Neurobiological activity of Parawixin 10, a novel anticonvulsant compound isolated from Parawixia bistriata spider venom (Araneidae: Araneae)

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Epilepsy & Behavior 22 (2011) 158–164

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Epilepsy & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ye b e h

Neurobiological activity of Parawixin 10, a novel anticonvulsant compound isolated from Parawixia bistriata spider venom (Araneidae: Araneae) Helene Aparecida Fachim a, b, Alexandra Olimpio Siqueira Cunha a, Adriana Colsera Pereira a, b, René Oliveira Beleboni c, Leonardo Gobbo-Neto d, Norberto Peporine Lopes d, Joaquim Coutinho-Netto e, Wagner Ferreira dos Santos a, b,⁎ a

Neurobiology and Venoms Laboratory, Department of Biology, FFCLRP, University of São Paulo, São Paulo, Brazil Instituto de Neurociências e Comportamento de Ribeirão Preto, São Paulo, Brazil c Department of Biotechnology, University of Ribeirão Preto, São Paulo, Brazil d Organic Chemistry Laboratory, Department of Physics and Chemistry, FCFRP, University of São Paulo, São Paulo, Brazil e Department of Biochemistry and Immunology, FMRP, University of São Paulo, São Paulo, Brazil b

a r t i c l e

i n f o

Article history: Received 30 March 2011 Revised 3 May 2011 Accepted 7 May 2011 Available online 16 July 2011 Keywords: Anticonvulsant Glutamate Glycine Parawixin 10 Spider venom Parawixia bistriata

a b s t r a c t The neurobiological activity of Parawixin 10, isolated from Parawixia bistriata spider venom, was investigated. Cannulas were implanted in the lateral ventricles of Wistar rats (200–250 g, n = 6–8 per group) to perform anticonvulsant and behavioral assays, and synaptosomes from cerebral cortices of male Wistar rats were used for neurochemical studies. The results indicate that pretreatment with Parawixin 10 prevents the onset of seizures induced with kainic acid, N-methyl-D-aspartate, and pentylenetetrazole in a dose–response manner. Lower doses of Parawixin 10 significantly increased the latency to onset of kainic acid-, pentylenetetrazole-, and N-methyl-D-aspartate-induced seizures. There were maximum increases of 79% in L-[ 3H]glutamine uptake and 40% in [3H]glycine uptake; [3H]GABA uptake did not change. The findings demonstrate that this novel compound from P. bistriata venom exerts a pharmacological effect on the glutamatergic and glycinergic systems. © 2011 Elsevier Inc. All rights reserved.

1. Introduction In recent decades, several molecules purified from venom, especially polyamines and peptides, have aided the elucidation of physiological intrinsic properties of ion channels, neurotransmitter receptors, and transporters [1–3]. However, despite their great potential as pharmacological tools, only small parts of these compounds have been studied and the vast majority of molecules remain unexplored. Parawixia bistriata spider venom has been the object of intense investigation by our laboratory [4,5]. When applied to the rat's central nervous system (CNS), P. bistriata crude venom induces limbic seizures [6], whereas deproteinized venom blocks generalized tonic–clonic seizures induced with the GABAergic antagonists bicuculline, picrotoxin, and pentylenetetrazole (PTZ). Moreover, the addition of P. bistriata spider venom to synaptosomes from rat cerebral cortex increases L-glutamate (L-Glu) uptake and inhibits GABA uptake [5,7].

⁎ Corresponding author at: Av. Bandeirantes, 3900, FFCLRP/USP, Departamento de Biologia, CEP: 14040–090, Ribeirão Preto, São Paulo, Brasil. Fax: + 55 16 3602 4886. E-mail address: [email protected] (W.F. dos Santos). 1525-5050/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2011.05.008

Fontana et al. [4] isolated and identified Parawixin 1 (formerly PbTx 1.2.3), which increases L-Glu uptake in rat cortical synaptosomes and protects retinal neurons from ischemic damage. Parawixin 1 promotes the direct and selective enhancement of L-Glu influx by the EAAT2 transporter subtype, but does not interfere with the affinity of the transporter for its co-substrates and Na + ions, as demonstrated using cloned liposomes and COS cells [8]. According to Salazar and Fahlke [9] the selectivity and specificity of Parawixin 1 make it a starting point in the design of small molecules to be used in the treatment of pathological conditions caused by alterations of L-Glu. The fractionation of P. bistriata venom also revealed a potent inhibitor of GABA and glycine uptake, namely, FrPbAII (2-amino-5ureidopentanamide). According to Beleboni and co-workers [5], FrPbAII acts directly on these transporters rather than on ion channels permeable to Na +, K +, or Ca 2+, GABA transaminase, or reverse transport. Bioassays have shown that FrPbAII (formerly PbTx 2.2.1) blocks tonic–clonic seizures induced with bicuculline [7], PTZ, kainic acid (KA), and pilocarpine [10]. Also, when injected into the substantia nigra and pars reticulata, FrPbAII inhibits generalized seizures induced by GABAergic blockade of the area tempestas in the pyriform cortex, and when injected into the dorsal hippocampus, it exerts anxiolytic effects [11]. FrPbAII is also neuroprotective after intravenous administration in Wistar rats submitted to experimental

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glaucoma in the outer and inner nuclear layers of the retina [5]. When injected into vitreous, it completely inhibits retinal layer neuronal death induced by ischemia and ischemia–reperfusion, being 100 times more potent than nipecotic acid (unpublished data). Considering that neurotransmitter transporter malfunction is closely associated with severe pathological conditions such as epilepsy, Alzheimer's disease, and ischemia, P. bistriata spider venom could be considered an interesting source of probes for novel therapeutic strategies. In the light of all these facts, the aim of this study was to identify a novel compound from P. bistriata venom that acts on neurotransmitter transport and inhibits seizures induced with chemoconvulsants.

159

Agener União, Brazil) and xylazine (8 mg/kg; Calier, Spain) anesthesia. The coordinates used were 0.9 mm posterior to bregma, 1.6 mm lateral from midline, and 3.4 mm ventral from the surface of the skull according to the atlas of Paxinos and Watson [12]. Next, animals were placed in a stereotactic frame (Stoelting, USA) and injected percutaneously before surgical incision with 0.1 mL of 2% lidocaine hydrochloride containing epinephrine at 1:200.000 (Astra Química, Brazil). Cannulas and stainless-steel screws, for anchoring, were fixed to the skull with dental acrylate. Each cannula was sealed with a stainless-steel wire to avoid obstruction. After surgery, animals were injected with flunixin meglumine (1 mg/kg; Schering–Plough Animal Health, Brazil) to minimize pain. The animals were then allowed to rest for 5–7 days to recover from surgery.

2. Material and methods 2.4. Anticonvulsant screening 2.1. Spider collection and venom fractionation Nests of P. bistriata collected in the rural areas neighboring Ribeirão Preto, São Paulo, Brazil, were frozen at −4 °C, taken to the laboratory, and kept at − 20 °C. Glands and venom reservoirs were removed, homogenized in CH3CN:H2O (1:1; v/v), and centrifuged at 8000 g for 10 minutes at 4 °C. Supernatants were collected and filtered in membranes with a 3000-Da cutoff (Millipore, Microcon, USA) under centrifugation at 8000 g and 4 °C until complete filtration. Next, the extract was lyophilized, weighed, and submitted to fractionation. Dry extract was dissolved in ultrapure water of Milli-Q grade containing 0.1% TFA. The solution was subjected to reverse-phase high-performance liquid chromatography (RP-HPLC; Shimadzu, Japan) using an ODS C18 column (15 μm, 20 × 250 mm; Phenomenex-Jupiter, USA) at the flow rate of 8.0 mL/min and a UV light detection system at 214 nm. Initially, an isocratic gradient was run with 1% CH3CN:H2O (v/v) containing 0.1% TFA for 10 minutes. Next, a linear gradient from 1 to 60% CH3CN for 60 minutes was performed. Fractions were collected, lyophilized, weighed, and used in bioassays. 2.2. Purity of fractions Dry fractions were dissolved in 50% CH3CN:H2O (v/v) containing 0.1% formic acid (v/v). Molecular masses were determined by positive electrospray ionization (ESI+) on a high-resolution spectrometer. Fractions were injected with the aid of an infusion pump at a flow of 10 kL/minute. ESI-MS spectra were acquired on an UltrOTOF apparatus (Bruker Daltonics, Billerica, USA) in the continuous acquisition mode, scanning from m/z 50 to 2000 with a scan time of 5 seconds. Calibrations were made using intact horse heart myoglobin (Sigma– Aldrich, USA) and its typical cone-voltage-induced fragments.

CD97 doses of chemoconvulsants were used, that is, the dose producing hindlimb tonic convulsions in 97% of animals. These doses were previously established in dose–response experiments for each convulsant [13]. Before injections, animals were placed in the open field for 10 minutes. Rats were divided into groups (n = 6–8). Three different doses of Parawixin 10 were tested with each convulsant: Parawixin 10 at 0.25, 0.5, 1.0, and 1.5 μg/μL, 10 minutes before KA (0.8 μg/μL); Parawixin 10 at 1.0, 2.0, and 5.0 μg/μL, 10 minutes before NMDA (20 μg/μL); Parawixin 10 at 1.0, 2.0, 3.0, and 6.0 μg/μL, 10 minutes before PTZ (85 mg/kg, 0.2 mL, ip), all injected intracerebroventricularly in a volume of 1 μL. In separate groups of animals, one-milliliter volumes of diazepam (2, 5, and 10 mg/kg) and saline were injected intraperitoneally. The final volume for drugs delivered by the intracerebroventricular route was 1 μL over a 1-minute period, whereas PTZ was injected in a volume of 0.2 mL into the loose fold of the neck. After drug administration, animals were placed in the arena (open field) and filmed for 30 minutes. Next, rats were packed in individual cages until total recovery. Kainic acid- and NMDA-induced seizures were scored as follows: 0 = no seizure activity; 1 = jaw movements; 2 = head myoclonus; 3 = hindlimb myoclonus; 4 = elevation; 5 = elevation and fall; 6 = ear and head myoclonus, clonic movements, sequential events of elevation and fall; 7 = rolling, violent jumps, and vocalization; 8 = all behaviors of class 7 followed by periods of hypertonus [14]. Latency to the onset of seizures and percentage of animals protected against score 8 seizures were the parameters used to analyze the anticonvulsant effects. Seizures induced by PTZ administration were analyzed according to the Lamberty and Klitgaard [15] index, considering behavior of the convulsing animals as well as latency to onset of tonic–clonic seizures.

2.3. Animals and surgery 2.5. Spontaneous locomotor activity and behavioral assays Male Wistar rats (220–250 g), from the animal house of the Campus Universitarius of the University of São Paulo at Ribeirão Preto were used in the assays. The animals were kept in pairs in wire-mesh cages in a room with a 12-hour dark/light cycle (lights on at 7:00 AM) with water and food cubes ad libitum. Animals were maintained in accordance with the Brazilian Society for Neuroscience and Behavior ethical statements that follow the guidelines for animal care prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council (USA). Luminosity and temperature (22 °C) were kept constant in the housing and experiment rooms. Efforts were made to minimize the potential suffering of experimental subjects. Approval was obtained from the Ethics Committee for Care and Use of Laboratory Animals from Campus Universitarius of the University of São Paulo at Ribeirão Preto (CEUA No. 10.1.619.53.3). All animals were implanted with stainless-steel guide cannulas (10 mm) in the right lateral ventricle under ketamine (60 mg/kg;

On the morning of the experiments, Wistar rats (n = 6, 200–250 g) were transferred to the experimental rooms and allowed a 10-minute period for acclimation. Next, they were randomly assigned to treatment groups and injected intracerebroventricularly with either Parawixin 10 (2.5, 5.0, or 10 μg/μL) or saline 15 mM, with the aid of a Hamilton syringe moved by an infusion pump (Insight, Brazil) injecting a volume of 1 μL/1 minute. After 10 minutes, rats were gently placed in an open field consisting of an acrylic arena 60 cm in diameter and 12 cm high. Animals were monitored by a video camera interfaced with a VCR and a monitor placed in the adjacent room for 20 minutes, after which they were returned to their home cages and remained under observation for 2 hours. Spontaneous locomotor activity of the animals was determined by counting the line crossings in the open field of each animal along four time windows (0–5, 5–10, 10–15, and 15–20 minutes). Moreover, the

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duration of each behavior was measured. Behavioral categories were grouped according to Speller and Westby [16] with modifications as follows: exploratory (EX), grooming (GR), rearing (RE), and inactivity (IN).

2.6. Motor impairment: ataxia To evaluate the potential animal motor impairment, Wistar rats (n = 6, 200–250 g) were assayed in the rotarod test (Ugo Basile, Italy), which consists of a rotating bar (Ø 5 cm) driven by a motor. The day before the test, rats were trained to maintain their equilibrium on the rotating rod. The training consisted of three consecutive 1-minute attempts at 4 rpm. On the morning of the test, rats were again tested on the rotarod and only animals able to maintain their equilibrium on the rod were retained for the experimental procedure. Parawixin 10 (7.5, 15, 30, 45, 60, and 75 μg/animal) or saline 15 mM was administered intracerebroventricularly in a volume of 1 μL/minute, 10 minutes before the test. The latencies to fall off the rod at 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, and 90 minutes were recorded. Moreover, the number of animals falling on three consecutive attempts was used to calculate the doses at which 50% of the animals displayed toxicity (TD50). After assays, rats were euthanized with overdoses of anesthetic.

2.7. Histology At the end of the behavioral experiments, the animals were killed with an overdose of sodium thiopental (Cristalia, Brazil) and injected intracerebroventricularly with 1 μL of toluidine blue dye to mark the correct site of injection. Brains were immediately removed and placed in 4% formaldehyde solution. Later, they were manually cut to check the position of the cannula. 2.8. [ 3H]Glutamate, [ 3H]glycine, and [ 3H]GABA uptake into cortical synaptosomes Cerebral cortices from male Wistar rats (200–250 g) were rapidly removed and homogenized in ice-cold 0.32 M sucrose using Potter– Elhvejen Labo Stirrer LS-50 Yamato-type equipment. The sample was centrifuged for 10 minutes at 1700 g (4 °C), and the supernatant centrifuged for 20 minutes at 21,200 g (4 °C). The pellet was resuspended in Krebs–phosphate buffer (in mM: NaCl 124, KCl 5, KH2PO4 1.2, CaCl2 0.75, MgSO4 1.2, Na2HPO4 20, glucose 10, pH 7.4), referred to as synaptosome P2 fraction, and used in the L-Glu, glycine, and GABA uptake assay. Protein content was determined by the Lowry et al. [17] method, as modified by Hartree [18]. Synaptosomes were resuspended in Krebs–phosphate buffer and pre-incubated for 5 minutes at 37 °C (for [ 3H]glutamate and [ 3H]glycine) or for 15 minutes at 25 °C (for [3H]GABA) in the absence or presence of 6–12 increasing concentrations of Parawixin 10 (from 10 –11 to 10 1 μg/mL). Uptake assays were initiated by addition of [ 3H]glutamate, [ 3H] glycine, and [ 3H]GABA (10 nM, final concentration: 1 mCi/mmol; Amersham Biosciences, USA) to synaptosomal suspensions (100 μg of protein ml –1), and incubation for 3 minutes at the aforementioned temperatures for each neurotransmitter, according to Pizzo et al. [19]. The final volume of each tube was 500 μL. All reactions were stopped by centrifugation (3000 g, 3 minutes at 4 °C). Supernatants were discarded; pellets were washed twice with ice-cold distilled water, homogenized in 10% trichloroacetic acid (TCA), and centrifuged (3000 g, 3 minutes at 4 °C). Aliquots of supernatants were transferred to scintillation vials containing 5 mL of the biodegradable scintillation cocktail ScintiVerse (Fisher Scientific, USA), and their radioactivity was quantified in a scintillation counter (LS-6800; Beckman Coulter, USA) with a counting efficiency of 50% for 3H.

2.9. Statistical analysis Probit analysis [20] was used to calculate ED50 and TD50 values (with 95% confidence intervals). All parametric data were submitted to one-way analysis of variance (ANOVA) followed by the Newman– Keuls post hoc test. The frequencies of protected animals in the anticonvulsant assays were analyzed using the χ 2 test and are expressed as the number of animals exhibiting score 8 seizures. We considered as significant P values b0.01. Data from neurochemistry assays are expressed as mean percentages of control and SEM. These data were submitted to one-way ANOVA followed by the Newman–Keuls post hoc test.

3. Results 3.1. Venom fractionation Reverse-phase HPLC fractionation of compounds with molecular masses lower than 3 KDa (net weight = 80 mg) resulted in the chromatographic profile shown in Fig. 1A. We eluted 19 fractions that were tested for neurochemical activity. In this work, we chose Parawixin 10, indicated in the chromatogram by an arrow. The ESI-MS spectrum of Parawixin 10 showed a major molecular ion peak at m/z 298.7363 Da (M + 2H + ) with an approximate molecular mass of 587.5 Da (Fig. 1B), thus confirming its high purity.

3.2. Anticonvulsant activity Data from anticonvulsant screening of Parawixin 10 for protecting animals against seizures are illustrated in Fig. 2. The data indicate that intracerebroventricular microinjection of Parawixin 10 into rats prior to the administration of kainic acid inhibits seizures in 40, 68, and 100% of animals at doses of 0.5, 1, and 1.5 μg/μL, respectively (χ 2[3] = 303.7, P b 0.001) (Fig. 2A), whereas intraperitoneal diazepam at 2 mg/ kg blocked KA-induced seizures in only one animal (16%); it did not protect animals at other doses (5 and 10 mg/kg of diazepam), and only decreased the severity of the seizures. In the NMDA assay, intracerebroventricular Parawixin 10 at 2.5 and 5 μg/μL blocked seizures in 50 and 100% of animals, respectively (χ 2[2] = 293.3, P b 0.0001) (Fig. 2B). No animal was protected against NMDA- or KA-induced seizures by intravenous diazepam (2, 5, and 10 mg/kg). Systemic administration of PTZ (80 mg/kg, ip) induced generalized seizures in all treated animals, which were blocked by preadministration of Parawixin 10 (2, 3, and 6 μg/μL) in 50, 7, and 100% of animals, respectively (χ 2[3] = 323.2, P b 0.0001) (Fig. 2C). In contrast, generalized seizures evoked by the GABA-mediated Cl – influx blockade with PTZ were completely abolished by the administration of diazepam (2 mg/kg), whereas the anticonvulsant effects of Parawixin 10 were observed only at higher doses. The anticonvulsant activity of Parawixin 10 exhibited a dose-dependent profile in all experiments. In addition, analysis of the latency to seizure onset of unprotected animals revealed that at lower doses, Parawixin 10 significantly increased latency to KA-induced [F(2,11) = 46.48, P b 0.01] and NMDA-induced [F(2,9) = 26,41, P = 0.0005] seizures. Latency to PTZ-induced seizures also increased after the administration of nonprotecting doses of Parawixin 10. In this case, 3 μg/μL Parawixin 10 increased the latency to seizure onset and generalization [F(3,20) = 47,70, P b 0.0001]. Latencies to seizure onset of seizing animals are listed in Table 1. It is important to note that the Parawixin 10 dose used to protect 100% of KA-induced seizures was the lowest dose used (1.5 μg/μL) to obtain this protection percentage relative to other convulsants.

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161

A 1,0

0,8

CH3CN (%)

Absorbance (214 nm)

60

Parawixin10

0,6

0,4

0,2

1 0,0 0

5

10

15

20

25

30

35

40

45

50

55

60

B 294,7363 Da (M+2H+)

Relative absorbance (%)

2.0

1.5

1.0

0.5

0.0 0

100

200

300

400

500

600

700

800

900

m/z

Fig. 1. (A) Reverse-phase HPLC chromatographic profile of low-molecular-weight compounds (N3000 Da) of the venom of the spider Parawixia bistriata. The arrow points to the compound Parawixin 10. (B) ESI-MS spectrum of Parawixin 10 showing a major peak at m/z 294.7363 (M + 2H+).

3.3. Spontaneous locomotor activity and behavioral assays Analysis of spontaneous locomotor activity in the open field revealed differences among the time periods (four time windows: 0–5, 5–10, 10–15, and 15–20 minutes) [F(3,9) = 30.369, P b 0.0001], Parawixin 10 treatments [F(3,11) = 13.542, P = 0.001], and treatment versus time window [F(9,23) = 4.158, P = 0.003] (Fig. 3A). A peak in exploratory activity was observed from 0 to 5 minutes after all treatments (P b 0.0001), followed by a decrease in exploratory activity. The administration of any dose of Parawixin 10 exerted a marked inhibitory effect on exploratory behavior, considering that there was a marked decrease in the number of line crosses from 5 to 20 minutes after injections (P b 0.001). Behavioral analysis revealed differences among treatments in the open-field experiments with respect to exploration [F(3,12) = 5.967, P = 0.009] and grooming [F(3,12) = 33.39, P b 0.0001], whereas

inactivity remained equal [F(3,12) = 0.573, P = 0.643] (Fig. 3B). Post hoc tests showed that rats treated with10 μg/μL Parawixin 10 spent more time grooming and did not explore the arena as much as physiological saline-treated control animals (P b 0.001). Lower doses of Parawixin 10 did not alter rat behavior in the open-field test.

3.4. Motor ataxia Parawixin 10 at any concentration used in the tests was able to induce motor impairments in the rotarod test. Animals did not fall off the rod after administration of Parawixin 10 at doses up to 75 μg/μL. Therefore, the estimated TD50 for Parawixin 10 is NN75 μg/μL, which is more than 37.5-fold higher than the ED50 for PTZ-induced seizures, more than 77-fold higher than the ED50 for NMDA-induced seizures, and more than 1071-fold higher than the ED50 for KA-induced seizures.

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A

Kainic Acid

***

85

50

*** 25

0.25 µg/ µL 0.5 µg/ µL

B

1 µg/ µL

NMDA

1.5 µg/ µL

Number of quadrants

***

saline

60

* 35

75

0-5

-15

***

50

B

5-10

* ** 10-15

15-20

time periods (min) Exploratory behavior

Grooming

Inactivity

750

25

0 saline

1 µg/ µL

C

2.5 µg/ µL

5 µg/ µL

PTZ

***

100

***

75

500

**

*

250

***

50

0

25 0

* ** **

10

***

100

Nonseizing animals (%)

Saline Parawixin 10 10ug/ul Parawixin 10 5ug/ul Parawixin 10 2.5ug/ul

75

0

Nonseizing animals (%)

A

Time spent (s)

Nonseizing animals (%)

100

Saline

2.5ug

5ug

10ug

Parawixin 10 saline

1 µg/ µL

2 µg/ µL

3 µg/ µL

6 µg/ µL

Fig. 2. Anticonvulsant effects of Parawixin 10 against (A) KA-, (B) NMDA-, and (C) PTZinduced seizures. Data are expressed as percentage of animals protected from tonic–clonic seizures induced with each chemoconvulsant versus Parawixin 10 concentration (log).

Fig. 3. (A) Spontaneous locomotor activity at different times in the open-field test of rats treated with different concentrations of Parawixin 10 or physiological saline. (B) Behavioral profile of animals in the open field. Data were analyzed using repeatedmeasures ANOVA (A) and one-way ANOVA (B) followed by Newman–Keuls post hoc test. P values b 0.05 were considered significant. **P b 0.01. Error bars represent SEM.

3.5. [ 3H]Glutamate, [ 3H]glycine, and [ 3H]GABA uptake into cortical synaptosomes

was added to synaptosomes. Finally, Parawixin 10 did not alter GABA uptake (Fig. 4C) at any of the concentrations used.

Figs. 4A–C are dose–response curves for the effect of Parawixin 10 on [ 3H]glutamate, [ 3H]glycine, and [ 3H]GABA uptake. The results indicate that in the presence of 10 ng/mL Parawixin 10, there was a maximum increase of 79% in glutamate uptake (Fig. 4A), whereas glycine (Fig. 4B) uptake increased by 40% when 1 ng/mL Parawixin 10

4. Discussion Previous studies on spider venom molecules have demonstrated the anticonvulsant and neuroprotective activities of these compounds in several animal models of seizure induction and neuronal damage

Table 1 Latency to onset of score 8 seizures evoked by chemoconvulsants after injection of Parawixin 10 and diazepam. Chemical convulsant (route, dose)

Kainic acid (icv, 2.4 μg/μL) PTZ (ip, 85 mg/kg) NMDA (icv, 17 μg/μL)

Latency to sezirure onset (s) Saline

218.85 ± 46.65 59.88 ± 6.42 130 ± 39.16

Diazepam

Parawixin 10

2 mg/kg

5 mg/kg

10 mg/kg

0.5 μg

1 μg

2 μg

2 μg

162.5 ± 18.87a — 250 ± 56.86a

237.5 ± 30.65 — 332 ± 68.72a

271 ± 112.2 — 397 ± 125.0a

1030 ± 141b 829.5 ± 97.6a —

—c 754 ± 214.5a 503.5 ± 2.35b

— — 611.5 ± 13.87b

— 1383 ± 18.5b —

Note. Mean latencies (± SEM) were submitted to one-way analysis of variance followed by Student–Newman–Keuls post hoc test. a P b 0.01, compared with saline. b P b 0.001, compared with saline. c —, Absence of seizures.

H.A. Fachim et al. / Epilepsy & Behavior 22 (2011) 158–164

L-[3H]-Glu uptake (% of control)

A

163

200 150

*

100

*

*

*

*

*

10-7

10-6

*

*

*

10-4

10-3

*

* *

*

100

10

50 0

control 10-11 10-10

10-9

10-8

10-5

10-2

10-1

Parawixin 10 (µg / mL)

[3H]-Gly uptake (% of control)

B

200

150

*

*

10-4

10-3

100

50

0

control

10-5

10-2

10-1

100

10

10-1

100

10

Parawixin 10 (µg / mL)

[3H]-GABA uptake (% of control)

C

100

50

0

control

10-5

10-4

10-3

10-2

Parawixin 10 (µg / mL) Fig. 4. Analysis of the effects of different concentrations of Parawixin 10 added to synaptosomes incubated with (A) L-[3H]glutamate, (B) [3H]glycine, and (C) [3H]GABA. Data from five experiments carried out in triplicate (error bars represent SEM.) were analyzed using one-way ANOVA followed by the Newman–Keuls test. P values b 0.05 were considered significant.

(for review, see Rajendra et al. [21] and Mortari et al. [22]). This inhibitory activity might be attributed to the selective antagonism of glutamatergic receptors (JsTx toxin [23]), blockade of Na + (μ-Aga-I to μ-Aga-IV [24]), and Ca 2+ (FTX toxin [25]) cationic channels, inhibition of glutamate release (PnTx3–6 toxin [26]), enhancement of glutamate transporters (Parawixin 1 [4]), and inhibition of GABA and glycine transporters (FrPbAII [5]). The present work evaluated the effects of a novel compound isolated from P. bistriata spider venom on chemically induced seizures and synaptosomal neurotransmitter uptake. Our data showed that intracerebroventricular administration of Parawixin 10 blocks generalized seizures induced with KA, NMDA, and PTZ, and the anticonvulsant dose that blocked KA-induced seizures in 100% of animals was three times lower than the dose for NMDA-induced seizures and four times lower than the dose for PTZ-induced seizures. Notwithstanding the effect on L-Glu uptake in in vitro experiments, these findings might indicate a substrate preference in the mode of action of Parawixin 10, that is, inhibition of excitatory transmission mediated

by non-NMDA receptors. We emphasize that no animals exhibited seizure-related behaviors when there was protection, all protected animals had 0 scores with respect to seizures. Interestingly, Parawixin 10 does not induce the locomotor alterations commonly reported after the administration of glutamatergic receptor antagonists [27]. Another issue is pore formation in the synaptosomal membranes. However, we ruled out the presence of lesions on neuron membranes, because we know that boiled venom does not cause the formation of pores in these membranes. That was done many times in our laboratory, using electron microscopy and LDH assay. Moreover, with respect to synaptosomal preparation integrity, we guaranteed in each experiment where we used the neurotransmitters "cold" (nonradioactive) as a control, and these inhibited nonspecific uptake by 20%. In fact, our neurochemistry data indicate that addition of low concentrations of Parawixin 10 to synaptosomes from rat cerebral cortices increases L-Glu uptake in 79% of animals, suggesting a highaffinity mediation [28]. Moreover, we found an alteration in glycine

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uptake, although with a maximum increase of 40% at higher doses. As glutamate transporters regulate the extracellular concentrations of L-Glu, increases in the activity of these transporters might function as a compensatory mechanism to counteract chemoconvulsant-induced hyperexcitation. Several studies have delineated the role of L-Glu in pathological processes. In fact, the extrasynaptic concentration of this transmitter is tightly regulated by high-affinity transporters, the failure of which may trigger excitotoxicity processes that lead to neuronal death (for review, see Danbolt [29] and Gadea and López-Colomé [30]). The presence of excessive glutamate concentrations has been reported in early neurodegenerative processes in several pathologies such as ischemia, epilepsy, amyotrophic lateral sclerosis, and Alzheimer's disease [30,31]. Previous work from our laboratory identified Parawixin 1, another compound that acts allosterically over EAAT2, the predominant CNS LGlu receptor subtype [4,8]. According to Fontana and co-workers [4], endovenous administration of Parawixin I protects rat retinal neurons from ischemic damage in an experimental model of acute glaucoma. Because of the importance of EAAT2 in the regulation of extracellular concentrations of glutamate, drugs that target this subtype of transporter may prevent excitotoxicity without blocking L-Glu transmission [9]. Another important finding is that Parawixin 10 increases glycine uptake. In this regard, Raiteri and co-workers [32] showed that glycine uptake by the astrocytic transporter, GLYT-1 may induce GABA release from co-localized synapses, enhancing the inhibitory activity of Parawixin 10. Another point is the co-localization of GLYT-1 and NMDA receptors, which suggests that this transporter regulates the activity of this excitatory receptor [33,34]. Finally, glycine works as a necessary co-agonist of the NMDA receptor by binding to the strychnine-insensitive site at the NMDA complex, allowing further glutamate activation of this receptor [35]. Hence, the increase in glycine uptake in particular synapses may lead to enhancement an inhibitory activity and decrease of excitotoxic activity, respectively mediated by GABA and L-Glu. The most interesting result of our work is rat seizure inhibition, and many assays can be performed, such as neurochemistry experiments using specific L-Glu and Gly antagonists in synaptosomes from cerebral cortex, to determine whether there would be potentiation or competition for the same site. Still, it may be necessary, for instance, to run a full set of tests using Gly and glutamate transporters transfected in host cell lines such as COS-7 and MDCK. Preliminary magnetic resonance structural analyses of Parawixin 10 indicate that this compound is probably a polyamine. Our findings indicate that Parawixin 10 has an important mode of action that should be further investigated as it may be a novel tool in understanding the glutamatergic and glycinergic transmission systems and diseases associated with these systems. Acknowledgments This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) from the São Paulo and Brazilian governments. The authors are grateful to José Carlos Tomaz for technical assistance. We also thank Juliana Martus de Azevedo for English review. References [1] Usherwood PNR, Blagbrough IS. Spider toxins affecting glutamate receptors: polyamines in therapeutic neurochemistry. Pharmacol Ther 1991;52:245–68. [2] Lewis RJ, Garcia ML. Therapeutic potential of venom peptides. Nat Rev Drug Discov 2003;2:790–802.

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