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Article Type: Original Article
HYPERTHERMIA-INDUCED SEIZURES ALTER ADENOSINE A1 AND A2A RECEPTORS AND 5´-NUCLEOTIDASE ACTIVITY IN RAT CEREBRAL CORTEX
David Agustín León-Navarro*, José L. Albasanz and Mairena Martín
Departamento de Química Inorgánica, Orgánica y Bioquímica. Facultad de Ciencias y Tecnologías Químicas. Centro Regional de Investigaciones Biomédicas. Universidad de Castilla-La Mancha. Avenida Camilo José Cela, 10. 13071 Ciudad Real -SPAIN-
*Address correspondence and reprint requests to: Dr. David Agustín León Navarro. Área de Bioquímica. Departamento de Química Inorgánica, Orgánica y Bioquímica. Facultad de Ciencias y Tecnologías Químicas. Avenida Camilo José Cela, 10. 13071 Ciudad Real. Spain. e-mail: [email protected] Tel.: +34 926295300 Ext: 3432. Fax: +34 926295318.
Running title: Effect of fever seizures on adenosine receptors.
Keywords: Fever seizures, adenosine receptor, 5,-nucleotidase, postnatal, cerebral cortex.
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Abbreviations: ADA, adenosine deaminase. A1R, adenosine A1 receptor. A2AR, adenosine A2A receptor. ACII , adenylyl cyclase type II. CPA, N6-cyclopentyladenosine. [3H]DPCPX , cyclopentyl-1,3-dypropylxanthine,8-[dipropy-2,3-3H(N)] FS, febrile seizures. NTPDases, nucleoside triphosphate diphosphohydrolases. [3H]ZM241385, [2-3H](4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl]phenol).
ABSTRACT Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5´-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR and 5´-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [3H]DPCPX as radioligand, and mRNA coding A1 was observed 48 hours after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [3H]ZM241385 as radioligand, 48 hours after hyperthermiaevoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5´-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5´-
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ACKNOWLEDGEMENTS
This work was supported in part by grants from the Ministerio de Economía y Competitividad from Spain (BFU2011-23034). We thank María Angeles Ruíz and Inmaculada Iglesias for helping us with dissection neonates. Authors have no conflicts of interest to declare.
REFERENCES Adén U., O'Connor W.T., and Berman R.F. (2004) Changes in purine levels and adenosine receptors in kindled seizures in the rat. Neuroreport 15, 1585-9. Angelatou F., Pagonopoulou O. and Kostopoulos G (1990) Alterations of A1 adenosine receptors in different mouse brain areas after pentylentetrazol-induced seizures, but not in the epileptic mutant mouse 'tottering'. Brain Res. 534, 251-6. Angelatou F., Pagonopoulou O. and Kostopoulos G (1991) Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas. Neurosci. Lett. 132, 203-6. Augusto E., Matos M., Sévigny J., El-Tayeb A., Bynoe M.S., Müller C.E., Cunha R.A., and Chen J.F. (2013) Ecto-5'-nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A2A receptor functions. J. Neurosci. 33, 11390-9. Biber K., Lubrich B., Fiebich B.L., Boddeke H.W. and van Calker D. (2001) Interleukin-6 enhances expression of adenosine A(1) receptor mRNA and signaling in cultured rat cortical astrocytes and brain slices. Neuropsychopharmacology 24, 86-96. Biber K., Pinto-Duarte A., Wittendorp M.C., Dolga A.M., Fernandes C.C., Von Frijtag Drabbe Künzel J., Keijser J.N., de Vries R., Ijzerman A.P., Ribeiro J.A., Eisel U., Sebastião A.M. and Boddeke H.W.
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has a proconvulsive effect on piriform cortex-kindled seizures and play a significant role in the pentylenetetrazol-induced kindling of seizures (Hosseinmardi ei al., 2007; El Yacoubi et al., 2009).
Extracellular adenosine can be formed by the combined action of a group of membrane-bound enzymes known as ectonucleotidases, which includes nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5´-nucleotidase, which hydrolizes 5´-AMP to adenosine (Zimmermann et al., 2012). Different works have shown changes in the activity of 5’-nucleotidase in several models of epilepsy (Bonan et al., 2000; Oses et al., 2007; de Paula Cognato et al., 2005).
Although several works have revealed significant changes in the density of adenosine receptors in different animal models of epilepsy as well in human epileptic tissues (see review Pagonopoulou et al., 2006), there are no studies, at least to our knowledge, analysing the effect of fever seizures on adenosine receptors. The aim of this study, therefore, was to examine the density of adenosine A1 and A2A receptors and the corresponding mRNA levels at different times (48 hours, 5 days and 20 days) after fever-induced seizures. The effect of fever-induced seizures on 5´-nucleotidase activity was also investigated.
MATERIALS AND METHODS 1.1. Materials Cyclopentyl-1,3-dypropylxanthine,8-[dipropy-2,3-3H(N)] ([3H]DPCPX 120 Ci/mmol) was from Amersham Biosciences (Buckinghamshire, UK) and [2-3H](4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo[2,3a][1,3,5]triazin-5-ylamino]ethyl]phenol) ([3H]ZM241385 27.4 Ci/mmol) from Tocris (Bristol, UK). Calf intestine
adenosine
deaminase,
theophylline,
N6-cyclopentyladenosine
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(CPA),
levamisole
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The duration of heating was 28 ± 2 minutes. Controls included a normothermic group which were separated from the dams for the same duration and placed in a chamber at room temperature and a hyperthermic control group which were subjected to the same degree and duration of hyperthermia but in which seizures were blocked by treating orally with potassium bromide (KBr) dissolved in saline (1800 mg/Kg) 90 minutes before to the induction of hyperthermia. Potassium bromide is a classical antiepileptic drug which has been shown to be effective against hyperthermia-induced seizures in rats (Hayashi et al., 2011). Rectal temperatures in hyperthermic control group were not significantly different from hyperthermia-associated seizure group which suggest that potassium bromide did not substantially decrease the temperature of neonates at the time of heating. Four of the eleven pups subjected to hyperthermia after KBr intake showed falling over associated with hindlimb clonus seizures and did not include in the work. No changes on the behavioural excitability could be observed after hyperthermic induction.
1.4 Cerebral cortical membranes isolation Cerebral cortical membranes were isolated following the protocol described by León and coworkers (2004) with modifications. Briefly, the brain cortex from pups were homogenized in 20 volumes of isolation buffer (50 mM Tris-HCl, pH 7.4 containing 10 mM MgCl2 and protease inhibitors) in a Dounce homogenizer using pestle A (10 strokes) and B (10 strokes). After homogenization, brain preparations were centrifuged for 5 min at 1000xg in a Beckman JA 21 centrifuge (Coulter España, Madrid, Spain). Supernatant was centrifuged for 20 min at 27000xg and the pellet was finally resuspended in isolation buffer. Protein concentration was measured by the Lowry method, using bovine serum albumin (BSA) as standard. Each cerebral cortical membrane isolation corresponds to one pup per litter.
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1.5 [3H]DPCPX and [3H]ZM241385 binding assays to cortical membranes The effect of fever seizures on A1R and A2AR was studied performing radioligand binding assays to cortical membranes from neonates following protocols described previously (López-Zapata et al., 2011) with minor modifications. Cerebral cortical membranes were incubated with 5 U/mg adenosine deaminase (ADA) in 50 mM Tris, 2 mM MgCl2, pH 7.4, for 30 min at 25º C, to remove endogenous adenosine. This reaction buffer was supplemented with 100 mM NaCl in [3H]ZM241385 binding assays. Then, cerebral cortical membranes (50-100 μg of protein) were incubated with [3H]DPCPX or [3H]ZM241385 for 2 h at 25ºC. Saturation assays were carried out at different [3H]DPCPX and [3H]ZM241385 concentrations (0.5–20 nM) using cyclopentyladenosine (CPA) at a concentration 104 times higher than [3H]DPCPX and 5 mM theophylline, respectively, to obtain nonspecific binding. Binding assays were stopped by rapid filtration through Whatman GF/B filters, previously pre-incubated overnight with 0.3% polyethylenimine using a FilterMate Harvester (PerkinElmer). Filters were then transferred to vials and scintillation liquid was added to measure the radioactivity in a Microbeta Trilux Jet scintillation counter.
1.6 Measurement of 5´-nucleotidase activity 20 μg of cerebral cortical membranes were preincubated in 180 μl of the reaction medium containing: 50 mM Tris, MgCl2 5 mM, pH 9, at 37ºC for 10 minutes. Then, the reaction was initiated by the addition of 20 μl AMP (final concentrations, 1μM - 3 mM) and stopped 10 minutes later by adding 200 μl of 10% trichloroacetic acid (TCA). The samples were chilled on ice for 10 minutes and then centrifuged at 12000xg for 4 minutes at 4ºC. The supernatants were used to measure inorganic phosphate released following the protocol described by Chan and coworkers (1986) using KH2PO4 as Pi standard. Non enzymatic hydrolysis of AMP was corrected by adding cerebral cortical membranes after TCA. Incubation times and protein concentration were selected in order to ensure the linearity
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of the reactions. All samples were run in triplicate. Enzyme activity is expressed as nmol Pi released/min/mg of protein.
1.7 Preparation of total RNA and cDNA Total RNA was extracted from cortex brain using an ABI 6100 Nucleic Acid PrepStation according to the manufacturer's protocol. All chemicals for the ABI 6100 were purchased from Applied Biosystems (Foster City, CA). Total RNA from animals was isolated and stored individually at -80°C. Ratio of A260/A280 (purity of RNA) was in the range 1.9–2.1. RNA concentrations were determined from the A260. One microgram of total RNA was reverse transcribed using Applied Biosystems HighCapacity cDNA Archive Kit. Parallel reactions for each RNA sample were run in the absence of MultiScribe Reverse Transcriptase to assess the degree of contaminating genomic DNA.
1.8 Quantitative Real Time RT-PCR Analysis Quantitative real time RT-PCR analysis (Higuchi et al., 1993) was performed with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan® universal PCR master mix according to the manufacturer’s specifications (Applied Biosystems Inc., Foster City, CA) as previously described (León et al., 2009). The validated TaqMan probes and primers for A1 (assay ID: Rn00567668_m1), A2A (assay ID: Rn00583935_m1), A2B (assay ID: Rn00567697_m1), A3 (assay ID: Rn00563680_m1), type II adenylyl cyclase (Rn00578713_m1) and β-actin (Rn00667869_m1) were assay-on-demand gene expression products (Applied Biosystems). The TaqMan® primer and probe sequences are packaged together in a 20x solution. Rat β-actin gene was used as endogenous control. The gene-specific probes were labeled using reporter dye FAM. A non-fluorescent quencher and the minor groove binder were linked at the 3’ end of probe as quenchers. The thermal cycler conditions were as follows: hold for 20 s at 95 °C, followed by two-step PCR for 40 cycles of 95 °C for
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3 s, followed by 60 °C for 30 s. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems) according to the 2-ΔΔCt method. Briefly, expression results for a gene were normalized to internal control β-actin relative to a calibrator, consisting of the mean expression level of the corresponding gene in control samples as follows: 2ΔΔCt
= 2-((Ct receptor gene - Ct actin)sample - (Ct receptor gene - Ct actin) calibrator). The results from three to five independent
repeat assays, performed on different plates each using different cDNAs from the animals analyzed, were averaged to produce a single mean quantity value.
1.9 Statistical and data analysis Saturation (Bmax, KD) binding curves were analyzed performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program (GraphPad Software, San Diego, CA, USA). In the case of 5´-nucleotidase activity, the Km and Vmax values were calculated using a non-linear Michaelis-Menten curve fitting with the GraphPad Prism 5 program. The effect of fever seizures on Bmax, KD, Vmax and Km and on mRNA level coding adenosine receptors were analyzed using unpaired two-tailed Student´s t test or one way ANOVA following Tukey post-hoc test.
RESULTS Status of A1R and A2AR 48 hours after hyperthermia-induced seizures In order to analyze the effect of hyperthermia-induced seizures on adenosine A1 and A2A receptors from neonatal brain, radioligand binding assays using [3H]DPCPX and [3H]ZM21680, respectively, were carried out. Data obtained could be best fitted to one binding site model. As shown in Figure 1A and table 1, a significant increase in A1R was observed in neonates 48 hours after hyperthermic seizures as compared to control animals (0.559 ± 0.097 pmol/mg protein vs 0.244 ± 0.076 pmol/mg
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protein, p < 0.05). No significant variation in A1R was observed in neonates pretreated with KBr as anticonvulsant before seizures as compared to control rats (0.127 ± 0.012 pmol/mg protein vs 0.244 ± 0.076 pmol/mg protein, p > 0.05). The statistical comparison of KD value between groups failed to find significant variations (control: 5.1 ± 4.7 nM; HIS: 2.1 ± 1.7 nM; HIS + KBr: 0.3 ± 0.2 nM, p > 0.05) suggesting that receptor affinity did not result changed by seizures evoked by hyperthermia.
Concerning to adenosine A2AR, a significant decrease in receptor density was detected in animals submitted to hyperthermia-induced seizures (0.124 ± 0.019 pmol/mg protein vs. 0.271 ± 0.051 pmol/mg protein, p < 0.05) (Figure 1B and table 1). This effect was not present in neonates pretreated with KBr before hyperthermic seizures (0.264 ± 0.036 pmol/mg protein vs. 0.271 ± 0.051 pmol/mg protein, p > 0.05). Similarly to A1R, the KD value of A2A receptor remained unchanged after seizures (control: 20.6 ± 8.3 nM; HIS: 7.6 ± 3.4 nM; HIS + KBr: 9.5 ± 3.5 nM, p > 0.05).
Status of A1R and A2AR 5 days after hyperthermia-induced seizures As illustrated in Figure 2A and table 1, after 5 days of hyperthermia-induced seizures no significant differences were found in the Bmax value of A1R among control animals and submitted to seizures (0.149 ± 0.025 pmol/mg protein vs 0.265 ± 0.073 pmol/mg protein, p > 0.05). The analysis of KD value also failed to find any significant differences (control: 3.9 ± 2.3 nM; hyperthermia-induced seizures group: 7.1 ± 4.9 nM; p > 0.05). The analysis of the results corresponding to A2AR showed a similar tendency. Thus, neither Bmax (control: 0.241 ± 0.058 pmol/mg protein; hyperthermia-induced seizures group: 0.192 ± 0.026 pmol/mg protein, p> 0.05) nor KD values (control: 21.2 9 ± 10.5 nM; hyperthermia-induced seizures group: 8.3 ± 3.2 nM; p > 0.05) were significantly altered by seizures induction (Figure 2B and table 1).
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Status of A1R and A2AR 20 days after seizures evoked by hyperthermia Finally, the binding parameters of A1 and A2A receptors were studied 20 days after hyperthermiainduced seizures. As shown in Figure 3A and table 1, [3H]-DPCPX bound with a Bmax of 0.151 ± 0.035 pmol/mg protein and a KD of 11.2 ± 5.4 nM to cerebral cortical membranes in control animals. Both values were not significantly altered by seizures. Thus, the analysis of the radioligand binding assays revealed that [3H]-DPCPX bound with a Bmax of 0.096 ± 0.014 pmol/mg protein (p > 0.05) and a KD of 3.2 ± 1.7 nM (p > 0.05) to cerebral cortical membranes in animals submitted to hyperthermiainduced seizures.
Concerning to A2AR, the analysis of the Bmax showed a similar value in control (0.292 ± 0.038 pmol/mg protein) and hyperthermia- associated seizure group (0.250 ± 0.022 pmol/mg protein, p > 0.05). However, a significant decrease in KD could be observed in animals submitted to hyperthermic seizures (1.6 ± 0.98 nM vs 10.6 ± 3.6 nM, p < 0.05) suggesting an increase in A2AR affinity 20 days after hyperthermia-induced seizures.
Effect of hyperthermia-induced seizures on mRNA coding adenosine receptors and adenylyl cyclase type II To determine whether the increase in A1R density detected 48 hours after seizures evoked by hyperthermia was due to an increase in A1R transcript levels, we carried out quantitative PCR assays. As shown in Figure 4, mRNA level coding A1R was significantly increased 48 hours (26%, p < 0.05) and 5 days (60%, p < 0.05) after hyperthermia-induced seizures. No significant differences were found when mRNA level coding A1R was analysed 20 days after seizures.
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In the case of A2AR, the loss of total receptor number observed 48 hours after hyperthermia-induced seizures in the radioligand binding assays could not be explained by a decrease on mRNA level coding A2AR which allow suggests the involvement of post-transcriptional modifications. However, a significant increase in A2AR mRNA could be detected 5 days after hyperthermia-induced seizures (49%, p < 0.05) (Figure 4). Concerning mRNA coding A2B and A3 receptors, only a slight decrease in A2B mRNA could be observed 20 days after seizures (Figure 4).
Since the expression level of adenylyl cyclase, the main effector system of adenosine receptors, has been shown to be modulated in kindling model of epilepsy in rats (Iwasa et al., 2000) we decided to investigate whether mRNA coding adenylyl cyclase type II (ACII) could be also regulated in response to fever seizures. As shown in Figure 4, a significant increase in mRNA coding ACII was detected in hyperthermia- associated seizure group 5 days after convulsions.
Effect of hyperthermia-induced seizures on 5´-nucleotidase activity It has been suggested that adenosine which activates A2AR is formed by 5´-nucleotidase activity (Augusto et al., 2013; Rebola et al., 2008). Therefore, we decided to investigate whether the shortterm changes on A2AR density were accompanied by a regulation on 5´-nucleotidase activity. Thus, 5´-nucleotidase activity was assayed in cerebral cortical membranes from control and experimental animals 48 hours and 5 days after hyperthermia-induced seizures. In order to know the kinetic parameters, 5´-nucleotidase activity was determined at AMP concentrations ranging from 1 μM to 1 mM. The results obtained indicated that ecto-5´-nucleotidase activity increased with increasing AMP concentrations until saturation. As illustrated in Figure 5, data could be adequately fitted to Michaelis Menten model using nonlinear regression. The analysis of the kinetic parameters of 5´nucleotidase activity 48 hours after seizures evoked by hyperthermia showed a significant reduction
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in Vmax parameter (9.5 ± 0.4 nmol Pi/min/mg of protein vs. 11.3 ± 0.3 nmol Pi/min/mg of protein, p < 0.05). No significant variation was found in the Km parameter (control: 57.6 ± 7.2 μM; HIS: 64.4 ± 10.7 μM, p > 0.05) (Figure 5a). Both Vmax and Km parameters were not significantly distinct between control (Vmax: 10.7 ± 0.2 nmol Pi/min/mg of protein; Km: 83.1 ± 6 μM) and hyperthermiaassociated seizure group (Vmax: 10.6 ± 0.5 nmol Pi/min/mg of protein; Km: 89.7 ± 11.7 μM) after 5 days of hyperthermia-induced seizures.
In order to investigate the participation of alkaline phosphatase in AMP hydrolysis, the hydrolysis of 200 μM AMP was assayed in presence of 100 μM levamisole, a specific inhibitor of alkaline phosphatase using control membranes. No significant differences were found after comparing results obtained in both conditions which allow to suggest that alkaline phosphatase did not participate in AMP hydrolysis under our conditions (absence of levamisole: 10.3 ± 0.8 nmol Pi/min/mg of protein; presence of 100 μM levamisole: 8.2 ± 0.4 nmol Pi/min/mg of protein, p > 0.05).
DISCUSSION Results obtained in the present work have shown time-dependent changes in A1 and A2A receptors in neonatal brain cortex after hyperthermia-induced seizures. Specifically, the density of A1R and its mRNA were up-regulated 48 hours after hyperthermia-induced seizures. In the case of A2AR, a significant decrease in receptor density could be observed 48 hours after hyperthermia-evoked convulsions, whereas an increase in the mRNA coding A2AR could be detected 5 days after seizures. These short-term changes in the density of A1R and A2AR were also accompanied by a decrease in the 5´-nucleotidase activity. Finally, a higher affinity in A2A receptor was revealed on the 20th day after induction of hyperthermia in neonatal rats.
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The activation of A1R can contribute to terminating seizure by inhibiting presynaptic excitatory neurotransmitter release and by hyperpolarizating the postsynaptic cell membrane (Fredholm et al., 2005a). In this regard, the short-term upregulation of A1R observed in the present work could potentiate the neuroprotective role of A1R against subsequent seizures. Previous works have shown that A1R agonist administered acutely reduce seizures in animal models, whereas selective A1R antagonist increases the duration and severity of seizures (see review Dragunow, 2010; Tomé et al., 2010). In line with our data, other works have also shown a significant increase in A1R in rodent brain cortices after a short period (1-24 hours) of pentylentetrazol-induced seizures (Tchekalarova et al., 2005; Pagonopoulou et al., 1993; Angelatou et al., 1990; 1991). The short-term upregulation of A1R was also accompanied by an increase in the mRNA coding A1R. This effect may be related to cytokine interleukin-6 whose levels significantly increase in the brain under pathological conditions such as febrile seizures or ischemia (Hu et al., 2014; Loddick et al., 1998). Thus, previous works have suggested that IL-6 may be neuroprotective from chemically-induced seizures by increasing the expression of A1 receptor mRNA and enhancing adenosinergic signalling in the brain (Biber et al., 2001, 2008). In this regard, Fukuda and co-workers (2007) have also suggested that IL-6 attenuated hyperthermia-induced seizures in developing rats through the adenosine system.
The role played by A2AR in the seizures is not fully understood. However, different works using pharmacological blockade of A2AR or A2AR knockout mice have suggested that the blockade of A2AR can exert a protective role against seizures. Thus, using A2AR knock out mice, El Yacoubi and colleagues (2009) showed that adenosine can exacerbates limbic seizures through A2AR activation. Similarly, the pharmacological blockade of A2AR with selective antagonist, SCH58261 or ZM241385, has been shown to reduce seizure occurrence in the pilocarpine model (Rosim et al., 2011), be protective against kainic-induced seizures (Bortolatto et al., 2011) and decrease epileptiform activity in the hippocampus in vitro (Etherington and Frenguelli, 2004). Furthermore, Fukuda and co-workers
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(2011) have also shown that A2AR activation may enhance seizures associated to hyperthermia. In accordance with these works, it is tempting to suggest that the significant reduction of A2AR observed 48 hours after hyperthermia-induced seizures in the present study could represent a protective mechanism against hyperthermia-evoked seizures. Although the few studies that examine the changes in the density of adenosine A2AR following seizures have focused on the striatum region, these works have also found a similar reduction of A2AR following seizures. Thus, Doriat and coworkers (1999) observed a significant reduction in the Bmax value (~ 15%) in 25- day-old rats exposed to bicuculline-induced seizures on the 15th postnatal day. A significant reduction of A2AR was also detected following kindled seizures in rats (Aden et al., 2004).
There is a tight relationship between 5´-nucleotidase and A2AR. Thus, in a recent and elegant work, Augusto and colleagues (2013) have shown a colocalization and physical association of ecto-5´nucleotidase and A2AR in the striatum. Furthermore, they revealed that ecto-5´-nucleotidase activity produced adenosine, which activated striatal A2AR. This tight association between 5´-nucleotidase and A2AR is not restricted to the striatum and is observed also in other regions of the nervous system such as the hippocampus (Rebola et al., 2008), the neuromuscular junction (Correia de Sá et al., 1996; Cunha et al., 1996) and cultured granular cells (Boeck et al., 2007). The loss of 5´-nucleotidase activity 48 hours after hyperthermia-induced seizures, as observed in the present work, may, therefore, represent an additional protective mechanism to maintain A2AR with a low level of functionality.
In conclusion, hyperthermia-induced seizures trigger both short-term up-regulation of A1R and down-regulation of A2AR in the cerebral cortex accompanied by a loss of 5´-nucleotidase, suggesting the possible existence of a neuroprotective mechanism against seizures.
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ACKNOWLEDGEMENTS
This work was supported in part by grants from the Ministerio de Economía y Competitividad from Spain (BFU2011-23034). We thank María Angeles Ruíz and Inmaculada Iglesias for helping us with dissection neonates. Authors have no conflicts of interest to declare.
REFERENCES Adén U., O'Connor W.T., and Berman R.F. (2004) Changes in purine levels and adenosine receptors in kindled seizures in the rat. Neuroreport 15, 1585-9. Angelatou F., Pagonopoulou O. and Kostopoulos G (1990) Alterations of A1 adenosine receptors in different mouse brain areas after pentylentetrazol-induced seizures, but not in the epileptic mutant mouse 'tottering'. Brain Res. 534, 251-6. Angelatou F., Pagonopoulou O. and Kostopoulos G (1991) Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas. Neurosci. Lett. 132, 203-6. Augusto E., Matos M., Sévigny J., El-Tayeb A., Bynoe M.S., Müller C.E., Cunha R.A., and Chen J.F. (2013) Ecto-5'-nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A2A receptor functions. J. Neurosci. 33, 11390-9. Biber K., Lubrich B., Fiebich B.L., Boddeke H.W. and van Calker D. (2001) Interleukin-6 enhances expression of adenosine A(1) receptor mRNA and signaling in cultured rat cortical astrocytes and brain slices. Neuropsychopharmacology 24, 86-96. Biber K., Pinto-Duarte A., Wittendorp M.C., Dolga A.M., Fernandes C.C., Von Frijtag Drabbe Künzel J., Keijser J.N., de Vries R., Ijzerman A.P., Ribeiro J.A., Eisel U., Sebastião A.M. and Boddeke H.W.
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(2008) Interleukin-6 upregulates neuronal adenosine A1 receptors: implications for neuromodulation and neuroprotection, Neuropsychopharmacology 33, 2237-50. Boeck C.R., Kroth E.H., Bronzatto M.J. and Vendite D. (2007) Effect of the L- or D-aspartate on ecto5'nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A(2A) receptors. Amino Acids 33, 439-44. Boison D. (2013) Role of adenosine in status epilepticus: a potential new target? Epilepsia 6, 20-2. Bonan C.D., Walz R., Pereira G.S., Worm P.V., Battastini A.M., Cavalheiro E.A., Izquierdo I. and Sarkis J.J. (2000) Changes in synaptosomal ectonucleotidase activities in two rat models of temporal lobe epilepsy. Epilepsy Res. 39, 229-38. Bortolatto C.F., Jesse C.R., Wilhelm E.A. and Nogueira C.W. (2012) Selective blockade of A(2A) receptor protects against neurotoxicity induced by kainic acid in young rats. Fundam. Clin. Pharmacol. 26, 495-502. Chan K.M., Delfert D. and Junger K.D. (1986) A direct colorimetric assay for Ca2+ -stimulated ATPase activity. Anal. Biochem 157, 375-80. Correia-de-Sá P., Timóteo M.A. and Ribeiro J.A. (1996) Presynaptic A1 inhibitory/A2A facilitatory adenosine receptor activation balance depends on motor nerve stimulation paradigm at the rat hemidiaphragm. J. Neurophysiol. 76, 3910-9. Cunha R.A., Correia-de-Sá P., Sebastião A.M. and Ribeiro J.A. (1996) Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides. British J Pharmacol 119, 253-60. Cunha R.A. (2005) Neuroprotection by adenosine in the brain: From A(1) receptor activation to A (2A) receptor blockade. Purinergic Signal. 2, 111-34.
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de Paula Cognato G., Bruno A.N., Vuaden F.C., Sarkis J.J. and Bonan C.D (2005) Ontogenetic profile of ectonucleotidase activities from brain synaptosomes of pilocarpine-treated rats. Int. J. Dev. Neurosci. 23,703-9. De Sarro G., De Sarro A., Di Paola E.D. and Bertorelli R. (1999) Effects of adenosine receptor agonists and antagonists on audiogenic seizure-sensible DBA/2 mice. Eur. J. Pharmacol. 371, 137-45. Dobbing J. and Sands J. (1973) Quantitative growth and development of human brain. Arch. Dis. Child. 48, 757-67. Doriat J.F., Koziel V., Humbert A.C. and Daval J.L. (1999) Medium- and long-term alterations of brain A1 and A2A adenosine receptor characteristics following repeated seizures in developing rats. Epilepsy Res. 35, 219-28. Dragunow M. (2010) Purinergic mechanisms in epilepsy. The open neuroscience journal 4, 31-34. El Yacoubi M., Ledent C., Parmentier M., Costentin J. and Vaugeois J.M. (2009) Adenosine A2A receptor deficient mice are partially resistant to limbic seizures. Naunyn Schmiedebergs Arch. Pharmacol. 380, 223-32. Etherington L.A. and Frenguelli B.G. (2004) Endogenous adenosine modulates epileptiform activity in rat hippocampus in a receptor subtype-dependent manner. J. Neurosci. 19, 2539-50. Fredholm B.B., Chen J.F., Cunha R.A., Svenningsson P. and Vaugeois J.M. (2005a) Adenosine and brain function. Int. Rev. Neurobiol. 63, 191-270. Fredholm B.B., Chen J.F., Masino S.A. and Vaugeois J.M. (2005) Actions of adenosine at its receptors in the CNS: insights from knockouts and drugs. Annu. Rev. Pharmacol. Toxicol. 45, 385-412. Fredholm B.B., IJzerman A.P., Jacobson K.A., Klotz K.N. and Linden J. (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53, 527-52.
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Fukuda M., Suzuki Y., Hino H., Morimoto T. and Ishii E. (2011) Activation of central adenosine A(2A) receptors lowers the seizure threshold of hyperthermia-induced seizure in childhood rats. Seizure 20, 156-9. Fukuda M., Morimoto T., Suzuki Y., Shinonaga C. and Ishida Y. (2007) Interleukin-6 attenuates hyperthermia-induced seizures in developing rats. Brain Dev. 29, 644-8. Gottlieb A., Keydar I. and Epstein H.T. (1977) Rodent brain growth stages: an analytical review. Biol. Neonate 32, 166-76. Higuchi R., Fockler C., Dollinger G. and Watson R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030 Hosseinmardi N., Mirnajafi-Zadeh J., Fathollahi Y. and Shahabi P. (2007) The role of adenosine A1 and A2A receptors of entorhinal cortex on piriform cortex kindled seizures in rats. Pharmacol. Res. 56, 110-7. Hayashi K., Ueshima S., Ouchida M., Mashimo T., Nishiki T., Sendo T., Serikawa T., Matsui H. and Ohmori I. (2011) Therapy for hyperthermia-induced seizures in Scn1a mutant rats. Epilepsia 52, 1010-7 Hu M.H., Huang G.S., Wu C.T., Lin J.J., Hsia S.H., Wang H.S. and Lin K.L. (2014) Analysis of plasma multiplex cytokines for children with febrile seizures and severe acute encephalitis. J. Child Neurol. 29, 182-6 Iwasa H., Kikuchi S., Mine S., Miyagishima H., Sugita K., Sato T. and Hasegawa S. (2000) Upregulation of type II adenylyl cyclase mRNA in kindling model of epilepsy in rats. Neurosci. Lett. 282, 173-6.
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León D.A., Castillo C.A., Albasanz J.L. and Martín M. (2009) Reduced expression and desensitization of
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Rebola N., Lujan R., Cunha R.A. and Mulle C. (2008) Adenosine A2A receptors are essential for longterm potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses. Neuron 57, 121-34. Rebola N., Sebastião A.M., de Mendonca A., Oliveira C.R., Ribeiro J.A. and Cunha R.A. (2003) Enhanced adenosine A2A receptor facilitation of synaptic transmission in the hippocampus of aged rats. J. Neurophysiol. 90, 1295-303. Rosim F.E., Persike D.S., Nehlig A., Amorim R.P., de Oliveira D.M. and Fernandes M.J. (2011) Differential neuroprotection by A(1) receptor activation and A(2A) receptor inhibition following pilocarpine-induced status epilepticus. Epilepsy Behav. 2, 207-13. Shinnar S. and Glauser T.A. (2002) Febrile seizures. J. Child Neurol. 1, 44-52. Stafstrom C.E. (2002) Assessing the behavioral and cognitive effects of seizures on the developing brain. Prog. Brain Res. 135, 377-90. Tchekalarova J., Sotiriou E., Georgiev V., Kostopoulos G. and Angelatou F. (2005) Up-regulation of adenosine A1 receptor binding in pentylenetetrazol kindling in mice: effects of angiotensin IV. Brain Res. 1032, 94-103. Tomé A.R., Silva H. and Cunha R.A. (2010) Role of the purinergic neuromodulation system in epilepsy. The open neuroscience journal 4, 64-83. Zimmermann H., Zebisch M. and Sträter N. (2012) Cellular function and molecular structure of ectonucleotidases. Purinergic Signal. 8, 437-502.
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FIGURE LEGENDS Figure 1.- Saturation curves of [3H]DPCPX (A) and [3H]ZM241385 (B) binding to cerebral cortical membranes after 48 hours of seizures evoked by hyperthermia. Binding of [3H]DPCPX and [3H]ZM241385 to cerebral cortical membranes from control,
hyperthermic and hyperthermic
control groups were performed as described in Experimental procedures. Binding parameters are reflected in Table 1 and were determined performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program. Data are mean ± SEM of at least five experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
Figure 2.- Saturation curves of [3H]DPCPX (A) and [3H]ZM241385 (B) binding to cerebral cortical membranes after 5 days of seizures evoked by hyperthermia. Binding of [3H]DPCPX and [3H]ZM241385 to cerebral cortical membranes from control
and hyperthermic groups were
performed as described in Experimental procedures. Binding parameters are reflected in Table 2 and were determined performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program. Data are mean ± SEM of at least four experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
Figure 3.- Saturation curves of [3H]DPCPX (A) and [3H]ZM241385 (B) binding to cerebral cortical membranes after 20 days of seizures evoked by hyperthermia. Binding of [3H]DPCPX and [3H]ZM241385 to cerebral cortical membranes from control
and hyperthermic groups were
performed as described in Experimental procedures. Binding parameters are reflected in Table 2 and were determined performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program. Data are mean ± SEM of at least five experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
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Figure 4.- Effect of hyperthermia-induced seizures on adenosine receptors and adenylyl cyclase type II gene expression assayed by quantitative real time RT-PCR analysis. RNA was isolated from cortex of control and hyperthermic groups 48 hours, 5 days and 20 days after hyperthermia induction and quantitative real time RT-PCR assays were carried out following the protocol indicated in Experimental procedures. Histograms represent mean ± SEM values obtained from three to five separate experiments using different preparations. * p < 0.05, significantly different from control group. RQ, relative quantitation. HIS, hyperthermia-induced seizures.
Figure 5.- Effect of hyperthermia-induced seizures on 5´-nucleotidase activity. 5´-nucleotidase activity was measured in cerebral cortical membranes from neonates 48 hours and 5 days after hyperthermia induction following the protocol described in Experimental procedures. Kinetic parameters are reflected in Table 3 and were determined using nonlinear Michaelis-Menten curvefitting. Data are mean ± SEM of at least three experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
Table 1.-Binding parameters of [3H]DPCPX and [3H]ZM241385 binding to cerebral cortical membranes 48 hours after hyperthermia-induced seizures. Data represent Bmax and KD of binding data shown in Figure 1 and determined by nonlinear nonlinear regression of binding data with the GraphPad Prism 5 program. Data are means ± SEM from three-six independent experiments performed with different cerebral cortical membranes isolations. *p < 0.05, significantly different from control using one-way ANOVA and Tukey as post hoc test.
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Table 2.-Binding parameters of [3H]DPCPX and [3H]ZM241385 binding to cerebral cortical membranes 5 days and 20 days after hyperthermia-induced seizures. Data represent Bmax and KD of binding data shown in Figures 2 and 3 and determined by nonlinear nonlinear regression of binding data with the GraphPad Prism 5 program. Data are means ± SEM from at least four independent experiments performed with different cerebral cortical membranes isolations. HIS, hyperthermiainduced seizures. *p < 0.05, significantly different from control using unpaired two-tailed Student´s t test.
Table 3.- Kinetic parameters of 5´-nucleotidase activity assayed in cerebral cortical membranes 48 hours and 5 days after hyperthermia-induced seizures. Data represent Vmax and Km of data shown in Figure 5 and determined by nonlinear regression Michaelis-Menten curve-fitting. Data are means ± SEM from at least three independent experiments performed with different cerebral cortical membranes isolations. HIS, hyperthermia-induced seizures. *p < 0.05, significantly different from control using unpaired two-tailed Student´s t test.
Table 1.
48 hours A1 receptor Bmax (pmol/mg prot)
A2A receptor KD
Bmax
KD
(nM)
(pmol/mg prot)
(nM)
Control
0.244 ± 0.076
5.1 ± 4.7
0.271 ± 0.051
20.6 ± 8.3
HIS
0.559 ± 0.097 *
2.1 ± 1.7
0.124 ± 0.019 *
7.6 ± 3.4
HIS + KBr
0.127 ± 0.012
0.3 ± 0.2
0.264 ± 0.036
9.5 ± 3.5
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Table 2.
5 days A1 receptor Bmax (pmol/mg prot)
A2A receptor
KD
Bmax
KD
(nM)
(pmol/mg prot)
(nM)
Control
0.149 ± 0.025
3.9 ± 2.3
0.241 ± 0.058
21.2 ± 10.6
HIS
0.265 ± 0.073
7.1 ± 4.9
0.192 ± 0.026
8.34 ± 3.2
20 days A1 receptor Bmax (pmol/mg prot)
A2A receptor
KD
Bmax
KD
(nM)
(pmol/mg prot)
(nM)
Control
0.151 ± 0.035
11.2 ± 5.4
0.29 ± 0.038
10.6 ± 3.6
HIS
0.096 ± 0.014
3.2 ± 1.7
0.25 ± 0.022
1.6 ± 0.8*
Table 3. Control
HIS
Vmax
Km
Vmax
Km
(nmol/min · mg prot)
(μM)
(nmol/min · mg prot)
(μM)
48 hours
11.3 ± 0.3
57.5 ± 7
9.5 ± 0.4 *
64.4 ± 10.7
5 days
10.7 ± 0.2
83.1 ± 6
10.56 ± 0.5
59.7 ± 11
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Article Type: Original Article
HYPERTHERMIA-INDUCED SEIZURES ALTER ADENOSINE A1 AND A2A RECEPTORS AND 5´-NUCLEOTIDASE ACTIVITY IN RAT CEREBRAL CORTEX
David Agustín León-Navarro*, José L. Albasanz and Mairena Martín
Departamento de Química Inorgánica, Orgánica y Bioquímica. Facultad de Ciencias y Tecnologías Químicas. Centro Regional de Investigaciones Biomédicas. Universidad de Castilla-La Mancha. Avenida Camilo José Cela, 10. 13071 Ciudad Real -SPAIN-
*Address correspondence and reprint requests to: Dr. David Agustín León Navarro. Área de Bioquímica. Departamento de Química Inorgánica, Orgánica y Bioquímica. Facultad de Ciencias y Tecnologías Químicas. Avenida Camilo José Cela, 10. 13071 Ciudad Real. Spain. e-mail: [email protected] Tel.: +34 926295300 Ext: 3432. Fax: +34 926295318.
Running title: Effect of fever seizures on adenosine receptors.
Keywords: Fever seizures, adenosine receptor, 5,-nucleotidase, postnatal, cerebral cortex.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13130 This article is protected by copyright. All rights reserved.
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Abbreviations: ADA, adenosine deaminase. A1R, adenosine A1 receptor. A2AR, adenosine A2A receptor. ACII , adenylyl cyclase type II. CPA, N6-cyclopentyladenosine. [3H]DPCPX , cyclopentyl-1,3-dypropylxanthine,8-[dipropy-2,3-3H(N)] FS, febrile seizures. NTPDases, nucleoside triphosphate diphosphohydrolases. [3H]ZM241385, [2-3H](4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl]phenol).
ABSTRACT Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5´-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR and 5´-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [3H]DPCPX as radioligand, and mRNA coding A1 was observed 48 hours after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [3H]ZM241385 as radioligand, 48 hours after hyperthermiaevoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5´-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5´-
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ACKNOWLEDGEMENTS
This work was supported in part by grants from the Ministerio de Economía y Competitividad from Spain (BFU2011-23034). We thank María Angeles Ruíz and Inmaculada Iglesias for helping us with dissection neonates. Authors have no conflicts of interest to declare.
REFERENCES Adén U., O'Connor W.T., and Berman R.F. (2004) Changes in purine levels and adenosine receptors in kindled seizures in the rat. Neuroreport 15, 1585-9. Angelatou F., Pagonopoulou O. and Kostopoulos G (1990) Alterations of A1 adenosine receptors in different mouse brain areas after pentylentetrazol-induced seizures, but not in the epileptic mutant mouse 'tottering'. Brain Res. 534, 251-6. Angelatou F., Pagonopoulou O. and Kostopoulos G (1991) Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas. Neurosci. Lett. 132, 203-6. Augusto E., Matos M., Sévigny J., El-Tayeb A., Bynoe M.S., Müller C.E., Cunha R.A., and Chen J.F. (2013) Ecto-5'-nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A2A receptor functions. J. Neurosci. 33, 11390-9. Biber K., Lubrich B., Fiebich B.L., Boddeke H.W. and van Calker D. (2001) Interleukin-6 enhances expression of adenosine A(1) receptor mRNA and signaling in cultured rat cortical astrocytes and brain slices. Neuropsychopharmacology 24, 86-96. Biber K., Pinto-Duarte A., Wittendorp M.C., Dolga A.M., Fernandes C.C., Von Frijtag Drabbe Künzel J., Keijser J.N., de Vries R., Ijzerman A.P., Ribeiro J.A., Eisel U., Sebastião A.M. and Boddeke H.W.
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has a proconvulsive effect on piriform cortex-kindled seizures and play a significant role in the pentylenetetrazol-induced kindling of seizures (Hosseinmardi ei al., 2007; El Yacoubi et al., 2009).
Extracellular adenosine can be formed by the combined action of a group of membrane-bound enzymes known as ectonucleotidases, which includes nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5´-nucleotidase, which hydrolizes 5´-AMP to adenosine (Zimmermann et al., 2012). Different works have shown changes in the activity of 5’-nucleotidase in several models of epilepsy (Bonan et al., 2000; Oses et al., 2007; de Paula Cognato et al., 2005).
Although several works have revealed significant changes in the density of adenosine receptors in different animal models of epilepsy as well in human epileptic tissues (see review Pagonopoulou et al., 2006), there are no studies, at least to our knowledge, analysing the effect of fever seizures on adenosine receptors. The aim of this study, therefore, was to examine the density of adenosine A1 and A2A receptors and the corresponding mRNA levels at different times (48 hours, 5 days and 20 days) after fever-induced seizures. The effect of fever-induced seizures on 5´-nucleotidase activity was also investigated.
MATERIALS AND METHODS 1.1. Materials Cyclopentyl-1,3-dypropylxanthine,8-[dipropy-2,3-3H(N)] ([3H]DPCPX 120 Ci/mmol) was from Amersham Biosciences (Buckinghamshire, UK) and [2-3H](4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo[2,3a][1,3,5]triazin-5-ylamino]ethyl]phenol) ([3H]ZM241385 27.4 Ci/mmol) from Tocris (Bristol, UK). Calf intestine
adenosine
deaminase,
theophylline,
N6-cyclopentyladenosine
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(CPA),
levamisole
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The duration of heating was 28 ± 2 minutes. Controls included a normothermic group which were separated from the dams for the same duration and placed in a chamber at room temperature and a hyperthermic control group which were subjected to the same degree and duration of hyperthermia but in which seizures were blocked by treating orally with potassium bromide (KBr) dissolved in saline (1800 mg/Kg) 90 minutes before to the induction of hyperthermia. Potassium bromide is a classical antiepileptic drug which has been shown to be effective against hyperthermia-induced seizures in rats (Hayashi et al., 2011). Rectal temperatures in hyperthermic control group were not significantly different from hyperthermia-associated seizure group which suggest that potassium bromide did not substantially decrease the temperature of neonates at the time of heating. Four of the eleven pups subjected to hyperthermia after KBr intake showed falling over associated with hindlimb clonus seizures and did not include in the work. No changes on the behavioural excitability could be observed after hyperthermic induction.
1.4 Cerebral cortical membranes isolation Cerebral cortical membranes were isolated following the protocol described by León and coworkers (2004) with modifications. Briefly, the brain cortex from pups were homogenized in 20 volumes of isolation buffer (50 mM Tris-HCl, pH 7.4 containing 10 mM MgCl2 and protease inhibitors) in a Dounce homogenizer using pestle A (10 strokes) and B (10 strokes). After homogenization, brain preparations were centrifuged for 5 min at 1000xg in a Beckman JA 21 centrifuge (Coulter España, Madrid, Spain). Supernatant was centrifuged for 20 min at 27000xg and the pellet was finally resuspended in isolation buffer. Protein concentration was measured by the Lowry method, using bovine serum albumin (BSA) as standard. Each cerebral cortical membrane isolation corresponds to one pup per litter.
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1.5 [3H]DPCPX and [3H]ZM241385 binding assays to cortical membranes The effect of fever seizures on A1R and A2AR was studied performing radioligand binding assays to cortical membranes from neonates following protocols described previously (López-Zapata et al., 2011) with minor modifications. Cerebral cortical membranes were incubated with 5 U/mg adenosine deaminase (ADA) in 50 mM Tris, 2 mM MgCl2, pH 7.4, for 30 min at 25º C, to remove endogenous adenosine. This reaction buffer was supplemented with 100 mM NaCl in [3H]ZM241385 binding assays. Then, cerebral cortical membranes (50-100 μg of protein) were incubated with [3H]DPCPX or [3H]ZM241385 for 2 h at 25ºC. Saturation assays were carried out at different [3H]DPCPX and [3H]ZM241385 concentrations (0.5–20 nM) using cyclopentyladenosine (CPA) at a concentration 104 times higher than [3H]DPCPX and 5 mM theophylline, respectively, to obtain nonspecific binding. Binding assays were stopped by rapid filtration through Whatman GF/B filters, previously pre-incubated overnight with 0.3% polyethylenimine using a FilterMate Harvester (PerkinElmer). Filters were then transferred to vials and scintillation liquid was added to measure the radioactivity in a Microbeta Trilux Jet scintillation counter.
1.6 Measurement of 5´-nucleotidase activity 20 μg of cerebral cortical membranes were preincubated in 180 μl of the reaction medium containing: 50 mM Tris, MgCl2 5 mM, pH 9, at 37ºC for 10 minutes. Then, the reaction was initiated by the addition of 20 μl AMP (final concentrations, 1μM - 3 mM) and stopped 10 minutes later by adding 200 μl of 10% trichloroacetic acid (TCA). The samples were chilled on ice for 10 minutes and then centrifuged at 12000xg for 4 minutes at 4ºC. The supernatants were used to measure inorganic phosphate released following the protocol described by Chan and coworkers (1986) using KH2PO4 as Pi standard. Non enzymatic hydrolysis of AMP was corrected by adding cerebral cortical membranes after TCA. Incubation times and protein concentration were selected in order to ensure the linearity
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of the reactions. All samples were run in triplicate. Enzyme activity is expressed as nmol Pi released/min/mg of protein.
1.7 Preparation of total RNA and cDNA Total RNA was extracted from cortex brain using an ABI 6100 Nucleic Acid PrepStation according to the manufacturer's protocol. All chemicals for the ABI 6100 were purchased from Applied Biosystems (Foster City, CA). Total RNA from animals was isolated and stored individually at -80°C. Ratio of A260/A280 (purity of RNA) was in the range 1.9–2.1. RNA concentrations were determined from the A260. One microgram of total RNA was reverse transcribed using Applied Biosystems HighCapacity cDNA Archive Kit. Parallel reactions for each RNA sample were run in the absence of MultiScribe Reverse Transcriptase to assess the degree of contaminating genomic DNA.
1.8 Quantitative Real Time RT-PCR Analysis Quantitative real time RT-PCR analysis (Higuchi et al., 1993) was performed with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan® universal PCR master mix according to the manufacturer’s specifications (Applied Biosystems Inc., Foster City, CA) as previously described (León et al., 2009). The validated TaqMan probes and primers for A1 (assay ID: Rn00567668_m1), A2A (assay ID: Rn00583935_m1), A2B (assay ID: Rn00567697_m1), A3 (assay ID: Rn00563680_m1), type II adenylyl cyclase (Rn00578713_m1) and β-actin (Rn00667869_m1) were assay-on-demand gene expression products (Applied Biosystems). The TaqMan® primer and probe sequences are packaged together in a 20x solution. Rat β-actin gene was used as endogenous control. The gene-specific probes were labeled using reporter dye FAM. A non-fluorescent quencher and the minor groove binder were linked at the 3’ end of probe as quenchers. The thermal cycler conditions were as follows: hold for 20 s at 95 °C, followed by two-step PCR for 40 cycles of 95 °C for
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3 s, followed by 60 °C for 30 s. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems) according to the 2-ΔΔCt method. Briefly, expression results for a gene were normalized to internal control β-actin relative to a calibrator, consisting of the mean expression level of the corresponding gene in control samples as follows: 2ΔΔCt
= 2-((Ct receptor gene - Ct actin)sample - (Ct receptor gene - Ct actin) calibrator). The results from three to five independent
repeat assays, performed on different plates each using different cDNAs from the animals analyzed, were averaged to produce a single mean quantity value.
1.9 Statistical and data analysis Saturation (Bmax, KD) binding curves were analyzed performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program (GraphPad Software, San Diego, CA, USA). In the case of 5´-nucleotidase activity, the Km and Vmax values were calculated using a non-linear Michaelis-Menten curve fitting with the GraphPad Prism 5 program. The effect of fever seizures on Bmax, KD, Vmax and Km and on mRNA level coding adenosine receptors were analyzed using unpaired two-tailed Student´s t test or one way ANOVA following Tukey post-hoc test.
RESULTS Status of A1R and A2AR 48 hours after hyperthermia-induced seizures In order to analyze the effect of hyperthermia-induced seizures on adenosine A1 and A2A receptors from neonatal brain, radioligand binding assays using [3H]DPCPX and [3H]ZM21680, respectively, were carried out. Data obtained could be best fitted to one binding site model. As shown in Figure 1A and table 1, a significant increase in A1R was observed in neonates 48 hours after hyperthermic seizures as compared to control animals (0.559 ± 0.097 pmol/mg protein vs 0.244 ± 0.076 pmol/mg
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protein, p < 0.05). No significant variation in A1R was observed in neonates pretreated with KBr as anticonvulsant before seizures as compared to control rats (0.127 ± 0.012 pmol/mg protein vs 0.244 ± 0.076 pmol/mg protein, p > 0.05). The statistical comparison of KD value between groups failed to find significant variations (control: 5.1 ± 4.7 nM; HIS: 2.1 ± 1.7 nM; HIS + KBr: 0.3 ± 0.2 nM, p > 0.05) suggesting that receptor affinity did not result changed by seizures evoked by hyperthermia.
Concerning to adenosine A2AR, a significant decrease in receptor density was detected in animals submitted to hyperthermia-induced seizures (0.124 ± 0.019 pmol/mg protein vs. 0.271 ± 0.051 pmol/mg protein, p < 0.05) (Figure 1B and table 1). This effect was not present in neonates pretreated with KBr before hyperthermic seizures (0.264 ± 0.036 pmol/mg protein vs. 0.271 ± 0.051 pmol/mg protein, p > 0.05). Similarly to A1R, the KD value of A2A receptor remained unchanged after seizures (control: 20.6 ± 8.3 nM; HIS: 7.6 ± 3.4 nM; HIS + KBr: 9.5 ± 3.5 nM, p > 0.05).
Status of A1R and A2AR 5 days after hyperthermia-induced seizures As illustrated in Figure 2A and table 1, after 5 days of hyperthermia-induced seizures no significant differences were found in the Bmax value of A1R among control animals and submitted to seizures (0.149 ± 0.025 pmol/mg protein vs 0.265 ± 0.073 pmol/mg protein, p > 0.05). The analysis of KD value also failed to find any significant differences (control: 3.9 ± 2.3 nM; hyperthermia-induced seizures group: 7.1 ± 4.9 nM; p > 0.05). The analysis of the results corresponding to A2AR showed a similar tendency. Thus, neither Bmax (control: 0.241 ± 0.058 pmol/mg protein; hyperthermia-induced seizures group: 0.192 ± 0.026 pmol/mg protein, p> 0.05) nor KD values (control: 21.2 9 ± 10.5 nM; hyperthermia-induced seizures group: 8.3 ± 3.2 nM; p > 0.05) were significantly altered by seizures induction (Figure 2B and table 1).
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Status of A1R and A2AR 20 days after seizures evoked by hyperthermia Finally, the binding parameters of A1 and A2A receptors were studied 20 days after hyperthermiainduced seizures. As shown in Figure 3A and table 1, [3H]-DPCPX bound with a Bmax of 0.151 ± 0.035 pmol/mg protein and a KD of 11.2 ± 5.4 nM to cerebral cortical membranes in control animals. Both values were not significantly altered by seizures. Thus, the analysis of the radioligand binding assays revealed that [3H]-DPCPX bound with a Bmax of 0.096 ± 0.014 pmol/mg protein (p > 0.05) and a KD of 3.2 ± 1.7 nM (p > 0.05) to cerebral cortical membranes in animals submitted to hyperthermiainduced seizures.
Concerning to A2AR, the analysis of the Bmax showed a similar value in control (0.292 ± 0.038 pmol/mg protein) and hyperthermia- associated seizure group (0.250 ± 0.022 pmol/mg protein, p > 0.05). However, a significant decrease in KD could be observed in animals submitted to hyperthermic seizures (1.6 ± 0.98 nM vs 10.6 ± 3.6 nM, p < 0.05) suggesting an increase in A2AR affinity 20 days after hyperthermia-induced seizures.
Effect of hyperthermia-induced seizures on mRNA coding adenosine receptors and adenylyl cyclase type II To determine whether the increase in A1R density detected 48 hours after seizures evoked by hyperthermia was due to an increase in A1R transcript levels, we carried out quantitative PCR assays. As shown in Figure 4, mRNA level coding A1R was significantly increased 48 hours (26%, p < 0.05) and 5 days (60%, p < 0.05) after hyperthermia-induced seizures. No significant differences were found when mRNA level coding A1R was analysed 20 days after seizures.
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In the case of A2AR, the loss of total receptor number observed 48 hours after hyperthermia-induced seizures in the radioligand binding assays could not be explained by a decrease on mRNA level coding A2AR which allow suggests the involvement of post-transcriptional modifications. However, a significant increase in A2AR mRNA could be detected 5 days after hyperthermia-induced seizures (49%, p < 0.05) (Figure 4). Concerning mRNA coding A2B and A3 receptors, only a slight decrease in A2B mRNA could be observed 20 days after seizures (Figure 4).
Since the expression level of adenylyl cyclase, the main effector system of adenosine receptors, has been shown to be modulated in kindling model of epilepsy in rats (Iwasa et al., 2000) we decided to investigate whether mRNA coding adenylyl cyclase type II (ACII) could be also regulated in response to fever seizures. As shown in Figure 4, a significant increase in mRNA coding ACII was detected in hyperthermia- associated seizure group 5 days after convulsions.
Effect of hyperthermia-induced seizures on 5´-nucleotidase activity It has been suggested that adenosine which activates A2AR is formed by 5´-nucleotidase activity (Augusto et al., 2013; Rebola et al., 2008). Therefore, we decided to investigate whether the shortterm changes on A2AR density were accompanied by a regulation on 5´-nucleotidase activity. Thus, 5´-nucleotidase activity was assayed in cerebral cortical membranes from control and experimental animals 48 hours and 5 days after hyperthermia-induced seizures. In order to know the kinetic parameters, 5´-nucleotidase activity was determined at AMP concentrations ranging from 1 μM to 1 mM. The results obtained indicated that ecto-5´-nucleotidase activity increased with increasing AMP concentrations until saturation. As illustrated in Figure 5, data could be adequately fitted to Michaelis Menten model using nonlinear regression. The analysis of the kinetic parameters of 5´nucleotidase activity 48 hours after seizures evoked by hyperthermia showed a significant reduction
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in Vmax parameter (9.5 ± 0.4 nmol Pi/min/mg of protein vs. 11.3 ± 0.3 nmol Pi/min/mg of protein, p < 0.05). No significant variation was found in the Km parameter (control: 57.6 ± 7.2 μM; HIS: 64.4 ± 10.7 μM, p > 0.05) (Figure 5a). Both Vmax and Km parameters were not significantly distinct between control (Vmax: 10.7 ± 0.2 nmol Pi/min/mg of protein; Km: 83.1 ± 6 μM) and hyperthermiaassociated seizure group (Vmax: 10.6 ± 0.5 nmol Pi/min/mg of protein; Km: 89.7 ± 11.7 μM) after 5 days of hyperthermia-induced seizures.
In order to investigate the participation of alkaline phosphatase in AMP hydrolysis, the hydrolysis of 200 μM AMP was assayed in presence of 100 μM levamisole, a specific inhibitor of alkaline phosphatase using control membranes. No significant differences were found after comparing results obtained in both conditions which allow to suggest that alkaline phosphatase did not participate in AMP hydrolysis under our conditions (absence of levamisole: 10.3 ± 0.8 nmol Pi/min/mg of protein; presence of 100 μM levamisole: 8.2 ± 0.4 nmol Pi/min/mg of protein, p > 0.05).
DISCUSSION Results obtained in the present work have shown time-dependent changes in A1 and A2A receptors in neonatal brain cortex after hyperthermia-induced seizures. Specifically, the density of A1R and its mRNA were up-regulated 48 hours after hyperthermia-induced seizures. In the case of A2AR, a significant decrease in receptor density could be observed 48 hours after hyperthermia-evoked convulsions, whereas an increase in the mRNA coding A2AR could be detected 5 days after seizures. These short-term changes in the density of A1R and A2AR were also accompanied by a decrease in the 5´-nucleotidase activity. Finally, a higher affinity in A2A receptor was revealed on the 20th day after induction of hyperthermia in neonatal rats.
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The activation of A1R can contribute to terminating seizure by inhibiting presynaptic excitatory neurotransmitter release and by hyperpolarizating the postsynaptic cell membrane (Fredholm et al., 2005a). In this regard, the short-term upregulation of A1R observed in the present work could potentiate the neuroprotective role of A1R against subsequent seizures. Previous works have shown that A1R agonist administered acutely reduce seizures in animal models, whereas selective A1R antagonist increases the duration and severity of seizures (see review Dragunow, 2010; Tomé et al., 2010). In line with our data, other works have also shown a significant increase in A1R in rodent brain cortices after a short period (1-24 hours) of pentylentetrazol-induced seizures (Tchekalarova et al., 2005; Pagonopoulou et al., 1993; Angelatou et al., 1990; 1991). The short-term upregulation of A1R was also accompanied by an increase in the mRNA coding A1R. This effect may be related to cytokine interleukin-6 whose levels significantly increase in the brain under pathological conditions such as febrile seizures or ischemia (Hu et al., 2014; Loddick et al., 1998). Thus, previous works have suggested that IL-6 may be neuroprotective from chemically-induced seizures by increasing the expression of A1 receptor mRNA and enhancing adenosinergic signalling in the brain (Biber et al., 2001, 2008). In this regard, Fukuda and co-workers (2007) have also suggested that IL-6 attenuated hyperthermia-induced seizures in developing rats through the adenosine system.
The role played by A2AR in the seizures is not fully understood. However, different works using pharmacological blockade of A2AR or A2AR knockout mice have suggested that the blockade of A2AR can exert a protective role against seizures. Thus, using A2AR knock out mice, El Yacoubi and colleagues (2009) showed that adenosine can exacerbates limbic seizures through A2AR activation. Similarly, the pharmacological blockade of A2AR with selective antagonist, SCH58261 or ZM241385, has been shown to reduce seizure occurrence in the pilocarpine model (Rosim et al., 2011), be protective against kainic-induced seizures (Bortolatto et al., 2011) and decrease epileptiform activity in the hippocampus in vitro (Etherington and Frenguelli, 2004). Furthermore, Fukuda and co-workers
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(2011) have also shown that A2AR activation may enhance seizures associated to hyperthermia. In accordance with these works, it is tempting to suggest that the significant reduction of A2AR observed 48 hours after hyperthermia-induced seizures in the present study could represent a protective mechanism against hyperthermia-evoked seizures. Although the few studies that examine the changes in the density of adenosine A2AR following seizures have focused on the striatum region, these works have also found a similar reduction of A2AR following seizures. Thus, Doriat and coworkers (1999) observed a significant reduction in the Bmax value (~ 15%) in 25- day-old rats exposed to bicuculline-induced seizures on the 15th postnatal day. A significant reduction of A2AR was also detected following kindled seizures in rats (Aden et al., 2004).
There is a tight relationship between 5´-nucleotidase and A2AR. Thus, in a recent and elegant work, Augusto and colleagues (2013) have shown a colocalization and physical association of ecto-5´nucleotidase and A2AR in the striatum. Furthermore, they revealed that ecto-5´-nucleotidase activity produced adenosine, which activated striatal A2AR. This tight association between 5´-nucleotidase and A2AR is not restricted to the striatum and is observed also in other regions of the nervous system such as the hippocampus (Rebola et al., 2008), the neuromuscular junction (Correia de Sá et al., 1996; Cunha et al., 1996) and cultured granular cells (Boeck et al., 2007). The loss of 5´-nucleotidase activity 48 hours after hyperthermia-induced seizures, as observed in the present work, may, therefore, represent an additional protective mechanism to maintain A2AR with a low level of functionality.
In conclusion, hyperthermia-induced seizures trigger both short-term up-regulation of A1R and down-regulation of A2AR in the cerebral cortex accompanied by a loss of 5´-nucleotidase, suggesting the possible existence of a neuroprotective mechanism against seizures.
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ACKNOWLEDGEMENTS
This work was supported in part by grants from the Ministerio de Economía y Competitividad from Spain (BFU2011-23034). We thank María Angeles Ruíz and Inmaculada Iglesias for helping us with dissection neonates. Authors have no conflicts of interest to declare.
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FIGURE LEGENDS Figure 1.- Saturation curves of [3H]DPCPX (A) and [3H]ZM241385 (B) binding to cerebral cortical membranes after 48 hours of seizures evoked by hyperthermia. Binding of [3H]DPCPX and [3H]ZM241385 to cerebral cortical membranes from control,
hyperthermic and hyperthermic
control groups were performed as described in Experimental procedures. Binding parameters are reflected in Table 1 and were determined performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program. Data are mean ± SEM of at least five experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
Figure 2.- Saturation curves of [3H]DPCPX (A) and [3H]ZM241385 (B) binding to cerebral cortical membranes after 5 days of seizures evoked by hyperthermia. Binding of [3H]DPCPX and [3H]ZM241385 to cerebral cortical membranes from control
and hyperthermic groups were
performed as described in Experimental procedures. Binding parameters are reflected in Table 2 and were determined performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program. Data are mean ± SEM of at least four experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
Figure 3.- Saturation curves of [3H]DPCPX (A) and [3H]ZM241385 (B) binding to cerebral cortical membranes after 20 days of seizures evoked by hyperthermia. Binding of [3H]DPCPX and [3H]ZM241385 to cerebral cortical membranes from control
and hyperthermic groups were
performed as described in Experimental procedures. Binding parameters are reflected in Table 2 and were determined performing nonlinear regression analysis of binding data with the GraphPad Prism 5 program. Data are mean ± SEM of at least five experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
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Figure 4.- Effect of hyperthermia-induced seizures on adenosine receptors and adenylyl cyclase type II gene expression assayed by quantitative real time RT-PCR analysis. RNA was isolated from cortex of control and hyperthermic groups 48 hours, 5 days and 20 days after hyperthermia induction and quantitative real time RT-PCR assays were carried out following the protocol indicated in Experimental procedures. Histograms represent mean ± SEM values obtained from three to five separate experiments using different preparations. * p < 0.05, significantly different from control group. RQ, relative quantitation. HIS, hyperthermia-induced seizures.
Figure 5.- Effect of hyperthermia-induced seizures on 5´-nucleotidase activity. 5´-nucleotidase activity was measured in cerebral cortical membranes from neonates 48 hours and 5 days after hyperthermia induction following the protocol described in Experimental procedures. Kinetic parameters are reflected in Table 3 and were determined using nonlinear Michaelis-Menten curvefitting. Data are mean ± SEM of at least three experiments performed with different cerebral cortical membrane preparations. HIS, hyperthermia-induced seizures.
Table 1.-Binding parameters of [3H]DPCPX and [3H]ZM241385 binding to cerebral cortical membranes 48 hours after hyperthermia-induced seizures. Data represent Bmax and KD of binding data shown in Figure 1 and determined by nonlinear nonlinear regression of binding data with the GraphPad Prism 5 program. Data are means ± SEM from three-six independent experiments performed with different cerebral cortical membranes isolations. *p < 0.05, significantly different from control using one-way ANOVA and Tukey as post hoc test.
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Table 2.-Binding parameters of [3H]DPCPX and [3H]ZM241385 binding to cerebral cortical membranes 5 days and 20 days after hyperthermia-induced seizures. Data represent Bmax and KD of binding data shown in Figures 2 and 3 and determined by nonlinear nonlinear regression of binding data with the GraphPad Prism 5 program. Data are means ± SEM from at least four independent experiments performed with different cerebral cortical membranes isolations. HIS, hyperthermiainduced seizures. *p < 0.05, significantly different from control using unpaired two-tailed Student´s t test.
Table 3.- Kinetic parameters of 5´-nucleotidase activity assayed in cerebral cortical membranes 48 hours and 5 days after hyperthermia-induced seizures. Data represent Vmax and Km of data shown in Figure 5 and determined by nonlinear regression Michaelis-Menten curve-fitting. Data are means ± SEM from at least three independent experiments performed with different cerebral cortical membranes isolations. HIS, hyperthermia-induced seizures. *p < 0.05, significantly different from control using unpaired two-tailed Student´s t test.
Table 1.
48 hours A1 receptor Bmax (pmol/mg prot)
A2A receptor KD
Bmax
KD
(nM)
(pmol/mg prot)
(nM)
Control
0.244 ± 0.076
5.1 ± 4.7
0.271 ± 0.051
20.6 ± 8.3
HIS
0.559 ± 0.097 *
2.1 ± 1.7
0.124 ± 0.019 *
7.6 ± 3.4
HIS + KBr
0.127 ± 0.012
0.3 ± 0.2
0.264 ± 0.036
9.5 ± 3.5
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Table 2.
5 days A1 receptor Bmax (pmol/mg prot)
A2A receptor
KD
Bmax
KD
(nM)
(pmol/mg prot)
(nM)
Control
0.149 ± 0.025
3.9 ± 2.3
0.241 ± 0.058
21.2 ± 10.6
HIS
0.265 ± 0.073
7.1 ± 4.9
0.192 ± 0.026
8.34 ± 3.2
20 days A1 receptor Bmax (pmol/mg prot)
A2A receptor
KD
Bmax
KD
(nM)
(pmol/mg prot)
(nM)
Control
0.151 ± 0.035
11.2 ± 5.4
0.29 ± 0.038
10.6 ± 3.6
HIS
0.096 ± 0.014
3.2 ± 1.7
0.25 ± 0.022
1.6 ± 0.8*
Table 3. Control
HIS
Vmax
Km
Vmax
Km
(nmol/min · mg prot)
(μM)
(nmol/min · mg prot)
(μM)
48 hours
11.3 ± 0.3
57.5 ± 7
9.5 ± 0.4 *
64.4 ± 10.7
5 days
10.7 ± 0.2
83.1 ± 6
10.56 ± 0.5
59.7 ± 11
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