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Effects of steroid hormones on synaptosomal ectonucleotidase activities from hippocampus and cortex of adult female rats
General and Comparative Endocrinology 140 (2005) 94–100 www.elsevier.com/locate/ygcen
EVects of steroid hormones on synaptosomal ectonucleotidase activities from hippocampus and cortex of adult female rats Bárbara Rückera,¤, Daniela Pochmanna, Cristina Ribas Fürstenaua, Marcela Sorelli Carneiro-Ramosb, Ana Maria Oliveira Battastinia, Maria Luiza M. Barreto-Chavesc, João José Freitas Sarkisa a
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil c Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil Received 1 March 2004; revised 1 October 2004; accepted 12 October 2004 Available online 24 November 2004
Abstract Over the last few years, the eVects of steroid hormones on the brain have been intensively discussed. It has been demonstrated that ATP (acting as a neurotransmitter) is hydrolyzed to adenosine in the synaptic cleft by the conjugated action of ectonucleotidases, which include an enzyme of the E-NTPDase family (NTPDase3, apyrase, EC 3.6.1.5) and a 5⬘-nucleotidase (EC 3.1.3.5). The 5⬘nucleotidase enzyme is able to hydrolyze AMP as well as other monophosphate nucleotides. The importance of this enzyme in the central nervous system is to participate in the adenosine formation, a nucleoside with neuroprotective properties and modulatory eVects. However, several questions have been raised about the mechanisms of steroid hormones and the possible neuroprotective eVects of estrogen. Thus, we examined the eVects of gonadal steroid hormone deprivation, induced by ovary removal (OVX) and estradiol replacement therapy, on the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult rats. ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus did not change as a function of OVX and results demonstrated an increase in AMP hydrolysis (82%) in the animals submitted to OVX in cerebral cortex, but not in hippocampus, when compared to control and sham-operated groups. Estradiol replacement therapy reversed this eVect. RT-PCR analysis showed that the enhancement of enzyme activity in cerebral cortex could be explained by the higher expression of 5⬘-nucleotidase, following OVX. The hormones 17-estradiol (cyclodextrin-encapsulated 17-estradiol), DHEAS, and pregnenolone (1.0, 2.5, and 5.0 M) did not alter the nucleotide hydrolysis, in vitro, in synaptosomes from cortex and hippocampus of female adult rats. Results presented, herein, should be considered relevant for hormone replacement therapy, since much controversy exists surrounding this area and the relationship between adenosine and sex steroids is still poorly understood. 2004 Elsevier Inc. All rights reserved. Keywords: Adenosine; Steroid hormones; Ectonucleotidases; OVX; ERT
1. Introduction In recent years, an increasing number of studies have been performed demonstrating the role of steroid hor-
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Corresponding author. Fax: +55 51 33165535. E-mail address: [email protected] (B. Rücker).
0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.10.008
mones in several physiological and pathological responses (Garcia-Segura et al., 2000; Valverde and Parker, 2002). Estrogen has been associated with a decreased risk, delayed onset and progression, or enhanced recovery from numerous traumatic or chronic neurological and mental diseases (Garcia-Segura et al., 2000). The eYcacy of estrogen has been shown in several models of neurodegeneration and ischemic injury in vivo
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and in vitro. In ovariectomized rats, physiological concentrations of estradiol, administered during hormone therapy, attenuated the extent of brain damage caused by permanent cerebral ischemia (Dubal et al., 1998; Harms et al., 2001). In contrast, several studies have shown conXicting data regarding the protective eVects of estrogen. Recently, studies have indicated a lack of protection or increased health risks of hormone replacement therapy (HRT) in the occurrence of cerebrovascular stroke (Viscoli et al., 2001), cardiovascular disease (Hulley et al., 1998), Alzheimer’s disease (Mulnard et al., 2001), and invasive breast cancer (Writing Group for the Women’s Health Initiative Investigators, 2002). It has been suggested that steroid hormone deprivation can modulate the expression and activity of an ectoATPase in synaptic plasma membranes of hippocampus and caudate nucleus from central nervous system of rats (Nedeljkovic et al., 2000). There is also increasing evidence to indicate that ecto-ATPase and ecto-ATP diphosphohydrolase are co-localized in the central nervous system (Kegel et al., 1997; Nedeljkovic et al., 2003). Ecto-enzymes, able to hydrolyze ATP and ADP, are present in the central nervous system of several species (Sarkis et al., 1995). ATP released by synapses can be hydrolyzed by ectonucleotidases in a highly sophisticated pathway composed of ecto-enzymes of the E-NTPDase family, including NTPDase3 (ATP diphosphohydrolase, ecto-apyrase, CD39, EC 3.6.1.5) and NTPDase2 (ectoATPase, EC 3.6.1.3), which can transform ATP and ADP to AMP (Bonan et al., 2001; Zimmermann, 2001). The AMP produced is subsequently hydrolyzed to adenosine by an ecto 5⬘-nucleotidase (CD73, EC 3.1.3.5), which constitutes the rate-limiting step in this pathway (Battastini et al., 1995; Sarkis and Saltó, 1991; Zimmermann, 1992); the Wnal product being the nucleoside adenosine, an important endogenous neuromodulator. Following ATP receptor activation, the signal is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, and this metabolic step leads to adenosine generation. Adenosine is involved in a diverse array of functions in the central nervous system and may play a number of roles in physiological and pathological conditions. In brain, adenosine levels are signiWcantly elevated by a wide array of pathological stimuli (Dunwiddie and Masino, 2001). Adenosine mediates its eVects through four types of G-protein-coupled receptors: A1, A2A, A2B, and A3 (Hauber and Bareiß, 2001). Since results from a previous study suggest that steroid hormone deprivation may have an eVect on the ecto-ATPase of synaptic plasma membrane obtained from hippocampus and caudate nucleus (CNS) (Nedeljkovic et al., 2000), we hypothesized that the eVect described could be mediated by: (a) a true ecto-ATPase: (b) an ATP diphosphohydrolase or (c) on both enzymes. It may be speculated that the eVect may depend on the ATP diphosphohydrolase enzymatic chain plus 5⬘-nucle-
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otidase and, thus, aVect the adenosine production in the central nervous system, particularly in view of the fact that adenosine is a neuroprotective structure. Thus, the possible eVects of these hormones on the enzyme ATP diphosphohydrolase (co-expressed with the ectoATPase) were investigated. In addition, the eVects of these hormones on 5⬘-nucleotidase, involved in the complete hydrolysis of ATP to adenosine, were also studied in the central nervous system synaptic cleft. With regard to the mechanism of action of steroid hormones and their possible neuroprotective eVects, much controversy still surrounds this area. Thus, we examined the eVects of gonadal steroid hormone deprivation, induced by removal of ovaries (ovariectomy) and estradiol replacement therapy, on the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of rats. In addition to their well-documented genomic eVects, some evidence exists to indicate that neuroactive steroids may act as potent modulators of the plasma membrane receptors, which may interact with diVerent eVector systems in neuronal membranes (Zylinska and Legutko, 1998; Zylinska et al., 1999). Therefore, we evaluated whether the hormones 17-estradiol (cyclodextrinencapsulated 17-estradiol), DHEAS, and pregnenolone were able to directly modulate the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult female rats, in vitro.
2. Methods 2.1. Chemicals Nucleotides, 17-estradiol (cyclodextrin-encapsulated 17-estradiol), dehydroepiandrosterone sulfate (5-androsten-3-ol-17-one sulfate), pregnenolone (5-pregnen3-ol-20-one), and -estradiol 3-benzoato were obtained from Sigma Chemical (St. Louis, MO, USA). All others reagents were of analytical grade. 2.2. Animals Female Wistar rats (aged 60–75 days; 200–250g) were used in this study. They were housed Wve to a cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on 7:00 a.m.) at a temperature of 23 §1°C. In all animals that were ovariectomized and received the pellet containing the hormone, 120mg/kg ketamine HCl (Dopalen: Agribrands, Campinas, SP, Brazil) and 16mg/kg xylazine (Anasedan: Agribrands) were used as anesthesia. Only animals in the diestrus state were used. 2.3. Hormone deprivation Animals were randomly divided into three groups: control group, the group corresponding to sham-oper-
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ated animals, and the group submitted to a bilateral ovariectomy (OVX) by removal of the glands through one abdominal incision. The sham-operated group was submitted only to abdominal incision, but not to removal of ovaries. The sham-operated and OVX groups were compared to the control group in order to characterize the eVects of surgery on the nucleotide hydrolysis. Three weeks after surgery, all groups were sacriWced by decapitation and the synaptosomes were prepared. 2.4. Estradiol replacement therapy Animals were randomly divided into three groups. A control group, a vehicle group (OVX + silastic pellets with oil), and a third group submitted to estradiol replacement therapy (ERT) (OVX + silastic pellet with -estradiol 3-benzoato). Pellets with 15 mm medical grade tubing (1.02 mm i.d. £ 2.16 mm o.d.; Medicone, Multiplast, Porto Alegre, RS, Brazil) were Wlled with 10 L of 5% (w/v) -estradiol 3-benzoato (Sigma, St. Louis, MO) in corn oil and sealed with silicone. These pellets were soaked in sterile saline overnight and implanted subcutaneously between the scapulae (Gamaro et al., 2003). Previous studies show that these pellets generate physiological serum levels of estradiol between 17 and 32 pg/mL, and measurements at 10 and 30 days post-implantation have shown no signiWcant change in circulating levels over these times (Brown et al., 1990; Luine et al., 1998). The pellets were implanted 2 weeks after OVX and, 3 weeks after the implant, the animals were sacriWced by decapitation and the synaptosomes prepared as described below. 2.5. Synaptosomes preparation Animals were sacriWced by decapitation and the brain structures (entire bilateral cortex and hippocampus) were removed to an ice-cold medium solution (320 mM sucrose, 5 mM Hepes, pH 7.5, and 0.1 mM EDTA). Structures were gently homogenized in Wve volumes of ice-cold medium solution with a motor-driven TeXonglass homogenizer. The synaptosomes were isolated as described previously by Nagy and Delgado-Escueta (1984). BrieXy, 0.5 mL of the crude mitochondrial fraction was mixed with 4.0 mL of 8.5% Percoll solution and layered onto an isoosmotic Percoll/sucrose discontinuous gradient (10/16%). The synaptosomes that banded at the 10/16% Percoll interface were collected with wide tip disposable plastic transfer pipettes. The synaptosomal fractions were then washed twice at 15,000g for 20 min with the same ice-cold medium to remove the contaminating Percoll. The synaptosome pellet was resuspended to a Wnal protein concentration of approximately 0.5 mg/mL. The material was prepared fresh daily and maintained at 0–4 °C throughout preparation.
2.6. Hormones in vitro Synaptosomes obtained from cortex and hippocampus of control animals (non-OVX) in the diestrus phase were used in these experiments. The hormones 17-estradiol (cyclodextrin-encapsulated 17-estradiol), DHEAS, and pregnenolone were added to the incubation medium at three concentrations: 1.0, 2.5, and 5.0M. 17-estradiol and DHEAS were dissolved in water and pregnenolone in 80% ethanol. The synaptosomes were pre-incubated for 10 min with each one of the hormones and after this time the reaction was started. Control tubes contained an equivalent amount of ethanol alone were made. The Wnal concentration of ethanol was less than 0.1% and this concentration of ethanol was tested for the hydrolysis of all nucleotides. 2.7. Enzyme assays The reaction medium used to assay ATP and ADP hydrolysis was essentially as described previously (Battastini et al., 1991) and contained 5.0 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose, and 45 mM Tris–HCl buVer, pH 8.0, in a Wnal volume of 200 L. The reaction medium used to assay 5⬘-nucleotidase activity contained 10 mM MgCl2, 100 mM Tris–HCl, pH 7.5, and 0.15 M sucrose in a Wnal volume of 200 L (Heymann et al., 1984). For in vitro assays, hormones (cyclodextrin-encapsulated 17-estradiol, DHEAS, and pregnenolone) were added to the reaction mixture. The synaptosomal fractions (10–20 g protein) were added to the reaction mixture, pre-incubated for 10 min, and incubated for 20 min at 37 °C. The reaction was initiated by the addition of ATP, ADP or AMP to a Wnal concentration of 1.0 mM and stopped by the addition of 200 L of 10% trichloroacetic acid. The samples were chilled on ice for 10 min and samples were taken for the assay of released inorganic phosphate (Pi) (Chan et al., 1986). Incubation times and protein concentration were chosen in order to ensure the linearity of the reaction. Controls with the addition of the enzyme preparation after addition of trichloroacetic acid were used to correct nonenzymatic hydrolysis of the substrates. 2.8. Protein determination Protein was measured by the Coomassie blue method using bovine serum albumin as standard (Bradford, 1976). 2.9. RT-PCR Total RNA from the cortex of four female rats from each of control, sham-operated, and OVX groups were isolated with Trizol reagent (Life Technologies) in accordance with the manufacturer’s instructions. The cDNA
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sham-operated group, and OVX group. In other oneway ANOVA, we compared the following groups: control group, OVX-oil group, and ERT group. All analyses were performed with an IBM compatible computer using the SPSSPC software.
species were synthesized with SuperScript II (Life Technologies) from 2 g of total RNA in a total volume of 20 L with an oligo(dT) primer in accordance with the manufacturer’s instructions. cDNA reactions were performed for 1 h at 42 °C and stopped by boiling for 5 min. Two microliters of cDNA was used as a template for PCR with primers speciWc for ecto-5⬘-nucleotidase. As a control for cDNA synthesis, -actin-PCR was performed. Two microliters of the RT reaction mix were used for PCR in a total volume of 25 L using a concentration of 0.5 M of each primer indicated below and 50 M of dNTP and 1 U Taq polymerase (Life Technologies) in the supplied reaction buVer. The PCR cycling conditions were as follows: for ecto5⬘-nucleotidase 45 s at 94 °C, 45 s at 64 °C, and 1 min 30 s at 72 °C (ampliWcation product 403 bp) and the same conditions for -actin (ampliWcation product 210 bp). All PCRs were carried out for 35 cycles and included an initial 3 min denaturation step at 94 °C and a Wnal 10 min extension at 72 °C. Ten microliters of the PCR were analyzed on a 1.5% agarose gel. The following set of primers were used: for ecto-5⬘-nucleotidase: 5⬘ CCC GGG GGC CAC TAG CAC CTC A 3⬘ and 5⬘ GCC TGG ACC ACG GGA ACC TT 3⬘ and for -actin: 5⬘ TAT GCC AAC ACA GTG CTG TCT GG 3⬘ and 5⬘ TAC TCC TGC TTC CTG ATC CAC AT 3⬘. Oligonucleotides were obtained from Invitrogen, Brazil. The PCR for each sample of cortex was repeated three times.
3. Results The eVects of hormonal deprivation on ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus are shown in Table 1. When compared to controls, the sham-operated group did not show any signiWcant diVerence in ATP and ADP hydrolysis in both structures. Similarly, the animals submitted to ovariectomy (OVX) treatment did not show signiWcant changes in ATP and ADP hydrolysis for both structures. Thus, the ovariectomy was unable to alter NTPDase activity in the central nervous system. In contrast, results demonstrated an increase in AMP hydrolysis in the cortex of OVX animals, ANOVA [F (2, 15) D 36.137; Duncan test P < 0.0001]; but not in hippocampus, ANOVA [F (2, 15) D 0.394; P D 0.681], when compared to the control group and sham-operated group (Table 1). Since 5⬘-nucleotidase activity is involved in the hydrolysis of AMP to adenosine in the synaptic cleft, ovariectomy may result in an increase in 5⬘-nucleotidase activity in cortical synaptosomes. Considering the eVects of hormone deprivation, we also evaluated whether estradiol replacement therapy (ERT) with pellets containing -estradiol 3-benzoato exerted eVects on AMP hydrolysis (Table 1). In the ERT group, a signiWcant inhibition of AMP hydrolysis was observed when compared with the OVX-oil group in the cerebral cortex of female rats [F (2, 14) D 12.337; Duncan test P < 0.001]. There were no signiWcant diVerences in AMP hydrolysis between the ERT group and control group indicating that the hormone replacement reversed the eVect of ovariectomy.
2.10. Statistical analysis The data are expressed as means § SD. All data were analyzed by one-way ANOVA, followed by the Duncan multiple range test when appropriate. P < 0.05 was considered to represent a signiWcant diVerence with statistical analysis used. In the statistical analysis, separate ANOVAs were used to analyze data from the cortex and hippocampus. For each brain region, we analyzed by one-way ANOVA the following groups: control group,
Table 1 EVects of ovariectomy (OVX) and estradiol replacement therapy (ERT) on nucleotide hydrolysis in synaptosomes from cerebral cortex and hippocampus from female adults rats Cortex (nmol Pi/min/mg)
Hippocampus (nmol Pi/min/mg)
ATP
ADP
AMP
OVX Control group Sham-operated group OVX group
140.49 § 22.4 131.38 § 19.0 158.98 § 19.1
52.46 § 4.6 54.75 § 5.9 58.26 § 3.1
12.60 § 1.3 11.07 § 0.9 22.03 § 3.9*
ERT Control group OVX-oil group ERT group
103.31 § 15.9 110.71 § 5.1 109.16 § 21.9
49.77 § 6.7 52.34 § 4.2 50.08 § 4.4
17.01 § 2.5 24.45 § 3.0夽 19.06 § 1.4#
ATP
ADP
AMP
92.41 § 9.1 94.72 § 11.6 97.82 § 16.1
43.25 § 9.1 46.37 § 4.5 50.82 § 9.3
20.51 § 6.4 24.47 § 4.2 27.79 § 6.9
104.34 § 17.8 120.82 § 29.9 111.80 § 13.9
47.14 § 6.3 47.88 § 3.3 43.13 § 12.1
19.34 § 3.9 24.59 § 9.3 23.06 § 7.3
Results are expressed as means § SD. Analysis of group diVerences were carried out using one-way ANOVA and Duncan’s post hoc test. For OVX experiments: n D 6; P 6 0.05 was considered statistically diVerent; *diVerent from control and sham-operated groups. For ERT experiments: n D 5; P 6 0.05 was considered statistically diVerent; 夽diVerent from control; and #diVerence from OVX-oil.
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We also tested the eVects of ERT on ATP and ADP hydrolysis in cortex and hippocampus. There were no diVerences in the hydrolysis of both nucleotides in these structures when hormone therapy was administered. To characterize how hormone deprivation may interact with the enzyme, we evaluated possible variations in the expression of 5⬘-nucleotidase, using the RT-PCR method. An increase in 5⬘-nucleotidase expression in cerebral cortex was observed in the OVX group, ANOVA [F (2, 11) D 4.698, P < 0.05; Duncan test P < 0.040], when compared with control and sham-operated groups, as shown in Fig. 1. This result suggests that the increase in the enzyme activity could be attributed to an increase in its own synthesis.
Considering the recent Wndings showing that neuroactive steroids can also act separately from their genomic eVects, we tested the eVects of various hormones (cyclodextrin-encapsulated 17-estradiol, DHEAS, and pregnenolone) on in vitro ATP, ADP, and AMP hydrolysis at the following concentrations: 1.0, 2.5, and 5.0 M. There were no signiWcant diVerences in synaptosomal ectonucleotidase activities for both structures in the presence of any of the hormones tested (results not shown). The inhibitory eVect observed for pregnenolone was due to the presence of the solvent used, ethanol (results not shown).
4. Discussion
Fig. 1. Representative semi-quantitative RT-PCR mRNA for 5⬘ nucleotidase from cerebral cortex of adult female rats. C (control), S (shamoperated), and OVX (ovariectomy) (A). The expression was evaluated by 5⬘-nucleotidase (CD73) to -actin mRNA ratio (B). Note that there was an increase in ecto-5⬘-nucleotidase mRNA levels in OVX group compared to sham rats. Bars represent arbitrary units of densitometry and are relative to means § SD of 5⬘-nucleotidase (CD73) mRNA/actin mRNA ratio (C). n, Number of animals used in each group (n D 4). For each sample of cortex three experiments of RT-PCR were repeated. *SigniWcantly diVerent from the sham group. SigniWcance level determined by ANOVA (P < 0.05).
The study described, herein, demonstrates changes in the activity and expression of 5⬘-nucleotidase in synaptosomes from female rat cerebral cortex, but not from hippocampus, following chronic steroid hormone deprivation induced by removal of ovaries. OVX treatment increased the activity of 5⬘-nucleotidase in synaptosomes from cerebral cortex when compared to the sham-operated and diestrus control groups. On the other hand, no changes in ATP and ADP hydrolysis were observed in the cortex and hippocampus, using the same protocol. The changes observed previously (Nedeljkovic et al., 2000) for ecto-ATPase in ovariectomized rats could be attributed to the co-expression of this enzyme and an ecto-ATP diphosphohydrolase in central nervous system (Nedeljkovic et al., 2003). It is important to note that the results for ecto-ATPase were obtained using synaptic plasma membranes and not a synaptosomal vesicular preparation as used in the present study. The altered ATP hydrolysis observed in the central nervous system, in OVX rats, may be a characteristic of isolated membranes. Many physiological manipulations can increase extracellular adenosine levels. The regulation of the activity of key enzymes, such as 5⬘-nucleotidase, is central to the mechanisms by which diverse stimuli can elevate extracellular adenosine levels in the brain (Dunwiddie and Masino, 2001). In the OVX group, this enzyme may hydrolyze AMP more eYciently when cortex is used as a source of synaptosomes. AMP is a product of the hydrolysis of the neurotransmitter ATP, promoted by a NTPDase activity. Thus, the enzymatic pathway from ATP to adenosine (ATP diphosphohydrolase plus 5⬘-nucleotidase) in the synaptic cleft is activated by changing the activity of only one of these two enzymes. The change was observed only in AMP hydrolysis and only in cortex. The enzymes ATP diphosphohydrolase and 5⬘-nucleotidase are largely distributed in the brain area and the activities were diVerent to diVerent areas (Bonan et al., 1998, 2000; Pereira et al., 2002). Then the distribution can account for diVerences in the results with cortex and hippocampus described in this paper.
B. Rücker et al. / General and Comparative Endocrinology 140 (2005) 94–100
The levels of adenosine in the cortex of these ovariectomized female rats are probably higher than those of the control group. This Wnding may be signiWcant, since this nucleoside is considered an important neuroprotector and neuromodulator agent (Bonan et al., 2001; Cunha, 2001; Dunwiddie and Masino, 2001; Ribeiro et al., 2003). Adenosine modulates the activity of the nervous system presynaptically at the cellular level by inhibiting or facilitating transmitter released, post-synaptically by hyperpolarizing or depolarizing neurons and/or exerting non-synaptic eVects (Ribeiro et al., 2003). It is important to note that the eVects of this nucleoside will depend on the relative density of adenosine A1 and A2A receptor subtypes present in cerebral cortex (Moreau and Huber, 1999; Ribeiro, 1999). With regard to additional eVects of the hormone on the central nervous system, it may be postulated that estradiol acts at extracellular sites and, thus, modulates synaptic transmission, changing the Ca2+ transport in nerve endings (Nikezic et al., 1996). Therefore, the increase in adenosine levels might be an important compensatory mechanism to control a possible imbalance caused by the hormonal deprivation. To assess the eVects of OVX on the expression of 5⬘ nucleotidase in the cortex of female rats, we examined the expression of ecto-5⬘-nucleotidase in surgical ovariectomy by evaluating the mRNA levels with the use of RT-PCR. RT-PCR analysis suggests that the enhanced enzyme activity in the cortex could be explained by the higher expression of 5⬘-nucleotidase following OVX. This is consistent with the classical view that steroids modulate gene expression via their nuclear receptors (Beato et al., 1995; Parker and White, 1996). Moreover, the in vitro results (not shown) did not alter the nucleotide hydrolysis, suggesting that, in this case, the hormone does not act through non-genomic mechanisms. At the same time it is important to note that the main consequence of this enhance in AMP hydrolysis will result in an increase in adenosine levels in CNS, increasing the neuroprotection. This result is interesting in view of the fact that Rose’Meyer et al. (2003) demonstrated a downregulation in adenosine receptors in response to ovariectomy. However, these authors used total brain and not speciWc tissue such as the cortex, used in the present study. Following estradiol replacement therapy (ERT), our results demonstrate that the hormone reversed the enhancement of AMP hydrolysis, promoted by ovariectomy. Thus, the adenosine levels are probably lower than those achieved with hormone deprivation. Thus, this nucleoside, which is an important neuroprotector and neuromodulator, could change the compensatory natural mechanism to normalize the adenosine levels in female rat cortex in the ERT. The relationship between estrogen neurotrophic eVects and their neuroprotective action is unclear. Grow-
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ing evidence indicates that estradiol may inXuence the risk of cerebrovascular stroke and also the extent of brain injury after ischemic insult (Wise, 2003). The eVect of hormone replacement therapy seems to be complex. In the brain, it seems to be inXuenced by the type of hormone used, the duration of the treatment, the nature of the tests and by the brain region studied (Genazzani et al., 2002). The latter could explain the change in nucleotide hydrolysis observed for cerebral cortex, but not for hippocampus in synaptosomal fraction from female rats. Over the last few years, hormone replacement therapy has been exhaustively discussed due to a rapid increase in life expectancy, with women consequently spending a signiWcant portion of their lives in the post-menopausal state. Studies published during the past few years (Hulley et al., 1998; Mulnard et al., 2001; Viscoli et al., 2001; Writing Group for the Women’s Health Initiative Investigators, 2002) have reviewed the recommendation for hormone replacement therapy. The results presented in this study should be considered relevant for the discussion of hormone replacement therapy, since much controversy surrounds this area and the relationship between adenosine and sex steroids is still poorly understood. Acknowledgment This work was supported by PRONEX, CNPq, CAPES, and FAPESP. References Battastini, A.M.O., Rocha, J.B.T., Barcellos, C.K., Dias, R.D., Sarkis, J.J.F., 1991. Characterization of an ATP diphosphohydrolase (EC 3.6.1.5.) from rat brain synaptic plasma membranes. Biochem. Mol. Biol. Int. 37, 209–219. Battastini, A.M.O., Oliveira, E.M., Moreira, C.M., Bonan, C.D., Sarkis, J.J.F., Dias, R.D., 1995. Solubilization and characterization of an ATP diphosphohydrolase (EC 3.6.1.5.) from rat brain plasma membranes. Biochem. Mol. Biol. Int. 37, 209–219. Beato, M., Herrlich, P., Schutz, G., 1995. Steroid hormone receptors: many actors in search of a plot. Cell 83 (6), 851–857. Bonan, C.D., Schetinger, M.R.C., Battastini, A.M.O., Sarkis, J.J.F., 2001. Ectonucleotidases and synaptic plasticity: implications in physiological and pathological conditions. Drug Dev. Res. 52, 57– 65. Bonan, C.D., Roesler, R., Quevedo, J., Battastini, A.M.O., Izquierdo, I., Sarkis, J.J.F., 1998. EVects of suramin on hippocampal apyrase activity and inhibitory avoidance learning of rats. Pharmacol. Biochem. Behav. 63, 153–158. Bonan, C.D., Roesler, R., Pereira, G.S., Battastini, A.M.O., Izquierdo, I., Sarkis, J.J.F., 2000. Learning-speciWc decrease in synaptosomal ATP diphosphohydrolase activity from hippocampus and enthorhinal cortex of adult rats. Brain Res. 854, 253–256. Bradford, M.M., 1976. A rapid and sensitive method for the quantiWcation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 218–254. Brown, T.J., MacLusky, N.J., Shanabrough, M., Naftolin, F., 1990. Comparison of age- and sex-related changes in cell nuclear estro-
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ecto-ATPase in distinct brain regions of female rats. Physiol. Res. 49, 419–426. Nedeljkovic, N., Banjac, A., Horvat, A., Stojiljkovic, M., Nikezic, G., 2003. Ecto-ATPase and ecto-ATP-diphosphohydrolase are colocalized n rat hippocampal and caudate nucleus synaptic plasma membranes. Physiol. Res. 52, 797–804. Nikezic, G., Horvat, A., Nedeljkovic, N., Martinovic, J.V., 1996. 17 beta-estradiol in vitro aVects Na-dependent and depolarizationinduced Ca2+ transport in rat brain synaptosomes. Experimentia 52 (3), 217–220. Parker, M.G., White, R., 1996. Nuclear receptors spring into action. Nat. Struct. Biol. 3, 113–115. Pereira, G.S., Mello E Souza, T., Battastini, A.M.O., Izquierdo, I., Sakis, J.J.F., Bonan, C.D., 2002. EVects of inhibitory avoidance training and/or isolated foot-shock on ectonucleotidase activities in synaptosomes of the anterior and posterior cingulate cortex and the medial precentral area of adult rats. Behav. Brain Res. 128 (2), 121–127. Ribeiro, J.A., 1999. Adenosine A2A receptor interactions with receptors for other neurotransmitters and neuromodulators. Eur. J. Pharmacol. 375, 101–113. Ribeiro, J.A., Sebastião, A.M., de Mendonça, A., 2003. Adenosine receptors in the nervous system: pathophysiological implications. Prog. Neurobiol. 68, 377–392. Rose’Meyer, R.B., Mellick, A.S., Garnham, B.G., Harrison, G.J., Massa, H.M., GriYths, L.R., 2003. The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis. Brain Res. Brain Res. Protoc. 11 (1), 9–18. Sarkis, J.J.F., Saltó, C., 1991. Characterization of a synaptosomal ATP diphosphohydrolase from the electric organ of Torpedo marmorata. Brain Res. Bull. 26, 871–876. Sarkis, J.J.F., Battastini, A.M.O., Oliveira, E.M., Frasseto, S.S., Dias, R.D., 1995. ATP diphosphohydrolases: an overview. J. Braz. Assoc. Adv. Sci. 47 (3), 131–136. Valverde, M.A., Parker, M.G., 2002. Classical and novel steroid actions: a uniWed but complex view. Trends Biochem. Sci. 27 (4), 172–173. Viscoli, C.M., Brass, L.M., Kernan, W.N., Sarrel, P.M., Suissa, S., Horwitz, R.I., 2001. A clinical trial of estrogen-replacement therapy after ischemic stroke. N. Engl. J. Med. 345, 1243–1249. Wise, P.M., 2003. Estrogens: protective or risk factors in brain function?. Prog. Neurobiol. 69, 181–191. Writing Group for the Women’s Health Initiative Investigators, 2002. Risks and beneWts of estrogen plus progestin in healthy postmenopausal women. JAMA 288, 321–333. Zimmermann, H., 1992. 5⬘ nucleotidase: molecular structure and functional aspects. Biochem. J. 285, 345–365. Zimmermann, H., 2001. Ectonucleotidases: some recent developments and a note on nomenclature. Drug Dev. Res. 52, 44–56. Zylinska, L., Legutko, B., 1998. Neuroactive steroids modulate in vitro the Mg2+-dependent Ca2+-ATPase activity in cultured rat neurons. Gen. Pharmacol. 30 (4), 533–536. Zylinska, L., Gromadzinska, E., Lachowicz, L., 1999. Short-time eVects of neuroactive steroids on rat cortical Ca2+-ATPase activity. Biochem. Biophys. Acta 1437, 257–264.
EVects of steroid hormones on synaptosomal ectonucleotidase activities from hippocampus and cortex of adult female rats Bárbara Rückera,¤, Daniela Pochmanna, Cristina Ribas Fürstenaua, Marcela Sorelli Carneiro-Ramosb, Ana Maria Oliveira Battastinia, Maria Luiza M. Barreto-Chavesc, João José Freitas Sarkisa a
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil c Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil Received 1 March 2004; revised 1 October 2004; accepted 12 October 2004 Available online 24 November 2004
Abstract Over the last few years, the eVects of steroid hormones on the brain have been intensively discussed. It has been demonstrated that ATP (acting as a neurotransmitter) is hydrolyzed to adenosine in the synaptic cleft by the conjugated action of ectonucleotidases, which include an enzyme of the E-NTPDase family (NTPDase3, apyrase, EC 3.6.1.5) and a 5⬘-nucleotidase (EC 3.1.3.5). The 5⬘nucleotidase enzyme is able to hydrolyze AMP as well as other monophosphate nucleotides. The importance of this enzyme in the central nervous system is to participate in the adenosine formation, a nucleoside with neuroprotective properties and modulatory eVects. However, several questions have been raised about the mechanisms of steroid hormones and the possible neuroprotective eVects of estrogen. Thus, we examined the eVects of gonadal steroid hormone deprivation, induced by ovary removal (OVX) and estradiol replacement therapy, on the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult rats. ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus did not change as a function of OVX and results demonstrated an increase in AMP hydrolysis (82%) in the animals submitted to OVX in cerebral cortex, but not in hippocampus, when compared to control and sham-operated groups. Estradiol replacement therapy reversed this eVect. RT-PCR analysis showed that the enhancement of enzyme activity in cerebral cortex could be explained by the higher expression of 5⬘-nucleotidase, following OVX. The hormones 17-estradiol (cyclodextrin-encapsulated 17-estradiol), DHEAS, and pregnenolone (1.0, 2.5, and 5.0 M) did not alter the nucleotide hydrolysis, in vitro, in synaptosomes from cortex and hippocampus of female adult rats. Results presented, herein, should be considered relevant for hormone replacement therapy, since much controversy exists surrounding this area and the relationship between adenosine and sex steroids is still poorly understood. 2004 Elsevier Inc. All rights reserved. Keywords: Adenosine; Steroid hormones; Ectonucleotidases; OVX; ERT
1. Introduction In recent years, an increasing number of studies have been performed demonstrating the role of steroid hor-
*
Corresponding author. Fax: +55 51 33165535. E-mail address: [email protected] (B. Rücker).
0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.10.008
mones in several physiological and pathological responses (Garcia-Segura et al., 2000; Valverde and Parker, 2002). Estrogen has been associated with a decreased risk, delayed onset and progression, or enhanced recovery from numerous traumatic or chronic neurological and mental diseases (Garcia-Segura et al., 2000). The eYcacy of estrogen has been shown in several models of neurodegeneration and ischemic injury in vivo
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and in vitro. In ovariectomized rats, physiological concentrations of estradiol, administered during hormone therapy, attenuated the extent of brain damage caused by permanent cerebral ischemia (Dubal et al., 1998; Harms et al., 2001). In contrast, several studies have shown conXicting data regarding the protective eVects of estrogen. Recently, studies have indicated a lack of protection or increased health risks of hormone replacement therapy (HRT) in the occurrence of cerebrovascular stroke (Viscoli et al., 2001), cardiovascular disease (Hulley et al., 1998), Alzheimer’s disease (Mulnard et al., 2001), and invasive breast cancer (Writing Group for the Women’s Health Initiative Investigators, 2002). It has been suggested that steroid hormone deprivation can modulate the expression and activity of an ectoATPase in synaptic plasma membranes of hippocampus and caudate nucleus from central nervous system of rats (Nedeljkovic et al., 2000). There is also increasing evidence to indicate that ecto-ATPase and ecto-ATP diphosphohydrolase are co-localized in the central nervous system (Kegel et al., 1997; Nedeljkovic et al., 2003). Ecto-enzymes, able to hydrolyze ATP and ADP, are present in the central nervous system of several species (Sarkis et al., 1995). ATP released by synapses can be hydrolyzed by ectonucleotidases in a highly sophisticated pathway composed of ecto-enzymes of the E-NTPDase family, including NTPDase3 (ATP diphosphohydrolase, ecto-apyrase, CD39, EC 3.6.1.5) and NTPDase2 (ectoATPase, EC 3.6.1.3), which can transform ATP and ADP to AMP (Bonan et al., 2001; Zimmermann, 2001). The AMP produced is subsequently hydrolyzed to adenosine by an ecto 5⬘-nucleotidase (CD73, EC 3.1.3.5), which constitutes the rate-limiting step in this pathway (Battastini et al., 1995; Sarkis and Saltó, 1991; Zimmermann, 1992); the Wnal product being the nucleoside adenosine, an important endogenous neuromodulator. Following ATP receptor activation, the signal is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, and this metabolic step leads to adenosine generation. Adenosine is involved in a diverse array of functions in the central nervous system and may play a number of roles in physiological and pathological conditions. In brain, adenosine levels are signiWcantly elevated by a wide array of pathological stimuli (Dunwiddie and Masino, 2001). Adenosine mediates its eVects through four types of G-protein-coupled receptors: A1, A2A, A2B, and A3 (Hauber and Bareiß, 2001). Since results from a previous study suggest that steroid hormone deprivation may have an eVect on the ecto-ATPase of synaptic plasma membrane obtained from hippocampus and caudate nucleus (CNS) (Nedeljkovic et al., 2000), we hypothesized that the eVect described could be mediated by: (a) a true ecto-ATPase: (b) an ATP diphosphohydrolase or (c) on both enzymes. It may be speculated that the eVect may depend on the ATP diphosphohydrolase enzymatic chain plus 5⬘-nucle-
95
otidase and, thus, aVect the adenosine production in the central nervous system, particularly in view of the fact that adenosine is a neuroprotective structure. Thus, the possible eVects of these hormones on the enzyme ATP diphosphohydrolase (co-expressed with the ectoATPase) were investigated. In addition, the eVects of these hormones on 5⬘-nucleotidase, involved in the complete hydrolysis of ATP to adenosine, were also studied in the central nervous system synaptic cleft. With regard to the mechanism of action of steroid hormones and their possible neuroprotective eVects, much controversy still surrounds this area. Thus, we examined the eVects of gonadal steroid hormone deprivation, induced by removal of ovaries (ovariectomy) and estradiol replacement therapy, on the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of rats. In addition to their well-documented genomic eVects, some evidence exists to indicate that neuroactive steroids may act as potent modulators of the plasma membrane receptors, which may interact with diVerent eVector systems in neuronal membranes (Zylinska and Legutko, 1998; Zylinska et al., 1999). Therefore, we evaluated whether the hormones 17-estradiol (cyclodextrinencapsulated 17-estradiol), DHEAS, and pregnenolone were able to directly modulate the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult female rats, in vitro.
2. Methods 2.1. Chemicals Nucleotides, 17-estradiol (cyclodextrin-encapsulated 17-estradiol), dehydroepiandrosterone sulfate (5-androsten-3-ol-17-one sulfate), pregnenolone (5-pregnen3-ol-20-one), and -estradiol 3-benzoato were obtained from Sigma Chemical (St. Louis, MO, USA). All others reagents were of analytical grade. 2.2. Animals Female Wistar rats (aged 60–75 days; 200–250g) were used in this study. They were housed Wve to a cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on 7:00 a.m.) at a temperature of 23 §1°C. In all animals that were ovariectomized and received the pellet containing the hormone, 120mg/kg ketamine HCl (Dopalen: Agribrands, Campinas, SP, Brazil) and 16mg/kg xylazine (Anasedan: Agribrands) were used as anesthesia. Only animals in the diestrus state were used. 2.3. Hormone deprivation Animals were randomly divided into three groups: control group, the group corresponding to sham-oper-
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ated animals, and the group submitted to a bilateral ovariectomy (OVX) by removal of the glands through one abdominal incision. The sham-operated group was submitted only to abdominal incision, but not to removal of ovaries. The sham-operated and OVX groups were compared to the control group in order to characterize the eVects of surgery on the nucleotide hydrolysis. Three weeks after surgery, all groups were sacriWced by decapitation and the synaptosomes were prepared. 2.4. Estradiol replacement therapy Animals were randomly divided into three groups. A control group, a vehicle group (OVX + silastic pellets with oil), and a third group submitted to estradiol replacement therapy (ERT) (OVX + silastic pellet with -estradiol 3-benzoato). Pellets with 15 mm medical grade tubing (1.02 mm i.d. £ 2.16 mm o.d.; Medicone, Multiplast, Porto Alegre, RS, Brazil) were Wlled with 10 L of 5% (w/v) -estradiol 3-benzoato (Sigma, St. Louis, MO) in corn oil and sealed with silicone. These pellets were soaked in sterile saline overnight and implanted subcutaneously between the scapulae (Gamaro et al., 2003). Previous studies show that these pellets generate physiological serum levels of estradiol between 17 and 32 pg/mL, and measurements at 10 and 30 days post-implantation have shown no signiWcant change in circulating levels over these times (Brown et al., 1990; Luine et al., 1998). The pellets were implanted 2 weeks after OVX and, 3 weeks after the implant, the animals were sacriWced by decapitation and the synaptosomes prepared as described below. 2.5. Synaptosomes preparation Animals were sacriWced by decapitation and the brain structures (entire bilateral cortex and hippocampus) were removed to an ice-cold medium solution (320 mM sucrose, 5 mM Hepes, pH 7.5, and 0.1 mM EDTA). Structures were gently homogenized in Wve volumes of ice-cold medium solution with a motor-driven TeXonglass homogenizer. The synaptosomes were isolated as described previously by Nagy and Delgado-Escueta (1984). BrieXy, 0.5 mL of the crude mitochondrial fraction was mixed with 4.0 mL of 8.5% Percoll solution and layered onto an isoosmotic Percoll/sucrose discontinuous gradient (10/16%). The synaptosomes that banded at the 10/16% Percoll interface were collected with wide tip disposable plastic transfer pipettes. The synaptosomal fractions were then washed twice at 15,000g for 20 min with the same ice-cold medium to remove the contaminating Percoll. The synaptosome pellet was resuspended to a Wnal protein concentration of approximately 0.5 mg/mL. The material was prepared fresh daily and maintained at 0–4 °C throughout preparation.
2.6. Hormones in vitro Synaptosomes obtained from cortex and hippocampus of control animals (non-OVX) in the diestrus phase were used in these experiments. The hormones 17-estradiol (cyclodextrin-encapsulated 17-estradiol), DHEAS, and pregnenolone were added to the incubation medium at three concentrations: 1.0, 2.5, and 5.0M. 17-estradiol and DHEAS were dissolved in water and pregnenolone in 80% ethanol. The synaptosomes were pre-incubated for 10 min with each one of the hormones and after this time the reaction was started. Control tubes contained an equivalent amount of ethanol alone were made. The Wnal concentration of ethanol was less than 0.1% and this concentration of ethanol was tested for the hydrolysis of all nucleotides. 2.7. Enzyme assays The reaction medium used to assay ATP and ADP hydrolysis was essentially as described previously (Battastini et al., 1991) and contained 5.0 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose, and 45 mM Tris–HCl buVer, pH 8.0, in a Wnal volume of 200 L. The reaction medium used to assay 5⬘-nucleotidase activity contained 10 mM MgCl2, 100 mM Tris–HCl, pH 7.5, and 0.15 M sucrose in a Wnal volume of 200 L (Heymann et al., 1984). For in vitro assays, hormones (cyclodextrin-encapsulated 17-estradiol, DHEAS, and pregnenolone) were added to the reaction mixture. The synaptosomal fractions (10–20 g protein) were added to the reaction mixture, pre-incubated for 10 min, and incubated for 20 min at 37 °C. The reaction was initiated by the addition of ATP, ADP or AMP to a Wnal concentration of 1.0 mM and stopped by the addition of 200 L of 10% trichloroacetic acid. The samples were chilled on ice for 10 min and samples were taken for the assay of released inorganic phosphate (Pi) (Chan et al., 1986). Incubation times and protein concentration were chosen in order to ensure the linearity of the reaction. Controls with the addition of the enzyme preparation after addition of trichloroacetic acid were used to correct nonenzymatic hydrolysis of the substrates. 2.8. Protein determination Protein was measured by the Coomassie blue method using bovine serum albumin as standard (Bradford, 1976). 2.9. RT-PCR Total RNA from the cortex of four female rats from each of control, sham-operated, and OVX groups were isolated with Trizol reagent (Life Technologies) in accordance with the manufacturer’s instructions. The cDNA
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sham-operated group, and OVX group. In other oneway ANOVA, we compared the following groups: control group, OVX-oil group, and ERT group. All analyses were performed with an IBM compatible computer using the SPSSPC software.
species were synthesized with SuperScript II (Life Technologies) from 2 g of total RNA in a total volume of 20 L with an oligo(dT) primer in accordance with the manufacturer’s instructions. cDNA reactions were performed for 1 h at 42 °C and stopped by boiling for 5 min. Two microliters of cDNA was used as a template for PCR with primers speciWc for ecto-5⬘-nucleotidase. As a control for cDNA synthesis, -actin-PCR was performed. Two microliters of the RT reaction mix were used for PCR in a total volume of 25 L using a concentration of 0.5 M of each primer indicated below and 50 M of dNTP and 1 U Taq polymerase (Life Technologies) in the supplied reaction buVer. The PCR cycling conditions were as follows: for ecto5⬘-nucleotidase 45 s at 94 °C, 45 s at 64 °C, and 1 min 30 s at 72 °C (ampliWcation product 403 bp) and the same conditions for -actin (ampliWcation product 210 bp). All PCRs were carried out for 35 cycles and included an initial 3 min denaturation step at 94 °C and a Wnal 10 min extension at 72 °C. Ten microliters of the PCR were analyzed on a 1.5% agarose gel. The following set of primers were used: for ecto-5⬘-nucleotidase: 5⬘ CCC GGG GGC CAC TAG CAC CTC A 3⬘ and 5⬘ GCC TGG ACC ACG GGA ACC TT 3⬘ and for -actin: 5⬘ TAT GCC AAC ACA GTG CTG TCT GG 3⬘ and 5⬘ TAC TCC TGC TTC CTG ATC CAC AT 3⬘. Oligonucleotides were obtained from Invitrogen, Brazil. The PCR for each sample of cortex was repeated three times.
3. Results The eVects of hormonal deprivation on ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus are shown in Table 1. When compared to controls, the sham-operated group did not show any signiWcant diVerence in ATP and ADP hydrolysis in both structures. Similarly, the animals submitted to ovariectomy (OVX) treatment did not show signiWcant changes in ATP and ADP hydrolysis for both structures. Thus, the ovariectomy was unable to alter NTPDase activity in the central nervous system. In contrast, results demonstrated an increase in AMP hydrolysis in the cortex of OVX animals, ANOVA [F (2, 15) D 36.137; Duncan test P < 0.0001]; but not in hippocampus, ANOVA [F (2, 15) D 0.394; P D 0.681], when compared to the control group and sham-operated group (Table 1). Since 5⬘-nucleotidase activity is involved in the hydrolysis of AMP to adenosine in the synaptic cleft, ovariectomy may result in an increase in 5⬘-nucleotidase activity in cortical synaptosomes. Considering the eVects of hormone deprivation, we also evaluated whether estradiol replacement therapy (ERT) with pellets containing -estradiol 3-benzoato exerted eVects on AMP hydrolysis (Table 1). In the ERT group, a signiWcant inhibition of AMP hydrolysis was observed when compared with the OVX-oil group in the cerebral cortex of female rats [F (2, 14) D 12.337; Duncan test P < 0.001]. There were no signiWcant diVerences in AMP hydrolysis between the ERT group and control group indicating that the hormone replacement reversed the eVect of ovariectomy.
2.10. Statistical analysis The data are expressed as means § SD. All data were analyzed by one-way ANOVA, followed by the Duncan multiple range test when appropriate. P < 0.05 was considered to represent a signiWcant diVerence with statistical analysis used. In the statistical analysis, separate ANOVAs were used to analyze data from the cortex and hippocampus. For each brain region, we analyzed by one-way ANOVA the following groups: control group,
Table 1 EVects of ovariectomy (OVX) and estradiol replacement therapy (ERT) on nucleotide hydrolysis in synaptosomes from cerebral cortex and hippocampus from female adults rats Cortex (nmol Pi/min/mg)
Hippocampus (nmol Pi/min/mg)
ATP
ADP
AMP
OVX Control group Sham-operated group OVX group
140.49 § 22.4 131.38 § 19.0 158.98 § 19.1
52.46 § 4.6 54.75 § 5.9 58.26 § 3.1
12.60 § 1.3 11.07 § 0.9 22.03 § 3.9*
ERT Control group OVX-oil group ERT group
103.31 § 15.9 110.71 § 5.1 109.16 § 21.9
49.77 § 6.7 52.34 § 4.2 50.08 § 4.4
17.01 § 2.5 24.45 § 3.0夽 19.06 § 1.4#
ATP
ADP
AMP
92.41 § 9.1 94.72 § 11.6 97.82 § 16.1
43.25 § 9.1 46.37 § 4.5 50.82 § 9.3
20.51 § 6.4 24.47 § 4.2 27.79 § 6.9
104.34 § 17.8 120.82 § 29.9 111.80 § 13.9
47.14 § 6.3 47.88 § 3.3 43.13 § 12.1
19.34 § 3.9 24.59 § 9.3 23.06 § 7.3
Results are expressed as means § SD. Analysis of group diVerences were carried out using one-way ANOVA and Duncan’s post hoc test. For OVX experiments: n D 6; P 6 0.05 was considered statistically diVerent; *diVerent from control and sham-operated groups. For ERT experiments: n D 5; P 6 0.05 was considered statistically diVerent; 夽diVerent from control; and #diVerence from OVX-oil.
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We also tested the eVects of ERT on ATP and ADP hydrolysis in cortex and hippocampus. There were no diVerences in the hydrolysis of both nucleotides in these structures when hormone therapy was administered. To characterize how hormone deprivation may interact with the enzyme, we evaluated possible variations in the expression of 5⬘-nucleotidase, using the RT-PCR method. An increase in 5⬘-nucleotidase expression in cerebral cortex was observed in the OVX group, ANOVA [F (2, 11) D 4.698, P < 0.05; Duncan test P < 0.040], when compared with control and sham-operated groups, as shown in Fig. 1. This result suggests that the increase in the enzyme activity could be attributed to an increase in its own synthesis.
Considering the recent Wndings showing that neuroactive steroids can also act separately from their genomic eVects, we tested the eVects of various hormones (cyclodextrin-encapsulated 17-estradiol, DHEAS, and pregnenolone) on in vitro ATP, ADP, and AMP hydrolysis at the following concentrations: 1.0, 2.5, and 5.0 M. There were no signiWcant diVerences in synaptosomal ectonucleotidase activities for both structures in the presence of any of the hormones tested (results not shown). The inhibitory eVect observed for pregnenolone was due to the presence of the solvent used, ethanol (results not shown).
4. Discussion
Fig. 1. Representative semi-quantitative RT-PCR mRNA for 5⬘ nucleotidase from cerebral cortex of adult female rats. C (control), S (shamoperated), and OVX (ovariectomy) (A). The expression was evaluated by 5⬘-nucleotidase (CD73) to -actin mRNA ratio (B). Note that there was an increase in ecto-5⬘-nucleotidase mRNA levels in OVX group compared to sham rats. Bars represent arbitrary units of densitometry and are relative to means § SD of 5⬘-nucleotidase (CD73) mRNA/actin mRNA ratio (C). n, Number of animals used in each group (n D 4). For each sample of cortex three experiments of RT-PCR were repeated. *SigniWcantly diVerent from the sham group. SigniWcance level determined by ANOVA (P < 0.05).
The study described, herein, demonstrates changes in the activity and expression of 5⬘-nucleotidase in synaptosomes from female rat cerebral cortex, but not from hippocampus, following chronic steroid hormone deprivation induced by removal of ovaries. OVX treatment increased the activity of 5⬘-nucleotidase in synaptosomes from cerebral cortex when compared to the sham-operated and diestrus control groups. On the other hand, no changes in ATP and ADP hydrolysis were observed in the cortex and hippocampus, using the same protocol. The changes observed previously (Nedeljkovic et al., 2000) for ecto-ATPase in ovariectomized rats could be attributed to the co-expression of this enzyme and an ecto-ATP diphosphohydrolase in central nervous system (Nedeljkovic et al., 2003). It is important to note that the results for ecto-ATPase were obtained using synaptic plasma membranes and not a synaptosomal vesicular preparation as used in the present study. The altered ATP hydrolysis observed in the central nervous system, in OVX rats, may be a characteristic of isolated membranes. Many physiological manipulations can increase extracellular adenosine levels. The regulation of the activity of key enzymes, such as 5⬘-nucleotidase, is central to the mechanisms by which diverse stimuli can elevate extracellular adenosine levels in the brain (Dunwiddie and Masino, 2001). In the OVX group, this enzyme may hydrolyze AMP more eYciently when cortex is used as a source of synaptosomes. AMP is a product of the hydrolysis of the neurotransmitter ATP, promoted by a NTPDase activity. Thus, the enzymatic pathway from ATP to adenosine (ATP diphosphohydrolase plus 5⬘-nucleotidase) in the synaptic cleft is activated by changing the activity of only one of these two enzymes. The change was observed only in AMP hydrolysis and only in cortex. The enzymes ATP diphosphohydrolase and 5⬘-nucleotidase are largely distributed in the brain area and the activities were diVerent to diVerent areas (Bonan et al., 1998, 2000; Pereira et al., 2002). Then the distribution can account for diVerences in the results with cortex and hippocampus described in this paper.
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The levels of adenosine in the cortex of these ovariectomized female rats are probably higher than those of the control group. This Wnding may be signiWcant, since this nucleoside is considered an important neuroprotector and neuromodulator agent (Bonan et al., 2001; Cunha, 2001; Dunwiddie and Masino, 2001; Ribeiro et al., 2003). Adenosine modulates the activity of the nervous system presynaptically at the cellular level by inhibiting or facilitating transmitter released, post-synaptically by hyperpolarizing or depolarizing neurons and/or exerting non-synaptic eVects (Ribeiro et al., 2003). It is important to note that the eVects of this nucleoside will depend on the relative density of adenosine A1 and A2A receptor subtypes present in cerebral cortex (Moreau and Huber, 1999; Ribeiro, 1999). With regard to additional eVects of the hormone on the central nervous system, it may be postulated that estradiol acts at extracellular sites and, thus, modulates synaptic transmission, changing the Ca2+ transport in nerve endings (Nikezic et al., 1996). Therefore, the increase in adenosine levels might be an important compensatory mechanism to control a possible imbalance caused by the hormonal deprivation. To assess the eVects of OVX on the expression of 5⬘ nucleotidase in the cortex of female rats, we examined the expression of ecto-5⬘-nucleotidase in surgical ovariectomy by evaluating the mRNA levels with the use of RT-PCR. RT-PCR analysis suggests that the enhanced enzyme activity in the cortex could be explained by the higher expression of 5⬘-nucleotidase following OVX. This is consistent with the classical view that steroids modulate gene expression via their nuclear receptors (Beato et al., 1995; Parker and White, 1996). Moreover, the in vitro results (not shown) did not alter the nucleotide hydrolysis, suggesting that, in this case, the hormone does not act through non-genomic mechanisms. At the same time it is important to note that the main consequence of this enhance in AMP hydrolysis will result in an increase in adenosine levels in CNS, increasing the neuroprotection. This result is interesting in view of the fact that Rose’Meyer et al. (2003) demonstrated a downregulation in adenosine receptors in response to ovariectomy. However, these authors used total brain and not speciWc tissue such as the cortex, used in the present study. Following estradiol replacement therapy (ERT), our results demonstrate that the hormone reversed the enhancement of AMP hydrolysis, promoted by ovariectomy. Thus, the adenosine levels are probably lower than those achieved with hormone deprivation. Thus, this nucleoside, which is an important neuroprotector and neuromodulator, could change the compensatory natural mechanism to normalize the adenosine levels in female rat cortex in the ERT. The relationship between estrogen neurotrophic eVects and their neuroprotective action is unclear. Grow-
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ing evidence indicates that estradiol may inXuence the risk of cerebrovascular stroke and also the extent of brain injury after ischemic insult (Wise, 2003). The eVect of hormone replacement therapy seems to be complex. In the brain, it seems to be inXuenced by the type of hormone used, the duration of the treatment, the nature of the tests and by the brain region studied (Genazzani et al., 2002). The latter could explain the change in nucleotide hydrolysis observed for cerebral cortex, but not for hippocampus in synaptosomal fraction from female rats. Over the last few years, hormone replacement therapy has been exhaustively discussed due to a rapid increase in life expectancy, with women consequently spending a signiWcant portion of their lives in the post-menopausal state. Studies published during the past few years (Hulley et al., 1998; Mulnard et al., 2001; Viscoli et al., 2001; Writing Group for the Women’s Health Initiative Investigators, 2002) have reviewed the recommendation for hormone replacement therapy. The results presented in this study should be considered relevant for the discussion of hormone replacement therapy, since much controversy surrounds this area and the relationship between adenosine and sex steroids is still poorly understood. Acknowledgment This work was supported by PRONEX, CNPq, CAPES, and FAPESP. References Battastini, A.M.O., Rocha, J.B.T., Barcellos, C.K., Dias, R.D., Sarkis, J.J.F., 1991. Characterization of an ATP diphosphohydrolase (EC 3.6.1.5.) from rat brain synaptic plasma membranes. Biochem. Mol. Biol. Int. 37, 209–219. Battastini, A.M.O., Oliveira, E.M., Moreira, C.M., Bonan, C.D., Sarkis, J.J.F., Dias, R.D., 1995. Solubilization and characterization of an ATP diphosphohydrolase (EC 3.6.1.5.) from rat brain plasma membranes. Biochem. Mol. Biol. Int. 37, 209–219. Beato, M., Herrlich, P., Schutz, G., 1995. Steroid hormone receptors: many actors in search of a plot. Cell 83 (6), 851–857. Bonan, C.D., Schetinger, M.R.C., Battastini, A.M.O., Sarkis, J.J.F., 2001. Ectonucleotidases and synaptic plasticity: implications in physiological and pathological conditions. Drug Dev. Res. 52, 57– 65. Bonan, C.D., Roesler, R., Quevedo, J., Battastini, A.M.O., Izquierdo, I., Sarkis, J.J.F., 1998. EVects of suramin on hippocampal apyrase activity and inhibitory avoidance learning of rats. Pharmacol. Biochem. Behav. 63, 153–158. Bonan, C.D., Roesler, R., Pereira, G.S., Battastini, A.M.O., Izquierdo, I., Sarkis, J.J.F., 2000. Learning-speciWc decrease in synaptosomal ATP diphosphohydrolase activity from hippocampus and enthorhinal cortex of adult rats. Brain Res. 854, 253–256. Bradford, M.M., 1976. A rapid and sensitive method for the quantiWcation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 218–254. Brown, T.J., MacLusky, N.J., Shanabrough, M., Naftolin, F., 1990. Comparison of age- and sex-related changes in cell nuclear estro-
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