Invertebrate Endocrinology and Hormonal Heterophily. Springer-Verlag, New York. Catalin R. E., Aragones M. D. and Martinez A. M. (1979a) Effect of ecdvsterone on cvclic AMP and cvclic GMP in mouse plasma. Biochem.. biophys. Res. &ommun. 87, 1018-1023. Catalan R. E., Aragones M. D. and Martinez A. M. (1979b) Effect of ecdysterone on the cyclic AMP-protein kinase system in mouse liver. Biochem. biophys. Res. Commun. 89, 44-49. Catalan R. E., Aragones M. D. and Martinez A. M. (1979~) Somatostatin effect on cyclic AMP and cyclic GMP levels in rat brain. Biochim. biophys. Acta 586, 213-216. Catalan R. E., Martinez A. M. and Aragones M. D. (1980) Heterophylic action of ecdysterone. Effect on the cyclic AMP-protein kinase system in mouse. Naturwissenschafien 67, 520. Catalan R. E., Martinez A. M., Mata F. and Aragones M. D. (1981) Effect of insulin on acetylcholinesterase activity. Biochem. biophys. Res. Commun. 101, 121~1220. Catalan R. E., Martinez A. M. and Aragones M. D. (1982a) In oitro effect of ecdysterone on protein kinase activity. Comp. Biochem. Physiol. 71B, 301-303. Catalan R. E., Martinez A. M., Aragones M. D., Miguel B. G., Robles A. and Godoy J. E. (1982b) Effect of ecdysterone treatment on the cyclic AMP-protein kinase system in adipose tissue. J. Steroid Biochem. 16, 573-576. Cherbas P., Cherbas L. and Williams C. M. (1977) Induction of acetylcholinesterase activity by p-ecdysone in a Drosophila cell line. Science 197, 275-277. Cohen E. (1981) Acetylcholinesterase activity in Aedes aegypri cell line. Experientia 37, 429430. Csaba G., Darvas Z., Laszlo V., Juvancz I., Vragha P., Bodrogi L. and Feher T. (1978) Long-lastine effect of single ecdysone injections in newborn rats. Biol. Neonate 33, 170-173. Ellman G. L., Courtney K. D., Andres V. Jr. and Featherstone R. H. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmat. 7, 88-95. Iramain C. A., Owasoyo J. 0. and Egbunike G. N. (1980) Influence of estradiol on acetylcholinesterase activity in several female rat brain areas and adenohypophysis. Neurosci. Lett. 16, 81-84. James T. C. and Kanungo M. S. (1978) Effect of sex steroids on choline acetyltransferase and acetylcholinesterase of cerebral hemisphere of male rats of various ages. Biochim. biophys. Acta 538, 205-2 11. Lowry 0. H., Rosebrough R. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Morton R. A. and Singh R. S. (1980) Variation in Drosophila acetylcholinesterase. Biochem. Genetics l&439-454. Moudgil V. K. and Kanungo M. S. (1973) Induction of acetylcholinesterase by 17-p-estradiol in the brain of rats of various ages. Biochem. biophys. Res. Commun. 52, 725-730.
Camp. Biochem. f’h~siol. Vol. 78C, No. 1. pp. 193-195, 1984 Printed in Great Britain
ECDYSTERONE
R. E.
CATALAN’,
INDUCES ACETYLCHOLINESTERASE IN MAMMALIAN BRAIN M.
D. ARAGONES~,
J. E. GODLY*
and A. M.
MARTINEZ’
‘Departamento de Bioquimica y Biologia Molecular, Centro de Biologia Molecular (CSIC-UAM), Universidad Autbnoma de Madrid, Madrid-34, Spain and 2Departamento de Bioquimica, Colegio Universitario Arcos de Jal6n, Madrid-17, Spain
(Received 4 August 1983) Abstract-l. The effects of ecdysterone on brain acetylcholinesterase of both sexes have been studied in in citro conditions.
(AChE)
in immature
and adult rats
2. Ecdysterone produced an increase of AChE in rat brain slices. The most remarkable effect was found in immature male rats. In vitro assay using a purified AChE from electric eel showed no effect. 3. Pretreatment with cycloheximide or actinomycin D abolished the ecdysterone action on brain AChE. 4. These results suunort the idea that induction of AChE may be involved in the heterophilic actlon .. of ecdysterone.
INTRODUCTION
MATERIALS
The concept of hormonal heterophily, that invertebrate hormones have some effects on metabolic and molecular events in vertebrates, has been reviewed before (Burdette, 1974). Thus, the heterophilic action of some insect-metamorphosing steroids has been described and a certain degree of regulation by these steroids on the protein, lipid and carbohydrate metabolism has been suggested. More recently, we have demonstrated that a close relationship exists between ecdysterone and the cyclic AMP-protein kinase system in some mammalian tissues (Catalin et al., 1979a; Catalan et al., 1979b; Catalin et al., 1980; Catalan et al., 1982a; Catalin et al., 1982b). On the other hand, it has been suggested that cholinergic neuronal functions can be regulated by steroid hormones. Thus, 17-P-estradiol increases the activity of acetylcholinesterase (AChE; EC 3.1.1.7) in vivo, in several female rat brain areas (Iramain et al., 1980), as well as in rats of various ages (Moudgil and Kanungo, 1973). In addition, testosterone and estradiol increase the AChE and choline acetyltransferase of cerebral hemisphere of male rats of various ages (James and Kanungo, 1980). With the knowledge that steroid hormones are effective in regulating AChE, we became interested in investigating the possibility that ecdysterone also can influence this enzyme in the mammalian brain. This assumption can be further supported by the fact that ecdysterone induces AChE in Drosophila melanogaster cell-line (Cherbas et al., 1977; Best-Belpomme and Courgeon, 1977) and in Aedes aegypti cell-line (Cohen, 198 1). The present study was undertaken to investigate the heterophilic action of ecdysterone on AChE mammalian brain. The possibility that the effect of ecdysterone on AChE activity may be age- or sexdependent was tested by using immature and adult rats of both sexes. We have also investigated the possible resemblances between ecdysterone and sex hormones, in an attempt to extend the understanding of ecdysterone heterophilic action. c BP 7*11c M
AND METHODS
Animals Immature (5-week-old) and adult (20-week-old) albino rats of Wistar strain were used throughout these experiments. Animals were maintained on a standard laboratory diet with water ad libitum and housed in temperature and humidity controlled quarters with 14 hr light and 10 hr dark.
Incubation conditions Rat cerebral cortex slices were prepared and incubated in Krebs-Ringer-bicarbonate buffer (pH 7.4) as previously described (CatalBn et al., 1979~) with the tested steroids at the appropriate concentrations. The ecdysterone was dissolved in 0.9% NaCl and the other steroids in a methanolethylene glycol (1: 3, v/v) mixture. Controls were handled in the same way except that saline or ethanol-ethylene glycol was added instead of ecdysterone or sex hormones. The incubations were terminated by the collection of slices and their immediate homogenization in 155 mM uhosohate buffer @H 8) in a proportion of 1% (w/v). Aliiuots’were taken for AChE assay and protein determination. Experiments for &dying the involvement of protein synthesis in the ecdysterone action were carried out by preincubating the slices in the presence of actinomycin D (8 x 1O-6 M) or cycloheximide (10-j M) at 37°C for 30 min prior to the steroid treatment. In vitro experiments were also done by using a purified AChE from electric eel (Electrophorus electricus). A 0.05 pg sample of the enzyme was preincubated in 155 mM phosphate buffer (pH 8) with ecdysterone at the aourooriate concentrations to make a total volume of 1ml for 30 min. The substrate was added and the AChE assay was performed. I.
.
Analytical determinations AChE activity was measured essentially by the method of Ellman et al. (1961) as described (Catalin et al., 1981). Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard.
Statistical analysis Statistical significance of the experimental data was carried out with the Students t-test. A probability of 0.05 was used as the significant level. All results are expressed as the mean *SD for G3 determinations. 193
R. E. CATALAN et al.
194 Table
1. Effect of ecdysterone
on acetylcholinesterase
AChE 5 week Ecdysterone
Female
0 3 x IO-‘M 3 x lo-‘M 3 x lo-‘M
62.2 15.9 16.5 19.6
Assay conditions as described *P < 0.01, **P < 0.05.
k + + k
(AChE)
(nmol/mg
4.4 17.0 9.3’* 5.9**
estradiol
in brain slices
protein/min) 20 week
Male 83.5 100.2 114.4 125.3
in the Materials
Table 2. Effect of testosterone,
activity
f f + f
Female
13.9 -7.0** 14.8’ 23.8*
and Methods
Male
f 3.2 f 6.2 f 9.1 f7.8
on acetylcholinesterase (nmol/mg
Control Testosterone 3 x lo-‘M 3 x 10m6 M 3 x IO-‘M Estradiol 3 x lo-‘M 3 x 1O-6 M 3 x IO-‘M Progesterone 3 x lo-‘M 3 x 10-6M 3 x 1O-5 M
f f f f
3.2 5.8” 7.8** 11.7*
(AChE)
in brain slices
protein/min) 20 week
5 week Treatment
59.3 68.2 70.6 77.8
section. The data are given as mea” f SD (N = 5).
and progesterone AChE activity
42.3 40.0 45.9 51.1
Female
Male
Female
Male
62.2 + 4.4
83.5 f 13.9
4i.3 f 3.2
59.3 f 3.2
59.4 f 13.5 65.1 + 9.8 70.2+ II.4
93.5 f IO.8 99.4 f 5.1** 113.5 f 13.2’
35.0 f 13.5 34.5 f 9.2 58.2 ?r 3.7*
64.3 f 6.7 60.6 + 9.1 67.9 f 10.7
65.9 + 12.3 63.8 i 5.9 70.1 * 11.1
90.7 i 8.7 103.0 * 10.5 121.1 i 12.5’
51.0 + 13.3 49.8 + 7.8 56.7 + 13.0*’
72.3 + 8.8*’ 17.1 + 7.5* 89.0 + 12.9’
73.4 i 9.3 69.7 & 6.5 75.9 f 12.0”
93.5 i 7.9 88.5 + 8.4 99.1* 7.3”
48.8 + 10.1 45.9 ? 4.3 53.3 f 10.1”
65.2 + 4.7’* 70.6 7 6.7’ 72.9f8.1’
Assav conditions as described ;P < 0.01, **p < 0.05.
in the Materials and Methods section. The data are given as mean f SD (N = 5).
Reagents
Table
Ecdysterone, testosterone, 17-b-estradiol acetate, progesterone, 5,5’-dithio(bis)dinitrobenzoic acid, cycloheximide, actinomycin D and AChE from electric organ of Electrophorus electricus were obtained from Sigma Chemical Co., St. Louis, U.S.A. Acetylthiocholine iodide was obtained from Merck Co., Darmstadt, F.R.G. All other reagents were of the highest analytical grade available.
RESULTS
The variations in the AChE activity in cerebral cortex slices from immature and adult rats of both sexes after administration of ecdysterone are given in Table I. Ecdysterone was able to stimulate significantly AChE activity in immature as well as adult male rats in a dose-dependent manner in a range from 3 x lo-’ to 3 x 10m5M. Immature female rats were less sensitive to the ecdysterone action while adult females were totally unresponsive. To compare the ecdysterone action with that of other steroid hormones, experiments were performed with testosterone, estradiol and progesterone, in the same experimental conditons. The results obtained are summarized in Table 2. AChE from immature male rats was sensitive to the action of the three hormones tested, especially to the testosterone action. On the other hand, in female rats of the same age, only progesterone caused a significant effect. Estradiol as well as progesterone evoked an increase of AChE in adult rats of both sexes, whereas testosterone caused a similar effect only in female rats. To acquire a greater knowledge of the mechanism by which ecdysterone affects the brain AChE, two kinds of experiments were also carried out. In the first, a purified enzyme from electric eel was assayed in the presence of ecdysterone at the concentrations
3. Effect of ecdysterone on a purified acetylcholinesterase fAChEj from electric eel AChE activity (pmol/mg protein/min)
Treatment Control Ecdysterone
3 x lo-‘M 3 x 10-6M 3 x IO-sM
50.3 46.8 51.4 55.1
f f + *
5.4 6.1 8.3 3.4
Assay conditions as described in the Materials and Methods The data are given as mea” f SD (N = 5).
section.
described above. The results are shown in Table 3. The enzyme was found unaffected by ecdysterone in the range of concentrations tested. To learn whether the ecdysterone action on brain AChE represents enhanced enzyme synthesis, we performed a second experiment in the presence of cycloheximide or actinomycin D. As can be seen in Table 4, either cycloheximide or actinomycin D abolished the ecdysterone effect. DISCUSSION
In previous reports (Catalan et al., 1979a; Catalan et al., 1979b; Catalan et al., 1980; Catalan et al., 1982a; Catalan et al., 1982b) we have indicated that ecdysterone can exert some effects on mammalian metabolic systems. Granting that the steroid hor-
Table 4. Effect of ecdysterone on acetylcholinesterase (AChE) in brain slices in the presence of cycloheximide or actinomycin D Treatment Control Ecdysterone Ecdysterone Ecdysterone
3 x lo-’ M + Cycloheximide + Actinomycin D
AChE activity (nmol/mg protein/min) 72.5 106.6 76.0 74.3
f f + *
8.2 13.0’ 6.2 10.1
Assay conditions as described in the Materials and Methods section. The data are given as mean k SD (N = 5). *P < 0.01.
Ecdysterone induces acetylcholinesterase manes are known as regulators of brain AChE (Iramain et al., 1980; James and Kanungo, 1980; Moudgil and Kanungo, 1973) we sought to find out whether ecdysterone has a similar effect on AChE in mammalian brain. The results reported here indicate that ecdysterone is capable of increasing brain AChE, in the same way that the increase occurs in insects (Cherbas et al., 1977; Best-Belpomme and Courgeon, 1977; Cohen, 1981). This fact is not surprising since it has been described that the molecular structure of invertebrate AChE is quite analogous to the vertebrate type (Morton and Singh, 1980). We must point out that the effect of ecdysterone is greater in immature than in adult rats. This fact would seem to suggest that the steroid hormone receptors are susceptible to ecdysterone in the immature rats. These results are in agreement with those reported by Csaba et al. (1978) in newborn rats and with those of Ashburner (1980) who indicated that ecdysteroid receptors were similar to those for oestrogen or progesterone in vertebrates. Comparison of ecdysterone with sex hormones suggests that the invertebrate hormone behaves in a manner similar to that of progesterone in immature rats, due to the fact that this is the only sex hormone that affects the AChE either in immature male or female rats. Furthermore, the lack of effect showed by ecdysterone in adult rats suggests that in adult animals, the receptors are completely formed and they are unable to recognize alien steroids. Among the mechanisms which may explain the ecdysterone action on AChE, the following may be considered: a direct effect on AChE catalytic properties or an effect through stimulation of enzyme synthesis. The first possibility is a rather unlikely mechanism since no effect was observed on a purified enzyme. However, this consideration deserves further studies due to the source of the purified enzyme. In pursuing the second approach outlined above, we have presented here evidence that the ecdysterone action is due to an increase of the enzyme synthesis at the transcription and translation levels. These findings agree with the general fact that the effect of steroid hormones is mediated through a specific cytoplasmatic receptor protein which binds the hormone to form an activated steroid-receptor complex. It is then translocated to the nucleus where it binds to specific chromosomal sites, causing specific changes in gene expression. Our studies indicate that the changes in the AChE induced by estradiol, progesterone and testosterone generally agree with those of other researchers (Iramain et al., 1980; James and Kanungo, 1980; Moudgil and Kanungo, 1973) who have stated that the intraperitoneal administration of steroid hormones stimulates the brain AChE. Discrepancies found may be due to different experimental conditions. Thus, in in vivo conditions the effect of these hormones might be due to their metabolites, as suggested previously (James and Kanungo, 1978). On the other hand, our results confirm previous data (James and Kanungo, 1978; Moudgil and Kanungo, 1973) which demonstrate that AChE activity is higher in immature than in adult rats, probably due to higher metabolism of acetylcholine associated with learning.
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The outcomes of these and related investigations accomplished in our laboratory may represent an important advance in understanding the heterophilic action of ecdysterone. Acknowledgements-The skilful technical assistance of MS B. G. Miguel, Mr A. Robles, Mr J. E. Godoy and Mr J. Blizquez is gratefully appreciated. This work was supported in part by grants from the Fondo de Investigaciones Sanitarias. REFERENCES Ashburner M. (1980) Chromosomal action of ecdysterone. Nuturc, Lond. 285, 435-436. Best-Belpomme M. S. and Courgeon A. M. (1977) Ecdysterone and acetylcholinesterase activity in cultured Drosophila cells. FEBS Lett. 82, 345-347. Burdette W. J. (1974) Invertebrate Endocrinology and Hormonal Heterophily. Springer-Verlag, New York. Catalin R. E., Aragones M. D. and Martinez A. M. (1979a) Effect of ecdvsterone on cvclic AMP and cvclic GMP in mouse plasma. Biochem.. biophys. Res. &ommun. 87, 1018-1023. Catalan R. E., Aragones M. D. and Martinez A. M. (1979b) Effect of ecdysterone on the cyclic AMP-protein kinase system in mouse liver. Biochem. biophys. Res. Commun. 89, 44-49. Catalan R. E., Aragones M. D. and Martinez A. M. (1979~) Somatostatin effect on cyclic AMP and cyclic GMP levels in rat brain. Biochim. biophys. Acta 586, 213-216. Catalan R. E., Martinez A. M. and Aragones M. D. (1980) Heterophylic action of ecdysterone. Effect on the cyclic AMP-protein kinase system in mouse. Naturwissenschafien 67, 520. Catalan R. E., Martinez A. M., Mata F. and Aragones M. D. (1981) Effect of insulin on acetylcholinesterase activity. Biochem. biophys. Res. Commun. 101, 121~1220. Catalan R. E., Martinez A. M. and Aragones M. D. (1982a) In oitro effect of ecdysterone on protein kinase activity. Comp. Biochem. Physiol. 71B, 301-303. Catalan R. E., Martinez A. M., Aragones M. D., Miguel B. G., Robles A. and Godoy J. E. (1982b) Effect of ecdysterone treatment on the cyclic AMP-protein kinase system in adipose tissue. J. Steroid Biochem. 16, 573-576. Cherbas P., Cherbas L. and Williams C. M. (1977) Induction of acetylcholinesterase activity by p-ecdysone in a Drosophila cell line. Science 197, 275-277. Cohen E. (1981) Acetylcholinesterase activity in Aedes aegypri cell line. Experientia 37, 429430. Csaba G., Darvas Z., Laszlo V., Juvancz I., Vragha P., Bodrogi L. and Feher T. (1978) Long-lastine effect of single ecdysone injections in newborn rats. Biol. Neonate 33, 170-173. Ellman G. L., Courtney K. D., Andres V. Jr. and Featherstone R. H. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmat. 7, 88-95. Iramain C. A., Owasoyo J. 0. and Egbunike G. N. (1980) Influence of estradiol on acetylcholinesterase activity in several female rat brain areas and adenohypophysis. Neurosci. Lett. 16, 81-84. James T. C. and Kanungo M. S. (1978) Effect of sex steroids on choline acetyltransferase and acetylcholinesterase of cerebral hemisphere of male rats of various ages. Biochim. biophys. Acta 538, 205-2 11. Lowry 0. H., Rosebrough R. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Morton R. A. and Singh R. S. (1980) Variation in Drosophila acetylcholinesterase. Biochem. Genetics l&439-454. Moudgil V. K. and Kanungo M. S. (1973) Induction of acetylcholinesterase by 17-p-estradiol in the brain of rats of various ages. Biochem. biophys. Res. Commun. 52, 725-730.