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Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats
Neuroscience Letters 712 (2019) 134474
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Research article
Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats
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Nataša Mitrovića,1, , Milorad Dragićb, Marina Zarića, Dunja Drakulića, Nadežda Nedeljkovićb, Ivana Grkovića,1 a b
Department of Molecular Biology and Endocrinology, VINČA Institute of Nuclear Sciences, University of Belgrade, Mike Petrovića Alasa 12-14, 11001 Belgrade, Serbia Department for General Physiology and Biophysics, Faculty of Biology, University of Belgrade, Belgrade, Studentski trg 3, 11001 Belgrade, Serbia
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Ectonucleotidase activity Estradiol receptors Hippocampus Male rats
Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and adenosine, are among least investigated signaling factors that participate in 17β-estradiol (E2)-mediated synaptic rearrangements in rodent hippocampus. Their levels in the extrasynaptic space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases1-3 (NTPDase1-3)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, the aim of the present study was to get closer insight in the E2-induced decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors (ERs i.e. ERα, ERβ or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -β (ERβ) and G- protein coupled estrogen receptor 1 (GPER1) in the mediation of E2 actions in hippocampal synaptosomes of male rats. Synaptic NTPDase1-3 activities are modulated only through activation of ERβ, while activation of ERα, -β and/or non-classical GPER1 decreases synaptic eN activity. Since both ATP and
Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; E2, 17β-estradiol; NTPDase1-3, ecto-nucleoside triphosphate diphosphohydrolases1-3; eN, ecto-5'-nucleotidase; ERs, estradiol receptors; ER, estradiol receptor; ERβ, estradiol receptor β; GPER1, G protein-coupled estrogen receptor 1; GPI, glycosylphosphatidylinositol; PPT, estradiol receptor agonist 13,5-tris(4-hydroxyphenyl)-4-propyl-1 H-pyrazole; DPN, estradiol β receptor agonist 23-bis(4-hydroxyphenyl)-propionitrile; ICI, nonselective estrogen receptor antagonist 7a 17b-[9-[(4,4,5,5,5 Pentafluoropentyl)sulfinyl]nonyl]estra1,3,5(10)-triene-3,17-diol (ICI 182,780); G1, selective GPER1 agonist; G-15, selective GPER1 antagonist; BSA, bovine serum albumin; DMSO, dymethyl sulfoxide; NO, nitric oxide; OVX, ovariectomy ⁎ Corresponding author at: Department of Molecular Biology and Endocrinology, VINČA Institute of Nuclear Science, University of Belgrade, Mike Petrović Alasa 12-14, 11000 Belgrade, Vinča, Serbia. E-mail address: [email protected] (N. Mitrović). 1 These authors contributed equally. https://doi.org/10.1016/j.neulet.2019.134474 Received 1 March 2019; Received in revised form 23 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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adenosine function as neuromodulators in the hippocampal networks, influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.
1. Introduction
different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance [11,14]. Thus, the aim of this study was to get closer insight in the E2-mediated decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors responsible for the observed effects.
Adenosine triphosphate (ATP) and its nucleoside adenosine act as versatile extracellular signaling molecules in the central nervous system (CNS) and are implicated in regulation of hippocampal function and plasticity [1,2]. Actions of ATP are mediated via two classes of ligandgated P2X and G protein-coupled P2Y receptors which are abundantly expressed in the brain [1]. In contrast to ATP, adenosine acts as a retaliatory molecule and neuromodulator, which operates via four G protein-coupled adenosine receptors (A1R, A2A, A2B and A3) [2]. The levels of ATP and adenosine in the extracellular space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases (NTPDases)/ecto-5′-nucleotidase (eN) enzyme chain [3]. Three distinct membrane-bound NTPDases (NTPDase1-3) are expressed in the CNS which differ in their preference for the substrate [3,4]. NTPDase1/ CD39 hydrolyzes ATP and ADP equally well to AMP, NTPDase2 exclusively degrades ATP to ADP, while NTPDase3 is the functional intermediate which preferentially hydrolyzes ATP, with a transient accumulation of ADP [3]. Thus, NTPDases provide the substrate (AMP) for the final and rate-limiting step of extracellular ATP degradation, catalyzed by ecto-5'-nucleotidase (CD73, eN). Ecto-5′-nucleotidase is a Zn2+-binding glycosylphosphatidylinositol (GPI)-anchored protein, with its catalytic site facing the extracellular compartment. It shows an affinity for 5'-AMP in the micromolar range, thus producing extracellular adenosine [3]. Since NTPDases and eN control extracellular levels of ATP and adenosine, regulatory mechanisms involved in the control of Ntpd1-3 and Nt5e gene expression, translation, post-translational modifications, membrane expression and the catalytic activity are of high relevance for normal brain functioning. It has been known for decades that female sex hormones, 17β-estradiol (E2), in particular, affect hippocampal morphology, plasticity and memory in male as well as in female rodents [5]. A recent study showed that in vivo application of E2 increases density of dendritic spines in the hippocampus of adult gonadectomized males, rapidly as in females [6]. Also, E2 potentiates excitatory synapses in the hippocampus of both sexes [7,8]. Furthermore, NTPDase1–3 and eN are localized along with estradiol receptors (ERs) in the rat hippocampal synaptic compartment [5,9–14] and the female ovarian hormones modulate the activities of the enzymes in female, as well as in male rat brain [11,10–14]. While E2 induces marked increase in ATP/ADP/ AMPase activity in the hippocampus of female rats, a single systemic injection of E2 in the male rats results in marked attenuation of a complete chain of adenine nucleotide hydrolysis. Immunoblot analyses revealed that lower ATP and ADP hydrolysis probably result from decreased protein abundance of NTPDase1 and NTPDase2 in the synaptic compartment, while a decrease in AMP hydrolysis following E2 treatment is not the consequence of alteration in the protein level, but rather the inhibition of eN catalytic activity [14]. Kinetic analysis showed that E2 alters kinetic properties of eN and reduces the enzyme catalytic efficiency by decreasing the enzyme affinity toward AMP [14]. It is known that high extracellular levels of the upstream substrates, ADP and ATP, may competitively inhibit eN enzyme activity in a feed-forward manner [9]. Therefore, we hypothesized that in male hippocampal synaptic membranes E2 directly attenuates the activity of NTPDases, which leads to accumulation of ATP and ADP, thus consequently induce feed-forward inhibition of eN. The aforementioned decline in NTPDase/eN activity after E2 injection in vivo was accompanied with activation of the molecular machinery required for synaptogenesis and synaptic refinement, including mTOR-mediated signaling [14]. Altogether, our previous evaluation supports the idea of specific roles of
2. Materials and methods 2.1. Materials Analytical grade salts and buffer reagents, 17b-estradiol 3-benzoate (E2), b-estradiol 6-(o-carboxy-methyl) oxime: bovine serum albumin (E2–BSA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Estradiol receptor agonists 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1 H-pyrazole (PPT), 2,3-bis(4hydroxyphenyl)-propionitrile (DPN), G1 and nonselective ERα/β antagonist 7a, 17b-[9-[(4,4,5,5,5 Pentafluoropentyl)sulfinyl]nonyl]estra1,3,5(10)-triene-3,17-diol (ICI 182,780) and GPER1 selective antagonist (G-15) were purchased from Tocris Bioscience (Ellisville, MO, USA). E2-BSA was filtered before use to eliminate free E2. 2.2. Animals All experiments were conducted using 3-month old, gonadally intact male rats (300–350 g) of the Wistar strain, obtained from a local colony. The care was taken to alleviate the pain and discomfort of the animals. Animals were treated in accordance with the European Community Council Directive of 86/609/ EEC for animal experiment. Research procedures were approved by the Ethical Committee for the Use of Laboratory Animals of VINČA institute of nuclear sciences, University of Belgrade, Belgrade, Republic of Serbia (Licence No. 02/11; 323-0703832/2015-05/1). Animals were housed (3–4/cage) under standard conditions: 12 h light/dark regime, constant ambient temperature (22 ± 2 °C) and humidity with ad libitum access to food and water. 2.3. Experimental groups and treatments Animals were randomly divided into the following groups (6–9 rats/ group): Veh - animals which received an injection of DMSO (1 ml/kg) were used as control group; E2 - animals injected with 17b-estradiol 3benzoate (33.3 μg/kg); PPT - animals which received an injection of a selective ERα agonist PPT (2.5 mg/kg); DPN - animals which received selective ERβ agonist DPN (2.5 mg/kg); G1 – animals treated with one dose of GPER1 agonist G1 (5 μg); ICI - animals treated with a non-selective antagonist (ICI 182,780; 2 mg/kg) of ERα and ERβ, but not GPER1; ICI + E2 - animals which received injection of ICI 182,780, 2 h prior to E2 administration (2 mg/kg + 33.3 μg/kg); G-15 – animals treated with one dose of selective GPER1 antagonist G-15 (40 μg); G15+E2 - animals which received one dose of G-15 two hours prior to E2 (40 μg + 33,3 μg/kg). Animals were treated s.c. at 9:00 a.m. and sacrificed 24 h after the treatment. An additional group of gonadally intact male rats without any treatment was used to investigate the impact of the vehicle on investigated parameters (Control). The dosages of E2, PPT, DPN, ICI, G-15, and G1 were determined previously [13–16]. 2.4. Synaptosomes preparation After decapitation with a small animal guillotine (Harvard 2
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adenosine. It has been previously demonstrated that E2 induces a significant decrease in the eN activity (56.3 ± 1.5 nmol Pi/mg/min) in respect to Veh (76.3 ± 1.8 nmol Pi/mg/min, Fig. 2) [14]. As shown in Fig. 2 administration of selective agonists of ERα, ERβ, or GPER1 (PPT, DPN, or G1, respectively) results in lower rates of AMP hydrolysis (53.6 ± 2.5 nmol Pi/mg/min, p < 0.001, 61.03 ± 2.3 nmol Pi/mg/ min, p < 0.001, 55.4 ± 3.3 nmol Pi/mg/min, p < 0.05, respectively) in respect to Veh (76.3 ± 1.8 nmol Pi/mg/min). Administration of ICI 182,780 on its own did not affect the rate of AMP hydrolysis, while ICI + E2 treatment (ICI + E2; 63.5 ± 2.1 nmol Pi/mg/min, p < 0.05) results in a modest decrease in respect to Veh, but without significant difference between ICI + E2 and E2 group. However, in marked contrast to its effects on ADP and ATP hydrolysis, pre-treatment of E2injected animals with ICI 182,780 did not block the effects of E2, suggesting that the effects of E2 on AMP hydrolysis were not mediated only by ERα or ERβ. To test this assumption we examined the effects of the selective GPER1 antagonist G-15, which did not affect AMP hydrolysis rate (75.3 ± 2.6 nmol Pi/mg/min) compared to Veh. Similar to the effects of ICI 182,780, pre-treatment of E2-injected animals with G-15 did not block the effects of E2 (60.4 ± 2.5 nmol Pi/mg/min, p < 0.05). Also, no significant difference was observed between G15+E2 and E2 group.
Apparatus, Holliston, MA, USA), hippocampi were dissected and pooled (3/group) in ice-cold isolation medium (0.32 M sucrose, 5 mM Tris-HCl, pH 7.4) for immediate preparation of synaptosomes as previously described [13]. The protein content was determined using bovine serum albumin (BSA) as a standard. 2.5. Enzyme assay The level of ATP, ADP or AMP hydrolysis was evaluated by spectrophotometric determination of liberated inorganic phosphate as reported elsewhere [9,10]. The effect of treatments on nucleotide hydrolysis was evaluated in respect to DMSO-treated group (Veh). DMSO did not induce any significant change in respect to control animals (data not shown). All samples were run in triplicate from at least six separate measurements performed in three independent synaptosome preparations. The mechanism of E2 action and the involvement of ERs in nongenomic modulation of eN activity were assessed in vitro in fresh hippocampal synaptosomal preparations prepared from individual brains (n = 3), and treated with agonists and antagonists of ERs, as described previously [9]. Synaptosomes were pre-incubated for 10 min at 37 °C with one of the following ER ligands: a) 1 μM E2; b) 1 μM E2-BSA(concentration of free E2); 1 μM PPT, 1 μM DPN or 2.5 μM G1 (a specific agonist of G-protein-coupled estrogen receptor 1). The E2, PPT and DPN were dissolved in ethanol in a maximal final concentration of 0.5%, G1 was dissolved in DMSO in a maximal final concentration of 0.5%, while E2-BSA was dissolved in Tris-HCl, pH 7.4. The concentrations of ligands for in vitro experiments were previously evaluated [13,17–21]. The AMP hydrolysis was estimated as described above. Control tubes (C) contained an equivalent amount of final concentration of ethanol, DMSO or BSA alone (0.5% or less) which did not significantly influence eN activity compared to the untreated group (data not shown). 2.6. Data analysis The results are expressed as mean activity (nmol Pi/mg/ min) ± SEM. Data were analyzed with a one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison post hoc test or Student’s t test using Origin 8.0 software package. The values of p < 0.05 or less were considered statistically significant. 3. Results The activity of ectonucleotidase enzymes was already assessed in the hippocampal synaptosomal fractions isolated from Veh- and E2treated male rats [14]. E2 induced significant decrease in ATP (82.7 ± 1.2; 51.0 ± 1.9%, p < 0.001; Fig. 1A) and ADP (18.0 ± 1.6 nmol Pi/mg/min; 61.9 ± 6.1%, p < 0.001; Fig. 1B) hydrolysis compared to Veh (162.1 ± 5.5 nmol Pi/mg/min for ATP and 29.1 ± 1.2 nmol Pi/mg/min for ADP). The role of ERs in E2-mediated decrease of ATP/ADP hydrolysis levels was assessed in males that were systemically treated with the ERα- or ERβ-specific agonists, PPT or DPN, respectively. As shown in Fig. 1, only DPN showed the ability to mimic E2 effect and to decrease rates of ATP (72.5 ± 13.01 nmol Pi/ mg/min) and ADP (19.8 ± 1.2 nmol Pi/mg/min) hydrolysis in respect to Veh. To test whether the observed attenuation of ATP and ADP hydrolysis was mediated by estrogen receptors α and/or -β, animals were systemically treated with a nonselective estrogen receptor antagonist ICI 182.780 alone (ICI) or 2 h prior to E2 administration (ICI + E2). Since the treatments did not produce a significant change in the nucleotides hydrolysis, it was concluded that E2 affects ATPase/ADPase activity via ERβ activation. The last enzyme in the cascade of extracellular ATP hydrolysis is eN, which catalyzes the rate-limiting step of conversion of AMP to
Fig. 1. In vivo effect of E2 and ERs on synaptic NTPDase activity. The (A) ATP and (B) ADP hydrolysis were determined in hippocampal synaptosomes obtained from male rats after systemic administration of single doses of E2, PPT, DPN, ICI or ICI + E2. Bars represent mean activity (nmol Pi/mg/min) ± SEM. *p < 0.05 or less indicates difference compared to vehicle-treated control (Veh), analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test. 3
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in NTPDases activities attenuation in the synaptic compartment. According to previously published data, a decrease in ATP/ADP hydrolysis rates might be the result of reduced protein abundances of NTPDase1 and NTPDase2 in the hippocampal synaptosomes [14]. However, it is well known that extracellular ATP and ADP in the synaptic cleft are hydrolyzed by three distinct synaptic NTPDases [3,4,13,14] and it is difficult to separate the individual contribution of each enzyme to the total specific activity. Thus, whether the ERβ activation by its agonist DPN initiates the exact same alterations of protein abundance of NTPDases or other receptors are included, needs further and more complex characterization. Selective agonists of ERα and ERβ, PPT and DPN, respectively, completely imitate E2-mediated decrease of eN activity both in vivo and in vitro. The failure of G15 pre-treatment to affect E2-mediated reduction of AMP hydrolysis suggests that the effects of E2 on AMP hydrolysis were not mediated by GPER1. However, similar negative effects obtained after pre-treatment with the ERα/β selective antagonist ICI 182,780, suggest that the effects of E2 on AMP hydrolysis may occur through an ER-independent pathway. Also, treatment with the GPER1 agonist G1, similar to treatment with the ERα/β selective agonists PPT and DPN, is sufficient to reduce AMP activity. These results, while curious, are also consistent with ER-independent effects of E2 on AMP hydrolysis. Furthermore, it needs to be emphasized that in considering the magnitude of the enzyme response, application of agonists of either ERα, ERβ or GPER1 resulted in a similar decrease of AMP hydrolysis rates. Also, an inhibition of ERα/β or GPER1 by specific antagonists, applied before steroid, does not influence E2-induced decrease of synaptic eN activity. Thus, informations about the relative contribution of each investigated receptor is still unclear. Since several issues possibly influence the agonist/antagonist effects (i.e. dosage, endogenous levels of estradiol, level of receptor expression and localization in the synaptic compartments), the study designed to parse out the more precise involvement of each ER subtype in the regulation/modulation of synaptic eN activity needs to be caried out. Molecular mechanisms by which ERs influence synaptic ectonucleotidases activity are still unknown. Our previous study indicate that E2 does not change eN protein abundance [14]. According to present and previous results, we can assume that E2 (probably by activating ERβ) lowers the NTPDases activity, which might ultimately lead to higher extracellular concentration of ATP/ADP and inhibition of eN activity. It is previously demonstrated in several physiological contexts that the rate of eN activity does not necessarily correlate with the enzyme protein level [11,13,14,24]. In the extracellular space ATP/ADP hydrolysis arises from the activity of NTPDases and decrease of their individual activities may result in an increase in extracellular concentrations of ATP/ADP and increased ATP/adenosine ratio. ATP/ADP may consequently initiate P2X- and P2Y- mediated responses that are necessary for ongoing synaptic rearrangements observed in the male hippocampus after E2 administration [14]. On the other hand, higher concentrations of ATP/ADP in the extrasynaptic space may ultimately lead to inhibition of eN activity. As already demonstrated, ATP and ADP are competitive inhibitors of eN activity [9]. Thus, when the amounts of released ATP are high, AMP will have to accumulate due to the feedforward inhibition of eN. In the hippocampal terminals, the levels of ATP and/or ADP has to decrease below the average value of 5μM to allow adenosine formation in high transient amounts. Also, our previous work revealed that following E2 and subsequent decrease in synaptic NTPDase activity in the hippocampal synaptosomes of male rats, changes in eN activity probably resulted in changed kinetic parameters of the enzyme (i.e. reduced substrate affinity and decrease in eN catalytic efficiency) [14]. Besides the feed-forward inhibition, E2, by binding to ERα and/or GPER1, might also modulate eN activity in a way that does not include changes in ATP/ADP levels. Non-genomic action of classical and nonclassical ERs include interactions with other membrane proteins and membrane-initiated responses. Namely, ERs are functionally or
Fig. 2. In vivo effect of E2 and ERs on synaptic eN activity. The AMP hydrolysis was determined in hippocampal synaptosomes obtained from male rats after systemic administration of single doses of E2, PPT, DPN, G1, ICI, ICI + E2, G15, G-15+E2. Bars represent mean activity (nmol Pi/mg/min) ± SEM. *p < 0.05 or less indicates significant difference compared to vehicle-treated control (Veh), analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test.
The next set of experiments was conducted in order to gain mechanistic insight into the modulatory role of ERs in the extracellular AMP hydrolysis. Since our previous work showed that E2 does not influence eN protein abundance in vivo [14], we also performed in vitro study to evaluate whether activation of ERs might influence eN activity by non-genomic mechanisms. Purified hippocampal synaptosomes isolated from intact male rats were incubated in vitro with the selected ERs ligands (as described in the Material and Methods section). As expected, E2 induced a noticeable decrease in AMP hydrolysis (53.04 ± 17.18 nmol Pi/mg/min; p < 0.001) in respect to control (81.18 ± 13.81 nmol Pi/mg/min) (Fig. 3A). To evaluate the participation of putative membrane-associated estrogen receptors in the observed E2 action, the synaptosome preparations were incubated with E2-BSA, which is a membrane-impermeable E2 conjugate. The results shown in Fig. 3B, demonstrate that E2-BSA exhibited the potential to significantly decrease AMP hydrolysis (50.15 ± 14.01 nmol Pi/mg/ min; p < 0.001), in respect to control (84.08 ± 5.41 nmol Pi/mg/ min). To identify the ER subtype(s) involved in the reduction of AMP hydrolysis rate, the synaptosomal fractions were pretreated with a selective agonist of ERα (PPT), ERβ (DPN) or G-protein-coupled estrogen receptor 1 - GPER1 (G1). All applied ligands significantly attenuated AMP hydrolysis (PPT, 35.71 ± 13.91 nmol Pi/mg/min, p < 0.001; DPN, 44.88 ± 8.21 nmol Pi/mg/min, p < 0.001; G1, 43.84 ± 2.67 nmol Pi/mg/min, p < 0.001), in respect to the corresponding controls (Fig. 3). These results imply that all tested ER subtypes (ERα, ERβ and/or GPER1) have a role in the estrogen-induced decrease in eN activity. 4. Discussion Extracellular ATP and adenosine are important modulators of hippocampal function and plasticity [1,22,23]. According to previously published data, E2 attenuates the activity of whole ectonucleotidase enzyme chain responsible for extracellular ATP hydrolysis [14]. In the present study, we have shown that in hormone-responsive brain structures, such as hippocampus, both classical (ERα, ERβ) and nonclassical (GPER1) estradiol receptors appear to collaborate in the modulation of the NTPDase/eN activity. Through the action mediated by ERβ, E2 decreases ATP and ADP hydrolysis, thus probably mediated 4
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Fig. 3. In vitro effects of estradiol and estradiol receptors on synaptic eN activity. The AMP hydrolysis was assessed after incubation of hippocampal synaptosomes obtained from intact males with indicatedconcentrations of (A) E2, (B) E2-BSA, (C) PPT, (D) DPN and (E) G1. Bars represent mean activity (nmol Pi/mg/min) ± SEM. *p < 0.01 or less indicates significant difference compared to control (C), analyzed with a Student’s t test.
Results of the current and previous studies suggest sex-related differences in the E2 modulatory actions on ectonucleotidases activity. Our previous study has shown twice as high eN activity in rat male hippocampal samples than the samples obtained from ovariectomized rats (OVX) [12]. It has been also shown that eN activity fluctuates through the estrous cycle and that in OVX samples is under control of estrogen receptor-α and -β [13]. In female rats, E2 enhances eN activity through ERα-induced transcriptional up-regulation, whereas the actions through ERβ lead to an increase in the eN activity, without corresponding changes in the eN gene and protein expression. In both cases, to make the impact, E2 has to cross the cell membrane and to enter the synaptic compartment where it finds ERs [13]. In the sharp contrast, in the male hippocampal synaptosomes, E2 attenuates NTPDase and eN activities [14]. The present study demonstrated participation of all synaptic ERs in attenuation of ATP/ADP/AMPase activity. Taken together, the results are in line with the extensively described role of E2 and ERs in the regulation and maintaining of sex differences in models of neuroplasticity [5,32]. It is interesting to note that all the above-mentioned results indicate that E2-induced synaptic remodeling reflects sex differences in which the same endpoint is achieved through distinct mechanisms in males vs. females.
physically coupled to glutamate receptors, receptor tyrosine kinases and non-receptor kinases and activate Ca2+ and NO signaling and initiate different cellular effects [25]. Thus, the possibility that activation of common signaling cascade(s) may modulate ectonucleotidase activity by changing the enzyme protein conformation and/or topography cannot be excluded [3]. Furthermore, ectonucleotidases interaction with ERα/β or GPER1 is supported by their exact spatial distribution, as well as by their enrichment and physical proximity in the hippocampal synapses [25–29]. In our previous work, we partly documented that ERβ establishes interactions with eN in the synaptic membranes [13]. ERs may interact directly with the eN protein to induce allosteric modification of the enzyme activity [30,31]. It should be noted that our study has some limitations when it comes to the influence of GPER1 activation on eN activity. Namely, it is not clear whether the inhibitory activity of G1 is a result of selective binding to GPER1. Our pilot experiment showed that concentrations of G1 up to 1 μM, (those are the concentrations which do not activate classical ERs) [17]), had no effects on eN activity in vitro (data not shown). Since there are no data about binding specificity for our used concentration of G1 (2.5 μM) in respect to ERα and ERβ these results should be taken with appropriate caution. Since the precise mechanisms of ERα/β or GPER1 that coordinate the NTPDases and eN responses are still unknown as well as intracellular signaling activated with the enzymes, further analyses are necessary to address this issue and gain further mechanistic insight into modulatory effects of activated ERs.
5. Conclusion In the present study, we show participation of ERα, ERβ, and GPER1 5
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in the modulation of eN activity in the hippocampal synaptosomes of male rats, as the main adenosine-producing enzyme. Since both ATP and adenosine function as neuromodulators in the hippocampal networks influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.
[14]
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Author’s contributions NM and IG designed and conducted the experiment and statistical analysis, analyzed data and wrote the manuscript MD, MZ conducted the experiment DD contributed reagents NN wrote the manuscript All authors have read and approved the final manuscript
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[17]
Declaration of Competing Interest
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The authors declare no conflict of interest. [19]
Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, grants OI 173044 and III 41014.
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Research article
Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats
T
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Nataša Mitrovića,1, , Milorad Dragićb, Marina Zarića, Dunja Drakulića, Nadežda Nedeljkovićb, Ivana Grkovića,1 a b
Department of Molecular Biology and Endocrinology, VINČA Institute of Nuclear Sciences, University of Belgrade, Mike Petrovića Alasa 12-14, 11001 Belgrade, Serbia Department for General Physiology and Biophysics, Faculty of Biology, University of Belgrade, Belgrade, Studentski trg 3, 11001 Belgrade, Serbia
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Ectonucleotidase activity Estradiol receptors Hippocampus Male rats
Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and adenosine, are among least investigated signaling factors that participate in 17β-estradiol (E2)-mediated synaptic rearrangements in rodent hippocampus. Their levels in the extrasynaptic space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases1-3 (NTPDase1-3)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, the aim of the present study was to get closer insight in the E2-induced decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors (ERs i.e. ERα, ERβ or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -β (ERβ) and G- protein coupled estrogen receptor 1 (GPER1) in the mediation of E2 actions in hippocampal synaptosomes of male rats. Synaptic NTPDase1-3 activities are modulated only through activation of ERβ, while activation of ERα, -β and/or non-classical GPER1 decreases synaptic eN activity. Since both ATP and
Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; E2, 17β-estradiol; NTPDase1-3, ecto-nucleoside triphosphate diphosphohydrolases1-3; eN, ecto-5'-nucleotidase; ERs, estradiol receptors; ER, estradiol receptor; ERβ, estradiol receptor β; GPER1, G protein-coupled estrogen receptor 1; GPI, glycosylphosphatidylinositol; PPT, estradiol receptor agonist 13,5-tris(4-hydroxyphenyl)-4-propyl-1 H-pyrazole; DPN, estradiol β receptor agonist 23-bis(4-hydroxyphenyl)-propionitrile; ICI, nonselective estrogen receptor antagonist 7a 17b-[9-[(4,4,5,5,5 Pentafluoropentyl)sulfinyl]nonyl]estra1,3,5(10)-triene-3,17-diol (ICI 182,780); G1, selective GPER1 agonist; G-15, selective GPER1 antagonist; BSA, bovine serum albumin; DMSO, dymethyl sulfoxide; NO, nitric oxide; OVX, ovariectomy ⁎ Corresponding author at: Department of Molecular Biology and Endocrinology, VINČA Institute of Nuclear Science, University of Belgrade, Mike Petrović Alasa 12-14, 11000 Belgrade, Vinča, Serbia. E-mail address: [email protected] (N. Mitrović). 1 These authors contributed equally. https://doi.org/10.1016/j.neulet.2019.134474 Received 1 March 2019; Received in revised form 23 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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adenosine function as neuromodulators in the hippocampal networks, influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.
1. Introduction
different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance [11,14]. Thus, the aim of this study was to get closer insight in the E2-mediated decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors responsible for the observed effects.
Adenosine triphosphate (ATP) and its nucleoside adenosine act as versatile extracellular signaling molecules in the central nervous system (CNS) and are implicated in regulation of hippocampal function and plasticity [1,2]. Actions of ATP are mediated via two classes of ligandgated P2X and G protein-coupled P2Y receptors which are abundantly expressed in the brain [1]. In contrast to ATP, adenosine acts as a retaliatory molecule and neuromodulator, which operates via four G protein-coupled adenosine receptors (A1R, A2A, A2B and A3) [2]. The levels of ATP and adenosine in the extracellular space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases (NTPDases)/ecto-5′-nucleotidase (eN) enzyme chain [3]. Three distinct membrane-bound NTPDases (NTPDase1-3) are expressed in the CNS which differ in their preference for the substrate [3,4]. NTPDase1/ CD39 hydrolyzes ATP and ADP equally well to AMP, NTPDase2 exclusively degrades ATP to ADP, while NTPDase3 is the functional intermediate which preferentially hydrolyzes ATP, with a transient accumulation of ADP [3]. Thus, NTPDases provide the substrate (AMP) for the final and rate-limiting step of extracellular ATP degradation, catalyzed by ecto-5'-nucleotidase (CD73, eN). Ecto-5′-nucleotidase is a Zn2+-binding glycosylphosphatidylinositol (GPI)-anchored protein, with its catalytic site facing the extracellular compartment. It shows an affinity for 5'-AMP in the micromolar range, thus producing extracellular adenosine [3]. Since NTPDases and eN control extracellular levels of ATP and adenosine, regulatory mechanisms involved in the control of Ntpd1-3 and Nt5e gene expression, translation, post-translational modifications, membrane expression and the catalytic activity are of high relevance for normal brain functioning. It has been known for decades that female sex hormones, 17β-estradiol (E2), in particular, affect hippocampal morphology, plasticity and memory in male as well as in female rodents [5]. A recent study showed that in vivo application of E2 increases density of dendritic spines in the hippocampus of adult gonadectomized males, rapidly as in females [6]. Also, E2 potentiates excitatory synapses in the hippocampus of both sexes [7,8]. Furthermore, NTPDase1–3 and eN are localized along with estradiol receptors (ERs) in the rat hippocampal synaptic compartment [5,9–14] and the female ovarian hormones modulate the activities of the enzymes in female, as well as in male rat brain [11,10–14]. While E2 induces marked increase in ATP/ADP/ AMPase activity in the hippocampus of female rats, a single systemic injection of E2 in the male rats results in marked attenuation of a complete chain of adenine nucleotide hydrolysis. Immunoblot analyses revealed that lower ATP and ADP hydrolysis probably result from decreased protein abundance of NTPDase1 and NTPDase2 in the synaptic compartment, while a decrease in AMP hydrolysis following E2 treatment is not the consequence of alteration in the protein level, but rather the inhibition of eN catalytic activity [14]. Kinetic analysis showed that E2 alters kinetic properties of eN and reduces the enzyme catalytic efficiency by decreasing the enzyme affinity toward AMP [14]. It is known that high extracellular levels of the upstream substrates, ADP and ATP, may competitively inhibit eN enzyme activity in a feed-forward manner [9]. Therefore, we hypothesized that in male hippocampal synaptic membranes E2 directly attenuates the activity of NTPDases, which leads to accumulation of ATP and ADP, thus consequently induce feed-forward inhibition of eN. The aforementioned decline in NTPDase/eN activity after E2 injection in vivo was accompanied with activation of the molecular machinery required for synaptogenesis and synaptic refinement, including mTOR-mediated signaling [14]. Altogether, our previous evaluation supports the idea of specific roles of
2. Materials and methods 2.1. Materials Analytical grade salts and buffer reagents, 17b-estradiol 3-benzoate (E2), b-estradiol 6-(o-carboxy-methyl) oxime: bovine serum albumin (E2–BSA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Estradiol receptor agonists 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1 H-pyrazole (PPT), 2,3-bis(4hydroxyphenyl)-propionitrile (DPN), G1 and nonselective ERα/β antagonist 7a, 17b-[9-[(4,4,5,5,5 Pentafluoropentyl)sulfinyl]nonyl]estra1,3,5(10)-triene-3,17-diol (ICI 182,780) and GPER1 selective antagonist (G-15) were purchased from Tocris Bioscience (Ellisville, MO, USA). E2-BSA was filtered before use to eliminate free E2. 2.2. Animals All experiments were conducted using 3-month old, gonadally intact male rats (300–350 g) of the Wistar strain, obtained from a local colony. The care was taken to alleviate the pain and discomfort of the animals. Animals were treated in accordance with the European Community Council Directive of 86/609/ EEC for animal experiment. Research procedures were approved by the Ethical Committee for the Use of Laboratory Animals of VINČA institute of nuclear sciences, University of Belgrade, Belgrade, Republic of Serbia (Licence No. 02/11; 323-0703832/2015-05/1). Animals were housed (3–4/cage) under standard conditions: 12 h light/dark regime, constant ambient temperature (22 ± 2 °C) and humidity with ad libitum access to food and water. 2.3. Experimental groups and treatments Animals were randomly divided into the following groups (6–9 rats/ group): Veh - animals which received an injection of DMSO (1 ml/kg) were used as control group; E2 - animals injected with 17b-estradiol 3benzoate (33.3 μg/kg); PPT - animals which received an injection of a selective ERα agonist PPT (2.5 mg/kg); DPN - animals which received selective ERβ agonist DPN (2.5 mg/kg); G1 – animals treated with one dose of GPER1 agonist G1 (5 μg); ICI - animals treated with a non-selective antagonist (ICI 182,780; 2 mg/kg) of ERα and ERβ, but not GPER1; ICI + E2 - animals which received injection of ICI 182,780, 2 h prior to E2 administration (2 mg/kg + 33.3 μg/kg); G-15 – animals treated with one dose of selective GPER1 antagonist G-15 (40 μg); G15+E2 - animals which received one dose of G-15 two hours prior to E2 (40 μg + 33,3 μg/kg). Animals were treated s.c. at 9:00 a.m. and sacrificed 24 h after the treatment. An additional group of gonadally intact male rats without any treatment was used to investigate the impact of the vehicle on investigated parameters (Control). The dosages of E2, PPT, DPN, ICI, G-15, and G1 were determined previously [13–16]. 2.4. Synaptosomes preparation After decapitation with a small animal guillotine (Harvard 2
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adenosine. It has been previously demonstrated that E2 induces a significant decrease in the eN activity (56.3 ± 1.5 nmol Pi/mg/min) in respect to Veh (76.3 ± 1.8 nmol Pi/mg/min, Fig. 2) [14]. As shown in Fig. 2 administration of selective agonists of ERα, ERβ, or GPER1 (PPT, DPN, or G1, respectively) results in lower rates of AMP hydrolysis (53.6 ± 2.5 nmol Pi/mg/min, p < 0.001, 61.03 ± 2.3 nmol Pi/mg/ min, p < 0.001, 55.4 ± 3.3 nmol Pi/mg/min, p < 0.05, respectively) in respect to Veh (76.3 ± 1.8 nmol Pi/mg/min). Administration of ICI 182,780 on its own did not affect the rate of AMP hydrolysis, while ICI + E2 treatment (ICI + E2; 63.5 ± 2.1 nmol Pi/mg/min, p < 0.05) results in a modest decrease in respect to Veh, but without significant difference between ICI + E2 and E2 group. However, in marked contrast to its effects on ADP and ATP hydrolysis, pre-treatment of E2injected animals with ICI 182,780 did not block the effects of E2, suggesting that the effects of E2 on AMP hydrolysis were not mediated only by ERα or ERβ. To test this assumption we examined the effects of the selective GPER1 antagonist G-15, which did not affect AMP hydrolysis rate (75.3 ± 2.6 nmol Pi/mg/min) compared to Veh. Similar to the effects of ICI 182,780, pre-treatment of E2-injected animals with G-15 did not block the effects of E2 (60.4 ± 2.5 nmol Pi/mg/min, p < 0.05). Also, no significant difference was observed between G15+E2 and E2 group.
Apparatus, Holliston, MA, USA), hippocampi were dissected and pooled (3/group) in ice-cold isolation medium (0.32 M sucrose, 5 mM Tris-HCl, pH 7.4) for immediate preparation of synaptosomes as previously described [13]. The protein content was determined using bovine serum albumin (BSA) as a standard. 2.5. Enzyme assay The level of ATP, ADP or AMP hydrolysis was evaluated by spectrophotometric determination of liberated inorganic phosphate as reported elsewhere [9,10]. The effect of treatments on nucleotide hydrolysis was evaluated in respect to DMSO-treated group (Veh). DMSO did not induce any significant change in respect to control animals (data not shown). All samples were run in triplicate from at least six separate measurements performed in three independent synaptosome preparations. The mechanism of E2 action and the involvement of ERs in nongenomic modulation of eN activity were assessed in vitro in fresh hippocampal synaptosomal preparations prepared from individual brains (n = 3), and treated with agonists and antagonists of ERs, as described previously [9]. Synaptosomes were pre-incubated for 10 min at 37 °C with one of the following ER ligands: a) 1 μM E2; b) 1 μM E2-BSA(concentration of free E2); 1 μM PPT, 1 μM DPN or 2.5 μM G1 (a specific agonist of G-protein-coupled estrogen receptor 1). The E2, PPT and DPN were dissolved in ethanol in a maximal final concentration of 0.5%, G1 was dissolved in DMSO in a maximal final concentration of 0.5%, while E2-BSA was dissolved in Tris-HCl, pH 7.4. The concentrations of ligands for in vitro experiments were previously evaluated [13,17–21]. The AMP hydrolysis was estimated as described above. Control tubes (C) contained an equivalent amount of final concentration of ethanol, DMSO or BSA alone (0.5% or less) which did not significantly influence eN activity compared to the untreated group (data not shown). 2.6. Data analysis The results are expressed as mean activity (nmol Pi/mg/ min) ± SEM. Data were analyzed with a one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison post hoc test or Student’s t test using Origin 8.0 software package. The values of p < 0.05 or less were considered statistically significant. 3. Results The activity of ectonucleotidase enzymes was already assessed in the hippocampal synaptosomal fractions isolated from Veh- and E2treated male rats [14]. E2 induced significant decrease in ATP (82.7 ± 1.2; 51.0 ± 1.9%, p < 0.001; Fig. 1A) and ADP (18.0 ± 1.6 nmol Pi/mg/min; 61.9 ± 6.1%, p < 0.001; Fig. 1B) hydrolysis compared to Veh (162.1 ± 5.5 nmol Pi/mg/min for ATP and 29.1 ± 1.2 nmol Pi/mg/min for ADP). The role of ERs in E2-mediated decrease of ATP/ADP hydrolysis levels was assessed in males that were systemically treated with the ERα- or ERβ-specific agonists, PPT or DPN, respectively. As shown in Fig. 1, only DPN showed the ability to mimic E2 effect and to decrease rates of ATP (72.5 ± 13.01 nmol Pi/ mg/min) and ADP (19.8 ± 1.2 nmol Pi/mg/min) hydrolysis in respect to Veh. To test whether the observed attenuation of ATP and ADP hydrolysis was mediated by estrogen receptors α and/or -β, animals were systemically treated with a nonselective estrogen receptor antagonist ICI 182.780 alone (ICI) or 2 h prior to E2 administration (ICI + E2). Since the treatments did not produce a significant change in the nucleotides hydrolysis, it was concluded that E2 affects ATPase/ADPase activity via ERβ activation. The last enzyme in the cascade of extracellular ATP hydrolysis is eN, which catalyzes the rate-limiting step of conversion of AMP to
Fig. 1. In vivo effect of E2 and ERs on synaptic NTPDase activity. The (A) ATP and (B) ADP hydrolysis were determined in hippocampal synaptosomes obtained from male rats after systemic administration of single doses of E2, PPT, DPN, ICI or ICI + E2. Bars represent mean activity (nmol Pi/mg/min) ± SEM. *p < 0.05 or less indicates difference compared to vehicle-treated control (Veh), analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test. 3
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in NTPDases activities attenuation in the synaptic compartment. According to previously published data, a decrease in ATP/ADP hydrolysis rates might be the result of reduced protein abundances of NTPDase1 and NTPDase2 in the hippocampal synaptosomes [14]. However, it is well known that extracellular ATP and ADP in the synaptic cleft are hydrolyzed by three distinct synaptic NTPDases [3,4,13,14] and it is difficult to separate the individual contribution of each enzyme to the total specific activity. Thus, whether the ERβ activation by its agonist DPN initiates the exact same alterations of protein abundance of NTPDases or other receptors are included, needs further and more complex characterization. Selective agonists of ERα and ERβ, PPT and DPN, respectively, completely imitate E2-mediated decrease of eN activity both in vivo and in vitro. The failure of G15 pre-treatment to affect E2-mediated reduction of AMP hydrolysis suggests that the effects of E2 on AMP hydrolysis were not mediated by GPER1. However, similar negative effects obtained after pre-treatment with the ERα/β selective antagonist ICI 182,780, suggest that the effects of E2 on AMP hydrolysis may occur through an ER-independent pathway. Also, treatment with the GPER1 agonist G1, similar to treatment with the ERα/β selective agonists PPT and DPN, is sufficient to reduce AMP activity. These results, while curious, are also consistent with ER-independent effects of E2 on AMP hydrolysis. Furthermore, it needs to be emphasized that in considering the magnitude of the enzyme response, application of agonists of either ERα, ERβ or GPER1 resulted in a similar decrease of AMP hydrolysis rates. Also, an inhibition of ERα/β or GPER1 by specific antagonists, applied before steroid, does not influence E2-induced decrease of synaptic eN activity. Thus, informations about the relative contribution of each investigated receptor is still unclear. Since several issues possibly influence the agonist/antagonist effects (i.e. dosage, endogenous levels of estradiol, level of receptor expression and localization in the synaptic compartments), the study designed to parse out the more precise involvement of each ER subtype in the regulation/modulation of synaptic eN activity needs to be caried out. Molecular mechanisms by which ERs influence synaptic ectonucleotidases activity are still unknown. Our previous study indicate that E2 does not change eN protein abundance [14]. According to present and previous results, we can assume that E2 (probably by activating ERβ) lowers the NTPDases activity, which might ultimately lead to higher extracellular concentration of ATP/ADP and inhibition of eN activity. It is previously demonstrated in several physiological contexts that the rate of eN activity does not necessarily correlate with the enzyme protein level [11,13,14,24]. In the extracellular space ATP/ADP hydrolysis arises from the activity of NTPDases and decrease of their individual activities may result in an increase in extracellular concentrations of ATP/ADP and increased ATP/adenosine ratio. ATP/ADP may consequently initiate P2X- and P2Y- mediated responses that are necessary for ongoing synaptic rearrangements observed in the male hippocampus after E2 administration [14]. On the other hand, higher concentrations of ATP/ADP in the extrasynaptic space may ultimately lead to inhibition of eN activity. As already demonstrated, ATP and ADP are competitive inhibitors of eN activity [9]. Thus, when the amounts of released ATP are high, AMP will have to accumulate due to the feedforward inhibition of eN. In the hippocampal terminals, the levels of ATP and/or ADP has to decrease below the average value of 5μM to allow adenosine formation in high transient amounts. Also, our previous work revealed that following E2 and subsequent decrease in synaptic NTPDase activity in the hippocampal synaptosomes of male rats, changes in eN activity probably resulted in changed kinetic parameters of the enzyme (i.e. reduced substrate affinity and decrease in eN catalytic efficiency) [14]. Besides the feed-forward inhibition, E2, by binding to ERα and/or GPER1, might also modulate eN activity in a way that does not include changes in ATP/ADP levels. Non-genomic action of classical and nonclassical ERs include interactions with other membrane proteins and membrane-initiated responses. Namely, ERs are functionally or
Fig. 2. In vivo effect of E2 and ERs on synaptic eN activity. The AMP hydrolysis was determined in hippocampal synaptosomes obtained from male rats after systemic administration of single doses of E2, PPT, DPN, G1, ICI, ICI + E2, G15, G-15+E2. Bars represent mean activity (nmol Pi/mg/min) ± SEM. *p < 0.05 or less indicates significant difference compared to vehicle-treated control (Veh), analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test.
The next set of experiments was conducted in order to gain mechanistic insight into the modulatory role of ERs in the extracellular AMP hydrolysis. Since our previous work showed that E2 does not influence eN protein abundance in vivo [14], we also performed in vitro study to evaluate whether activation of ERs might influence eN activity by non-genomic mechanisms. Purified hippocampal synaptosomes isolated from intact male rats were incubated in vitro with the selected ERs ligands (as described in the Material and Methods section). As expected, E2 induced a noticeable decrease in AMP hydrolysis (53.04 ± 17.18 nmol Pi/mg/min; p < 0.001) in respect to control (81.18 ± 13.81 nmol Pi/mg/min) (Fig. 3A). To evaluate the participation of putative membrane-associated estrogen receptors in the observed E2 action, the synaptosome preparations were incubated with E2-BSA, which is a membrane-impermeable E2 conjugate. The results shown in Fig. 3B, demonstrate that E2-BSA exhibited the potential to significantly decrease AMP hydrolysis (50.15 ± 14.01 nmol Pi/mg/ min; p < 0.001), in respect to control (84.08 ± 5.41 nmol Pi/mg/ min). To identify the ER subtype(s) involved in the reduction of AMP hydrolysis rate, the synaptosomal fractions were pretreated with a selective agonist of ERα (PPT), ERβ (DPN) or G-protein-coupled estrogen receptor 1 - GPER1 (G1). All applied ligands significantly attenuated AMP hydrolysis (PPT, 35.71 ± 13.91 nmol Pi/mg/min, p < 0.001; DPN, 44.88 ± 8.21 nmol Pi/mg/min, p < 0.001; G1, 43.84 ± 2.67 nmol Pi/mg/min, p < 0.001), in respect to the corresponding controls (Fig. 3). These results imply that all tested ER subtypes (ERα, ERβ and/or GPER1) have a role in the estrogen-induced decrease in eN activity. 4. Discussion Extracellular ATP and adenosine are important modulators of hippocampal function and plasticity [1,22,23]. According to previously published data, E2 attenuates the activity of whole ectonucleotidase enzyme chain responsible for extracellular ATP hydrolysis [14]. In the present study, we have shown that in hormone-responsive brain structures, such as hippocampus, both classical (ERα, ERβ) and nonclassical (GPER1) estradiol receptors appear to collaborate in the modulation of the NTPDase/eN activity. Through the action mediated by ERβ, E2 decreases ATP and ADP hydrolysis, thus probably mediated 4
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Fig. 3. In vitro effects of estradiol and estradiol receptors on synaptic eN activity. The AMP hydrolysis was assessed after incubation of hippocampal synaptosomes obtained from intact males with indicatedconcentrations of (A) E2, (B) E2-BSA, (C) PPT, (D) DPN and (E) G1. Bars represent mean activity (nmol Pi/mg/min) ± SEM. *p < 0.01 or less indicates significant difference compared to control (C), analyzed with a Student’s t test.
Results of the current and previous studies suggest sex-related differences in the E2 modulatory actions on ectonucleotidases activity. Our previous study has shown twice as high eN activity in rat male hippocampal samples than the samples obtained from ovariectomized rats (OVX) [12]. It has been also shown that eN activity fluctuates through the estrous cycle and that in OVX samples is under control of estrogen receptor-α and -β [13]. In female rats, E2 enhances eN activity through ERα-induced transcriptional up-regulation, whereas the actions through ERβ lead to an increase in the eN activity, without corresponding changes in the eN gene and protein expression. In both cases, to make the impact, E2 has to cross the cell membrane and to enter the synaptic compartment where it finds ERs [13]. In the sharp contrast, in the male hippocampal synaptosomes, E2 attenuates NTPDase and eN activities [14]. The present study demonstrated participation of all synaptic ERs in attenuation of ATP/ADP/AMPase activity. Taken together, the results are in line with the extensively described role of E2 and ERs in the regulation and maintaining of sex differences in models of neuroplasticity [5,32]. It is interesting to note that all the above-mentioned results indicate that E2-induced synaptic remodeling reflects sex differences in which the same endpoint is achieved through distinct mechanisms in males vs. females.
physically coupled to glutamate receptors, receptor tyrosine kinases and non-receptor kinases and activate Ca2+ and NO signaling and initiate different cellular effects [25]. Thus, the possibility that activation of common signaling cascade(s) may modulate ectonucleotidase activity by changing the enzyme protein conformation and/or topography cannot be excluded [3]. Furthermore, ectonucleotidases interaction with ERα/β or GPER1 is supported by their exact spatial distribution, as well as by their enrichment and physical proximity in the hippocampal synapses [25–29]. In our previous work, we partly documented that ERβ establishes interactions with eN in the synaptic membranes [13]. ERs may interact directly with the eN protein to induce allosteric modification of the enzyme activity [30,31]. It should be noted that our study has some limitations when it comes to the influence of GPER1 activation on eN activity. Namely, it is not clear whether the inhibitory activity of G1 is a result of selective binding to GPER1. Our pilot experiment showed that concentrations of G1 up to 1 μM, (those are the concentrations which do not activate classical ERs) [17]), had no effects on eN activity in vitro (data not shown). Since there are no data about binding specificity for our used concentration of G1 (2.5 μM) in respect to ERα and ERβ these results should be taken with appropriate caution. Since the precise mechanisms of ERα/β or GPER1 that coordinate the NTPDases and eN responses are still unknown as well as intracellular signaling activated with the enzymes, further analyses are necessary to address this issue and gain further mechanistic insight into modulatory effects of activated ERs.
5. Conclusion In the present study, we show participation of ERα, ERβ, and GPER1 5
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in the modulation of eN activity in the hippocampal synaptosomes of male rats, as the main adenosine-producing enzyme. Since both ATP and adenosine function as neuromodulators in the hippocampal networks influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.
[14]
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Author’s contributions NM and IG designed and conducted the experiment and statistical analysis, analyzed data and wrote the manuscript MD, MZ conducted the experiment DD contributed reagents NN wrote the manuscript All authors have read and approved the final manuscript
[16]
[17]
Declaration of Competing Interest
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The authors declare no conflict of interest. [19]
Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, grants OI 173044 and III 41014.
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