Bucladesine

Effects of pentoxifylline and H-89 on epileptogenic activity of bucladesine in pentylenetetrazol-treated mice
Mahshid Sadat Hosseini-Zare a, Forouz Salehi a, Seyedeh Yalda Seyedi a,b, Kian Azami a, Tahereh Ghadiri c,
Mohammad Mobasseri a, Shervin Gholizadeh a, Cordian Beyer d, Mohammad Sharifzadeh a,c,d,⁎
a Department of Pharmacology and Toxicology, Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
c Department of Neuroscience, Faculty of Advanced Science and Technology in Medicine, Tehran University of Medical Sciences, Tehran, Iran
d Institute of Neuroanatomy, Faculty of Medicine, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany

a r t i c l e i n f o

Article history:
Received 6 February 2011
Received in revised form 31 August 2011 Accepted 7 September 2011
Available online 21 September 2011

Keywords:
Cyclic AMP Phosphodiesterase inhibitor Protein kinase A
GABA NMDA
Pentylenetetrazol Seizure
a b s t r a c t

The present study shows interactive effects of pentoxifylline (PTX) as a phosphodiesterase (PDE) inhibitor, H-89 as a protein kinase A (PKA) inhibitor and bucladesine (db-cAMP) as a cAMP agonist on pentylenetetrazol (PTZ)- induced seizure in mice. Different doses of pentoxifylline (25, 50, 100 mg/kg), bucladesine (50, 100, 300 nM/ mouse), and H-89 (0.05, 0.1, 0.2 mg/100 g) were administered intraperitoneally (i.p.), 30 min before intravenous (i.v.) infusion of PTZ (0.5% w/v). In combination groups, the first and second components were injected 45 and 30 min before PTZ infusion. In all groups, the control animals received an appropriate volume of vehicle. Single administration of PTX had no significant effect on both seizure latency and threshold. Bucladesine significantly decreased seizure latency and threshold only at a high concentration (300 nM/mouse). Intraperitoneal administration of H-89 (0.2 mg/100 g) significantly increased seizure latency and threshold in PTZ-treated animals. All applied doses of bucladesine in combination with PTX (50 mg/kg) caused a significant reduction in seizure latency. Pretreatment of animals with PTX (50 and 100 mg/kg) attenuated the anticonvulsant effect of H-89 (0.2 mg/100 g) in PTZ-exposed animals. H-89 (0.05, 0.2 mg/100 g) prevented the epileptogenic activity of bucladesine (300 nM) with significant increase of seizure latency and seizure threshold. In conclusion, we showed that seizure activities were affected by pentoxifylline, H-89 and bucladesine via interactions with intracellular cAMP and cGMP signaling pathways, cyclic nucleotide-dependent protein kinases, and related neurotransmitters.

© 2011 Elsevier B.V. All rights reserved.

⦁ Introduction

Epilepsy represents a chronic neurological disorder with a high in- cidence rate in human and is characterized by recurrent unprovoked seizures (Sander and Shorvon, 1996). Epileptic seizures are classified by their pattern of activities in the brain and their behavioral effects. Seizures can be described as either focal or generalized affecting the entire cortex. Although different animal species were used in the past to assess the mechanisms underlying seizure (Bradford, 1995; Fisher, 1989), the physiological basis of epileptic seizure is still vague. In vivo and in vitro studies show that cyclic adenosine monophosphate (cAMP) plays an important role in the pathophysiology of epileptic disor- ders (Ludvig and Moshe, 1987). It has been demonstrated that cAMP levels change during seizure in the brain (Ferrendelli and Gross, 1980). In addition, cAMP levels are elevated in the cerebral cortex (Ferrendelli

⁎ Corresponding author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Tel./fax: + 98 21 6648 2705.
E-mail address: [email protected] (M. Sharifzadeh).
and Gross, 1980) and in spinal fluid (Myllyla et al., 1975) after an epileptic attack. It was recently reported that intracellular cAMP signaling path- ways in cerebral cortical and hippocampal neurons are involved in sei- zure disorders (Naseer et al., 2010). Cyclic AMP causes a decrease in the spontaneous discharge rate of mammalian neurons (Boulton et al., 1993). Nevertheless, the role of cAMP during seizure is still controversial- ly discussed. Some studies showed that cAMP exhibits anticonvulsant effects by scavenging free radicals and suppressing inflammatory mechanisms (Ray et al., 2005; Whitcomb et al., 1990). On the other hand, cAMP appears to be a proconvulsant second messenger that ex- erts its effects particularly by activating cAMP-dependent protein ki- nase pathways. Cyclic AMP-induced protein kinase activation results in the phosphorylation of the cAMP response element binding protein (CREB), γ-aminobutyric acidA (GABAA), and N-methyl-D-aspartic acid (NMDA) receptors (Jancic et al., 2009; Lan et al., 2001; Poisbeau et al., 1999; Westphal et al., 1999), which play important roles for seizure- mediated processes (Bracey et al., 2009; Esteban et al., 2003; Lopez et
al., 2007; Poisbeau et al., 1999).
PDE enzymes consist of several subtypes that are distributed in different tissues. These enzymes play critical roles in cyclic nucleotide

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catalysis which make them predisposed as tools for pharmacological manipulation. PDE inhibitors affect different seizure-mediated path- ways by increasing intracellular levels of cyclic nucleotides (Bracey et al., 2009; Jeon et al., 2005).
PTX as a methyl xanthine agent inhibits various types of PDE en- zymes and elevates intracellular cyclic nucleotides levels (Cunha et al., 2002; Maurice et al., 2003). Adenosine that possesses a clear anticon- vulsant potential during pentylenetetrazol-induced seizures (Berman et al., 2000) acts as a neuro-protective agent for hypoxic stress, status epilepticus and catatonia with mainly inhibitory effects on neural activ- ity (Dunwiddie, 1999; Singh and Kulkarn, 2002; Thorat and Kulkarn, 1990). The effects of adenosine were pharmacologically antagonized by PTX (Fredholm and Persson, 1982). PTX effects on seizure activity appear to be dependent on different controversially discussed mecha- nisms (Tariq et al., 2008).
Bucladesine is a cell membrane permeable cyclic nucleotide ana- logue which mimics the action of endogenous cAMP and acts as a phosphodiesterase inhibitor (Azami et al., 2010; Campos-Toimil et al., 2008; Sharifzadeh et al., 2007b). This component is widely used to increase intracellular cAMP level (Neumann et al., 2002; Qiu et al., 2002). Besides provoking epileptic seizure, the injection of bucladesine into the amygdala (AM) causes neuronal damage at the injection site and in the CA1–3 hippocampal regions (Tojo et al., 1990). Increased excitability and enhancement of epileptiform activ- ity by cAMP analogues was also previously shown (Boulton et al., 1993). Moreover, the administration of bucladesine caused long la- tency limbic type behavioral seizure with epileptiform EEG events (Ludvig et al., 1992).
H-89 is a PKA inhibitor which competitively blocks the phosphor- ylation of serine and threonine residues of this enzyme (Hidaka et al., 1984; Sharifzadeh et al., 2005, 2007a). This chemical component has been frequently used to study PKA signaling pathways in neuronal tissues (Kaneishi et al., 2002; Kim et al., 2005). Previous studies have shown the possible involvement of protein kinases in apoptotic cell death induced by neurotoxic compounds (Koriyama et al., 2003; Ueda et al., 1996). In addition, the effect of PKA inhibitors on attenu- ation of apoptotic cell death has been shown in vitro (Ueda et al., 1996). Therefore, PKA inhibitors may represent prime candidates to attenuate PTZ-mediated seizure.
Considering the controversial roles of cyclic nucleotides in the oc- currence of seizure, this study aimed to clarify the effects of cyclic nucleotide-related compounds on PTZ-induced seizure in mice. For this reason, we used pharmacological tools such as PTX and db- cAMP to block PDE isozymes and increase intracellular levels of cAMP, respectively, and H-89 for inhibiting cAMP-dependent protein kinases.

⦁ Materials and methods

⦁ Animals

Male albino mice weighing 20–25 g were obtained from the Pasteur Institute. Animals were kept under a controlled light and dark cycle (12/12 h) and allowed free access to food and water ad libitum. All animal manipulations were performed according to the Ethical Committee for the use and care of laboratory animals of Faculty of Sciences of Tehran University (357; 8 November 2000). All efforts were made to minimize animal suffering.

⦁ Chemicals

All chemicals used in this study were purchased from Sigma (St Louis, MO, USA). The following chemical agents were used: bucladesine (50, 100 and 300 nM/mouse), pentoxifylline (25, 50, and 100 mg/kg),
H-89 (0.05, 0.1 and 0.2 mg/100 g) and pentylenetetrazol (0.5% w/v). Bucladesine (db-cAMP) and H-89 were dissolved in a solution of
DMSO (dimethyl sulfoxide) and distilled water (10%). The solvent for PTX was saline. PTZ was prepared in normal saline to obtain a concen- tration of 0.5% w/v for intravenous (i.v.) administration.

⦁ Routs of administration

Pentoxifylline (25, 50, 100 mg/kg), bucladesine (50, 100, 300 nM/ mouse) and H-89 (0.05, 0.1, 0.2 mg/100 g) were administered intra- peritoneally (i.p.) 30 min before intravenous (i.v.) infusion of PTZ. In combination groups, the first and second components were injected 45 and 30 min before PTZ infusion. In all groups, the respective con- trol animals received an appropriate volume of vehicle. For the i.v. in- fusion, the needle was inserted into the lateral tail vein, fixed to the tail vein by a narrow piece of adhesive tape, and the animal was allowed to move freely (Gholipour et al., 2008, 2009). PTZ solution was infused at a concentration rate of 1 ml/min.

⦁ Seizure threshold determination

Infusion was stopped at the onset of seizure exhibition. The threshold for the appearance of colonic seizures was calculated by the following formula (Dhir et al., 2011; Gholipour et al., 2009):

Rðml=sÞ × TðsÞ × Cðmg=mlÞ × 1000=B:W:ðgÞ

R: infusion rate of 0.5% (w/v) PTZ solution, i.e. 1 ml/min. T: time for onset of seizure (s), C: concentration of infused PTZ (mg/ml), B.W.: body weight of the animal (g).
Seizure threshold was measured by the following end points: 1. Initial myoclonic jerk. 2. Onset of generalized clonus with loss of righting reflex. 3. Onset of tonic extensor phase. The minimum time for the occurrence of these stages of convulsion was also recorded. The values of latency, weigh of the animal, and rate of PTZ infusion were substituted in the above mentioned formula. The amount of PTZ in mg/kg for induction of the first signs of each convulsion stage was measured as seizure threshold. In control groups, the laten- cy and value of administered PTZ to any seizure sign manifestation were recorded at the start of each convulsion phase.

⦁ Statistical analysis

All values were shown as means±S.E.M. of 7 animals per group. Data were analyzed by using one way analysis of variance (ANOVA) followed by Newman Keuls post-hoc test. A P value of 0.05 or less was considered statistically significant.

⦁ Results

⦁ Effects of PTZ on the onset and threshold of seizure

The administration of PTZ in mice tail vein was associated with a convulsion episode such as sudden fore- and hind limb constriction, hyperextension of tail, postural and myoclonic jerk losing. These signs of epilepsy were observed in PTZ-treated animals. The latency time for the onset of seizure and the value of seizure threshold were 21.24 ± 0.48 min and 72.07 ± 2.19 mg/kg respectively.

⦁ Effect of pentoxifylline pre-treatment on PTZ-induced seizures

Animals belonged to the PTX-treated group received PTX (25, 50 and 100 mg/kg, i.p) 30 min before PTZ (0.5% w/v, i.v.) infusion. Simi- larly, animals of the control group received saline (0.9%) as a PTX sol- vent 30 min before PTZ administration. None of the applied PTX doses had any significant effect on seizure threshold and seizure latency compared to control group (Table 1).

Table 1
Effect of pentoxifylline pre-treatment on seizure latency and threshold in PTZ-treated animals.

Treatment (mg/kg) Seizure latency (s) Seizure threshold (mg/kg)
Control 22.10 ± 0.71 73.55 ± 3.33
PTX 25 21.75 ± 1.29 84.11 ± 4.91
PTX 50 21.09 ± 0.89 57.74 ± 3.37
PTX 100 21.19 ± 0.82 76.36 ± 2.49
Administration of pentoxifylline did not cause any significant alterations in seizure latency and seizure threshold compared to control group. Data are expressed as means ±S.E.M. of seven animals in each group.

⦁ Effect of single administration of bucladesine on seizure parameters induced by PTZ

Bucladesine (50, 100 and 300 nM/mouse, i.p.) was administered 30 min before PTZ infusion. Control group received a vehicle using the same volume. Pretreatment of animals with bucladesine changed seizure parameters induced by PTZ (Fig. 1A and B). Significant re- sponse was obtained with a concentration of 300 nM/mouse which caused a decrease of seizure latency (***Pb 0.001) and threshold (**Pb 0.01) (Fig. 1A and B).

⦁ Effect of H-89 pre-treatment on PTZ-induced seizure

Effects of pretreatment with different doses of H-89 (0.05, 0.1 and
0.2 mg/100 g, i.p., 30 min) on PTZ (0.5% w/v i.v)-induced seizure are shown in Fig. 2A and B. The administration of H-89 at a dose of
0.2 mg/100 g significantly increased seizure latency and threshold

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compared to the control group (***Pb 0.001). No significant differ- ences were observed in seizure latency and threshold with two other doses of H-89 (0.05 and 0.1 mg/100 g) in comparison with con- trol animals (Fig. 2A and B).

⦁ Combinative effects of pentoxifylline and bucladesine pre-treatment on PTZ-induced seizure parameters

Single administration of either PTX (25, 50, and 100 mg/kg) or bucladesine (50 nM/mouse) had no effect on seizure threshold and latency. By applying bucladesine (50 nM/mouse) and pentoxifylline (50 mg/kg) 45 and 30 min before PTZ infusion, respectively, the la- tency decreased significantly compared to control group (**Pb 0.01) (Fig. 3A). However, this combination did not affect seizure threshold (Fig. 3B). There was no significant difference between combination group bucladesine (50 nM/mouse) and PTX (50 mg/kg) pretreated animals in both seizure latency and threshold (Fig. 3A and B). Howev- er, this combination decreased seizure latency compared to the db- cAMP (50 nM/mouse)-pretreated group (*Pb 0.05) (Fig. 3C), whereas no significant changes were observed in seizure threshold (data not shown).
In animals pretreated with combination of bucladesine (100 nM/ mouse, 45 min) and pentoxifylline (50 mg/kg, 30 min) before PTZ infu- sion, the seizure latency was significantly decreased in comparison with control (***P b 0.001) or PTX (50 mg/kg, **P b 0.01)-treated mice (Fig. 3A). The seizure threshold was also significantly (**P b 0.01) re- duced in this combination group compared to PTX (50 mg/kg)-treated animals (Fig. 3B). The statistical comparison between combination group bucladesine (100 nM/mouse) and PTX (50 mg/kg) with db- cAMP (100 nM/mouse) pretreated mice showed significant decrease only in seizure latency (*P b 0.05) (Fig. 3C).
Data obtained from animals which received a combination of bucladesine (high dose 300 nM/mouse, 45 min) and pentoxifylline (50 mg/kg, 30 min) before PTZ infusion showed significant differences in latency compared to control (***Pb 0.001) and PTX (50 mg/kg,
**Pb 0.01) groups (Fig. 3A). In addition, seizure latency was not affected by the combination group db-cAMP (300 nM/mouse) and (PTX 50 mg/kg) in comparison with db-cAMP (300 nM/mouse) pre- treated mice (Fig. 3C). The comparison between combination group, db-cAMP (300 nM/mouse) and (PTX 50 mg/kg) with db-cAMP (300 nM/mouse) did not show any significant alteration in seizure threshold (data not shown).

⦁ Effects of pre-treatment with pentoxifylline and H-89 in combination

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Control 50 100 300
Bucladesine (nM/mouse)
on PTZ-induced seizure in mice

All animals belonging to this combination group received PTX as the first component for 45 min and H-89 as the second one 30 min before PTZ infusion. Data obtained from groups that received PTX 50 mg/kg and H-89 0.2 mg/100 g, and PTX 100 mg/kg and H-89
0.2 mg/100 g, showed significant differences in seizure latency and threshold compared to controls (***Pb 0.001) (Fig. 4A and B). The ef- fect of H-89 (0.2 mg/100 g) on seizure threshold and latency was sig- nificantly attenuated by PTX (50 and 100 mg/kg) administration significantly (*Pb 0.05) (Fig. 4A and B).

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Control 50
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⦁ Effect of bucladesine and H-89 pre-treatment on PTZ-induced seizure
Bucladesine has initially been administered 15 min before H-89 administration and H-89 was injected 30 min before PTZ infusion.

Bucladesine (nM/mouse)
Fig. 1. Effect of bucladesine on seizure latency (A) and seizure threshold (B). Analysis shows significant differences in both latency (***P b 0.001) and threshold (**P b 0.01) of seizure in the bucladesine group (300 nM/mouse), compared to the control animals. Data represent the mean±S.E.M. of seven animals in each group.
H-89 (0.05 and 0.2 mg/100 g) prevented the epileptogenic effects of bucladesine (300 nM/mouse) and significantly increased the latency (***Pb 0.001) and threshold of seizure (***Pb 0.001) in com- parison with bucladesine group (Fig. 5A and B). Administration of bucladesine (300 nM/mouse) and H-89 (0.2 mg/100 g) in

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Control 0.05 0.1 0.2
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- 50 50 50 50 PTX (mg/kg)

Fig. 2. Effect of H-89 on latency (A) and seizure threshold (B). A significant elevation in
both latency (***Pb 0.001) and seizure threshold (***P b 0.001) was observed in the H- 10
89 group (0.2 mg/100 g), compared to the control mice. Data represent the mean ±S.E.
M. of seven animals in each group. 5

combination increased the seizure threshold significantly (*Pb 0.05), compared to control animals (Fig. 5B).

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⦁ Discussion

The present study aimed to investigate the effects of cAMP-related chemical components on latency and seizure threshold in PTZ- induced seizure. Our results failed to show clear-cut significant changes on both latency and seizure threshold in PTX-treated groups. The inhibition of PDEs, blockade of adenosine receptors, and scaveng- ing of free radicals are the well-known pharmacological mechanisms of PTX (Cunha et al., 2002; Davila-Esqueda and Martinez-Morales, 2004).
Several types of PDE enzymes which are implicated in the regulation of intracellular cAMP and cGMP levels are blocked by PTX (Essayan, 1999; Maurice et al., 2003). The levels of cAMP, cGMP and NO that are known to be important for seizure activity were increased by PDE inhi- bition (Ahern et al., 2002; Prast and Philippu, 2001). The interaction of NO/cGMP pathway with glutamate neurotransmission can also affect seizure activities (Garthwaite, 1991; Nandhakumar and Tyagi, 2008; Paul and Ekambaram, 2005; Prast and Philippu, 2001; Rajasekaran et al., 2003; Snyder and Bredt, 1991). In addition, cGMP-induced activa- tion of PKG decreased GABAergic function via phosphorylation of GABA receptor subtypes (Ahern et al., 2002). Moreover, PTX as an aden- osine receptor antagonist (Fredholm and Persson, 1982) has the poten- cy to interact with anticonvulsant activity of adenosine in PTZ-induced seizure (Berman et al., 2000). Some studies also showed that cAMP sup- presses seizure activity mechanisms involving free radicals (Ferrendelli and Kinscherf, 1977; Whitcomb et al., 1990).
Fig. 3. Effect of db-cAMP and PTX in combination on seizure latency (A and C) and threshold (B). The administration of db-cAMP (50 nM/mouse) and PTX (50 mg/kg) in combination reduced seizure latency in comparison with control (**P b 0.01, A) and db-cAMP (50 nM/mouse) (*P b 0.05, C) pretreated groups. Seizure latency was also re- duced in animals received combination of db-cAMP (100 or 300 nM/mouse) and PTX (50 mg/kg), compared to control (***P b 0.001, A) and PTX (50 mg/kg) (**P b 0.01, A) groups. Pretreatment of animals with a combination of db-cAMP (100 nM/mouse) and PTX (50 mg/kg) decreased seizure latency and threshold significantly in compari- son with db-cAMP (100 nM/mouse) (*P b 0.05, C) and PTX (50 mg/kg) (**P b 0.01, B), respectively.

Thus, based on our findings and by considering the different neuro- behavioral effects of PTX on seizure mechanisms (Davila-Esqueda and Martinez-Morales, 2004; Fredholm and Persson, 1982; Garthwaite, 1991; Nandhakumar and Tyagi, 2008; Snyder and Bredt, 1991), the ab- sence of any significant changes in the PTX-treated group was expected. Our data further demonstrate that bucladesine decreased seizure latency and seizure threshold. Bucladesine as a cAMP analogue and PDE3 inhibitor (Tilley and Maurice, 2002) raises the intracellular level of cAMP (Neumann et al., 2002). PDE3 inhibitors by increasing calcium influx (Cho et al., 2000) cause phosphorylation of intracellu- lar proteins such as ion channels, receptors, enzymes and transcrip- tion factors which stimulate neuronal excitability and induce epileptic seizure (Butler et al., 1995). It seems feasible that the effects of bucladesine on cAMP level and PDE3 function are probably in- volved in the epileptogenic function of bucladesine. Our results show that the threshold and latency of PTZ-induced seizure were sig- nificantly decreased by bucladesine. Nevertheless, our findings and results from other groups coincide in terms of an involvement of

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Control H-89 H-89+ (0.2mg/100g) PTX (50)

H-89+ PTX (100)

H-89+ PTX (100)

cAMP in epileptogenic excitatory processes of bucladesine. However, the type of seizure induction and the concentration of the adminis- tered drugs appear to be crucial for seizure parameters evaluation.
Here, we report that H-89 increased both seizure threshold and sei- zure latency. The interactions of cAMP-dependent PKA with GABA re- ceptors have been proven previously (Datta, 2007; Jancic et al., 2009; Naseer et al., 2010). PKA by phosphorylation of CREB and serine/threo- nine residues of GABA receptors impairs GABA signaling function and reduces the efficacy of GABAergic synaptic transmission (Jancic et al., 2009; Poisbeau et al., 1999). The GABAergic system plays an important role in the regulation of pathophysiological disorders such as seizure in the central nervous system (CNS) (Datta, 2007, Naseer et al., 2010). Considering the effects of PKA on GABA receptors, the inhibition of PKA by H-89 may decrease excitability through enhanced GABA receptor-mediated inhibition and therefore, increases seizure latency and threshold in PTZ-exposed rats.
A significant increase in intracellular cAMP level was shown in neu- rons treated with neurotoxic drugs (Prapong et al., 2001). In the present study, the PKA inhibitor H-89 prevented PTZ-mediated epileptic sei- zure. It is possible that the stimulatory effect of PTZ on neuronal PKA pathways is implicated in seizure activity. Such a hypothesis infers the role of PKA-induced activity of adenylyl cyclase and cAMP in PTZ ex- posed animals.
Another effect of PKA associated with the occurrence of seizure is the regulation of NMDA and glutamate functions (Lan et al., 2001;

Fig. 4. Effect of pentoxifylline and H-89 in combination on the latency (A) and seizure threshold (B). Data show that both latency and seizure threshold were significantly in- creased (***P b 0.001) in comparison with control animals. The application of pentoxi- fylline (50 and 100 mg/kg) and H-89 (0.2 mg/100 g) in combination caused an obvious increase in latency (***P b 0.001) and seizure threshold (***P b 0.001). The comparison of combination groups and H-89-treated mice showed that the effect of H-89 was de- creased (*P b 0.05) in PTX/H-89 animals. Data represent the means ±S.E.M. of seven animals per group.

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Westphal et al., 1999). NMDA receptors are widely distributed in the CNS and display an important role in seizure induction (Rice and Delorenzo, 1998). Interactions between cAMP-PKA pathway and the glutaminergic system are well described (Adolph et al., 2007; Rath et al., 2008).
The regulation of NMDA receptor activity by PKA contributes to the modulation of synaptic transmission (Westphal et al., 1999). PKA activation results in a rapid improvement of NMDA receptor cur- rents (Westphal et al., 1999) and provokes spontaneous glutamate release from hippocampal neurons (Chavez-Noriega and Stevens, 1994). PKA-induced NMDA receptor phosphorylation is also associat- ed with an enhancement of calcium influx through these receptors (Lan et al., 2001; Westphal et al., 1999). The consequence of this extra calcium influx is neuronal damage and seizure (Esteban et al., 2003). The crucial and novel role of PKA-mediated phosphorylation for preventing seizure spreading led to the development of new anti-

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Control Bucladesine Bucladesine Bucladesine 300 nM + H89 (0.05) + H89 (0.2)

Control Bucladesine Bucladesine Bucladesine 300 nM + H89 (0.05) + H89 (0.2)
epileptic drugs such as levetiracetam (Lee et al., 2008).
Our findings suggest that the neuroprotective effect of H-89 which causes an increase in seizure threshold and latency is possibly related to the above mentioned PKA effects on GABAergic, glutaminergic and cAMP signaling pathways.
The data further indicate that in mice received PTX and H-89 in combination, the effect of H-89 on seizure threshold and latency was reduced by PTX. Thus, it seems possible that the PTX-induced in- crease of cAMP and cGMP levels affects PKA activity and decreases the anticonvulsant effect of H-89.
Administration of PTX and bucladesine in combination decreased the seizure latency and threshold in PTZ-exposed animals. It has been shown that the levels of cGMP and cAMP are elevated in differ- ent parts of the brain prior and after the onset of clinical seizure activ- ity (Ferrendelli and Gross, 1980; Kochanek et al., 2006; Myllyla et al., 1975). The effects of cGMP and cAMP on seizure activity are related to their capacity for reducing the function of GABAergic system (Delmas et al., 1994; Lopez et al., 2007; Nandhakumar and Tyagi, 2008;

Fig. 5. Effect of bucladesine and H-89 in combination on seizure latency (A) and threshold (B). Data reveal that H-89 in both doses (0.05 and 0.2 mg/100 g) prevented epileptogenic effects of bucladesine (300 nM/mouse) (***Pb 0.001). The decrease of seizure latency in bucladesine group was returned to control level in combination treated animals. No signif- icant differences between combination groups and control animals were found in seizure latency. Groups which received bucladesine (300 nM/mouse) and H-89 (0.2 mg/100 g) in combination had a higher seizure threshold compared to control animals (B, *Pb 0.05). Data are expressed in means±S.E.M. of seven animals per group.
Poisbeau et al., 1999). By considering the effect of PTX and db-cAMP on GABAergic mechanisms, it is reasonable to deduce that the admin- istration of these two components in combination increases their in- dividual effects on epileptic activity by reducing seizure latency and threshold. We found that the combination of PTX with lower doses of db-cAMP resulted in a further reduction of seizure latency. Howev- er, the effect of high dose db-cAMP on seizure latency was not

affected by PTX. It appears that db-cAMP at a concentration of 300 nM/mouse produced maximal cAMP levels for inhibiting GABAergic system function which could not be further affected with PTX.
A major finding of our study is that the administration of H-89 prevents neurotoxicity of bucladesine in PTZ-treated animals. Re- garding the opposite effects of H-89 and bucladesine on PKA-related functions, it is feasible that H-89 decreases bucladesine-induced ex- citability through modulation of cAMP/PKA signaling pathways.
In conclusion, our study highlights the importance of intracellular cyclic nucleotides signaling cascades in seizure activity. This study further suggests that cyclic nucleotide-related compounds affect cell signaling elements in epileptic seizure via interaction with intracellu- lar messenger pathways, trans-membrane enzymes and protein kinases.

References
Adolph, O., Koster, S., Rath, M., Georgieff, M., Weigt, H.U., Engele, J., 2007. Rapid in- crease of glial glutamate uptake via blockade of the protein kinase A pathway. Glia 55, 1699–1707.
Ahern, G.P., Klyachko, V.A., Jackson, M.B., 2002. cGMP and S-nitrosylation: two routes for modulation of neuronal excitability by NO. Trends Neurosci. 25, 510–517.
Azami, K., Etminani, M., Tabrizian, K., Salar, F., Belarana, M., Hosseini, A., Hosseini-S, A., Sharifzadeh, M., 2010. The quantitative evaluation of cholinergic markers in spatial memory improvement induced by nicotine–bucladesine combination in rats. Eur. J. Pharmacol. 636, 102–107.
Berman, R.F., Fredholm, B.B., Aden, U., O’Connor, W.T., 2000. Evidence for increase dor- sal hippocampal adenosine release and metabolism during pharmacologically in- duced seizure in rats. Brain Res. 872, 44–53.
Boulton, C.L., Mccrohan, C.R., O’Shaughnessy, C.T., 1993. Cyclic AMP analogues increase excitability and enhance epileptiform activity in rat neocortex in vitro. Eur. J. Phar- macol. 236, 131–136.
Bracey, J.M., Kurz, J.E., Low, B., Churn, S.B., 2009. Prolonged seizure activity leads to in- creased protein kinase A activation in the rat pilocarpine model of status epilepti- cus. Brain Res. 4, 167–176.
Bradford, H.F., 1995. Glutamate, GABA and epilepsy. Prog. Neurobial. 47, 477–511. Butler, L.S., Silva, A.J., Abeliovich, A., Watanabe, Y., To-negawa, S., Mc Narama, J.O.,
1995. Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation. Proc. Natl. Acad. Sci. U. S. A. 15, 6852–6855.
Campos-Toimil, M., Keravis, T., Orallo, F., Takeda, K., Lugnier, C., 2008. Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in op- posing agonist-induced Ca (2+) responses in endothelial cells. Br. J. Pharmacol. 154, 82–92.
Chavez-Noriega, L.E., Stevens, C.F., 1994. Increase neurotransmitter release at excitato- ry synapses produced by direct activation of adenylate cyclase in rat hippocampal slices. J. Neuroci. 14, 310–317.
Cho, C.H., Cho, D.H., Seo, M.R., Juhnn, Y.S., 2000. Differential changes in the expression of cyclic nucleotide phosphodiesterase isoforms in rat brains by chronic treatment with electroconvulsive shock. Exp. Mol. Med. 32, 110–114.
Cunha, G.M.A., Farias, P.A.M., Viana, G.S.B., 2002. Evidence for the involvement of the muscarinic cholinergic system in the central actions of pentoxifylline. Behav. Phar- macol. 13, 149–156.
Datta, S., 2007. Activation of pedunculopontine tegmental PKA prevents GABA B recep- tor activation-mediated rapid eye movement sleep suppression in the freely mov- ing rat. J. Neurophysiol. 97, 3841–3850.
Davila-Esqueda, M.E., Martinez-Morales, F., 2004. Pentoxifylline diminishes the oxida- tive damage renal tissue induced by streptozotocin in the rat. Exp. Diabesity Res. 5, 245–251.
Delmas, V., Molina, C.A., Lalli, E., De Groot, R., Foulkes, N.S., Masquilier, D., Sassone- Corsi, P., 1994. Complexity and versatility of the transcriptional response to cAMP. Rev. Physiol. Biochem. Pharmac. 124, 1–28.
Dhir, A., Zolkowska, D., Murphy, R.B., Michael, A., Rogawski, M.A., 2011. Seizure protec- tion by intrapulmonary delivery of propofol hemisuccinate. JPET 336, 215–222.
Dunwiddie, T.V., 1999. Adenosine and suppression of seizures. Adv. Neurol. 79, 1001–1010.
Essayan, D.M., 1999. Cyclic nucleotide phosphodiesterase (PDE) inhibitors and immu- nomodulation. Biochem. Pharmacol. 57, 965–973.
Esteban, J.A., Shi, S.H., Wilson, C., Nuriya, M., Huganir, R.L., Malinow, R., 2003. PKA phosphorylation of AMPA receptor subunits control synaptic trafficking underlying plasticity. Nat. Neurosci. 6, 136–143.
Fredholm, B.B., Persson, C.G.A., 1982. Xanthine derivatives as adenosine receptor an- tagonists. Eur. J. Pharmacol. 81, 673–676.
Ferrendelli, J.A., Gross, R.A., 1980. Relationship between seizure activity and cyclic nucleotide level in the brain. Brain Res. 200, 93–103.
Ferrendelli, J.A., Kinscherf, D.A., 1977. Cyclic nucleotides in epileptic brain: effect of pentylenetetrazol on regional cyclic AMP and cyclic GMP levels in vitro. Epilepsia 18, 525–531.
Fisher, R.S., 1989. Animal models of the epilepsies. Brain Res. Rev. 14, 245–278.
Garthwaite, J., 1991. Glutamate, nitric oxide and cell–cell signaling, in the nervous sys- tem. Trends Neurosci. 14, 60–67.
Gholipour, T., Rasouli, A., Jabbarzadeh, A., Nazemi, B.G., Riazi, K., Sharifzadeh, M., Dehpour, A.R., 2009. The interaction of sildenafil with the anticonvulsant effect of diazepam. Eur. J. Pharmacol. 617, 79–83.
Hidaka, H., Inagaki, M., Kawamoto, S., Sasaki, Y., 1984. Isoquinoline sulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein ki- nase C. Biochemistry 23, 5036–5041.
Jancic, D., Lopez, d.A.M., Valor, L.M., Olivares, R., Barco, A., 2009. Inhibition of cAMP re- sponse element-binding protein reduces neuronal excitability and plasticity, and triggers neurodegeneration. Cereb. Cortex 19, 2535–2547.
Jeon, Y.H., Heo, Y.S., Kim, C.M., Hyun, Y.L., Lee, T.G., Ro, S., Cho, J.M., 2005. Phosphodiesterase: overview of protein structures, potential therapeutic applications and recent progress in drug development. Cell. Mol. Life Sci. 62, 1198–1220.
Kaneishi, K., Sakuma, Y., Kobayashi, H., Kato, M., 2002. 3-5-Cyclic adenosine monopho- sphate augments intracellular Ca2+ concentration and gonadotropin-releasing hormone (GnRH) release in immortalized GnRH neurons in a Na+-dependent manner. Endocrinology 143, 4210–4217.
Kochanek, P.M., Vagni, V.A., Janesko, K.L., Washington, C.B., Crumrine, P.K., Garman, R.H., Jenkins, L.W., Clark, R.S., Homanics, G.E., Dixon, C.E., Schnermann, J., Jackson, E.K., 2006. Adenosine A1 receptor knockout mice develop lethal status epilepticus after experimental traumatic brain injury. J. Cereb. Blood Flow Metab. 26, 565–575.
Kim, S.H., Won, S.J., Mao, X.O., Jin, K., Greenberg, D.A., 2005. Involvement of protein ki- nase A in cannabinoid receptor-mediated protection from oxidative neuronal inju- ry. J. Pharmacol. Exp. Ther. 313, 88–94.
Koriyama, Y., Chiba, K., Mohri, T., 2003. Propentofylline protects beta-amyloid protein- induced apoptosis in cultured rat hippocampal neurons. Eur J Pharmacol. 458, 235–241.
Lan, J.Y., Skeberdis, V.A., Jover, T., Grooms, S.Y., Lin, Y., Araneda, R.C., Zheng, X., Bennett, M.V., Zukin, R.S., 2001. Protein kinase C modulates NMDA receptor trafficking and gating. Nat. Neurosci. 4, 382–390.
Lee, C.H., Lee, C.Y., Tsai, T.S., Liou, H.H., 2008. PKA-mediated phosphorylation is a novel mechanism for levetiracetam, an antiepileptic drug, activating ROMK1 channels. Biochem. Pharmacol. 76, 225–235.
Lopez, D.A.M., Jancic, D., Olivares, R., Alarcon, J.M., Kandel, E.R., Barco, A., 2007. cAMP response element-binding protein-mediated gene expression increases the intrin- sic excitability of CA1 pyramidal neurons. J. Neurosci. 27, 13909–13918.
Ludvig, N., Moshe, S.L., 1987. Cyclic AMP derivatives injected into the inferior colliculus induce audiogenic seizure-like phenomena in normal rats. Brain res. 437, 193–196. Ludvig, N., Mishra, P.K., Jobe, P.C., 1992. Dibutyryl cyclic AMP has epileptogenic poten- tial in the hippocampus of freely behaving rats: a combined EEG-intracerebral
microdialysis study. Neurosci. Lett. 141, 187–191.
Maurice, D.H., Palmer, D., Tilley, D.G., Dunkerley, H.A., Netherton, S.J., Raymond, D.R., Elbatarny, H.S., Jimmo, S.L., 2003. Cyclic nucleotide phosphodiesterase activity, ex- pression, and targeting in cells of the cardiovascular system. Mol. Pharmacol. 64, 533–546.
Myllyla, V.V., Heikkinen, E.R., Vapaatalo, H., Hokkanen, E., 1975. Cyclic AMP concentra- tion and enzyme activities of cerebrospinal fluid in patients with epilepsy or cen- tral nerves system damage. Eur. Neurol. 13, 123–130.
Nandhakumar, J., Tyagi, M.G., 2008. Evaluation of cyclic nucleotide phosphodiesterase III inhibitors in animal models of epilepsy. Biomed. Res. 19, 13–17.
Naseer, M.I., Lee, H.Y., Ullah, N., Ullah, I., Park, M.S., Kim, S.H., Kim, M.O., 2010. Ethanol and PTZ effects on siRNA-mediated GABAB1 receptor: down regulation of intracel- lular signaling pathway in prenatal rat cortical and hippocampal neurons. Synaps 65, 181–190.
Neumann, S., Bradke, F., Tessier-Lavigne, M., Basbaum, A.I., 2002. Regeneration of sen- sory axons within the injured spinal cord induced by intraganglionic cAMP eleva- tion. Neuron 13, 885–893.
Paul, V., Ekambaram, P., 2005. Effects of sodium nitroprusside, a nitric oxide donor, on γ-aminobutyric acid concentration in the brain and on picrotoxin-induced convul- sions in combination with phenobarbitone in rats. Pharmacol. Biochem. Behav. 80, 363–370.
Poisbeau, P., Cheney, M.C., Browning, M.D., Mody, I., 1999. Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons. J. Neurosci. 19, 647–683.
Prapong, T., Uemura, E., Hsu, W.H., 2001. G protein and cAMP-dependent protein ki- nase mediate amyloid [beta]-peptide inhibition of neuronal glucose uptake. Exp. Neurol. 167, 59–64.
Prast, H., Philippu, A., 2001. Nitric oxide as modulator of neuronal function. Prog. Neurobiol.
64, 51–68.
Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P.N., Bregman, B.S., Filbin, M.T., 2002. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34, 895–903.
Rajasekaran, K., Jayakumar, R., Venkatachalam, K., 2003. Increased neuronal nitric oxide synthase (nNOS) activity triggers picrotoxin-induced seizures in rats and ev- idence for participation of nNOS mechanism in the action of antiepileptic drugs. Brain Res. 979, 85–97.
Rath, M., Fohr, K.J., Weigt, H.U., Gauss, A., Engele, J., Georgieff, M., Kster, S., Adolph, O., 2008. Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA. Br. J. Pharmacol. 155, 925–933.
Ray, A., Gulati, K., Anand, S., Vijayan, V.K., 2005. Pharmacological studies on mech- anisms of aminophylline-induced seizures in rats. Indian J. Exp. Biol. 43, 849–853.
Rice, A.C., DeLorenzo, R.J., 1998. NMDA receptor activation during status epilepticus is required for the development of epilepsy. Brain Res. 782, 240–247.
Sander, J.W., Shorvon, S.D., 1996. Epidemiology of the epilepsies. J. Neurol. Neurosurg.
Psychiatry 61, 433–443.

Sharifzadeh, M., Aghsami, M., Gholizadeh, S., Tabrizian, K., Soodi, M., Khalaj, S., Ranjbar, A., Hosseini-Sharifabad, A., Roghani, A., Karimfar, M.H., 2007a. Protective effects of chronic lithium treatment against spatial memory retention deficits induced by the protein kinase AII inhibitor H-89 in rats. Pharmacology 80, 158–165.
Sharifzadeh, M., Naghdi, N., Ghahremani, M.H., Sharifzadeh, K., Roghani, A., 2005. Post training intrahippocampal infusion of protein kinase AII inhibitor impairs spatial memory retention in rats. Neurosci. Res. 379, 392–400.
Sharifzadeh, M., Zamanian, A.R., Gholizadeh, S., Tabrizian, K., Etminani, M., Khalaj, S., Zarrindast, M.R., Roghani, A., 2007b. Post-training intrahippocampal infusion of nicotine-bucladesine combination causes a synergistic enhancement effect on spa- tial memory retention in rats. Eur. J. Pharmacol. 562, 212–220.
Singh, A., Kulkarn, S.K., 2002. Role of adenosine in drug induced catatonia in mice.
Indian J. Exp. Biol. 40, 882–888.
Snyder, S.H., Bredt, D.S., 1991. Nitric oxide as a neuronal messenger. Trends Pharmacol.
Sci. 12, 125–128.
Tariq, M., Ahmad, M., Moutaery, K.A., Deeb, S.A., 2008. Pentoxifylline ameliorates lithium-pilocarpine induced status epilepticus in young rats. Epilepsy Behav. 12, 354–365.
Thorat, S.N., Kulkarn, S.K., 1990. Effect of MK-801 and its interaction with adenosiner- gic agents and carbamazepine against hypoxic stress-induced convulsion and death in mice. Meth. Find. Exp. Clin. Pharmacol. 12, 595–600.
Tilley, D.G., Maurice, D.H., 2002. Vascular smooth muscle cell phosphodiesterase (PDE) 3 and PDE4 activities and levels are regulated by cyclic AMP in vivo. Mol. Pharma- col. 62, 497–506.
Tojo, Y., Kurokouchi, A., Mori, N., Kumashiro, H., 1990. Neuroexcitotoxic action of db- cAMP: lesioning of neuronal cell bodies while sparing fibers of passage. No To Shinkei 42, 231–237.
Ueda, K., Yagami, T., Kageyama, H., Kawasaki, K., 1996. Protein kinase inhibitor attenu- ates apoptotic cell death induced by amyloid beta protein in culture of the rat ce- rebral cortex. Neurosci. Lett. 203, 175–178.
Westphal, R.S., Steven, J.T., Jerry, W.L., Neal, M.A., Iain, D.C.F., Lorene, K., Langeberg, Morgan, S., John, D.S., 1999. Regulation of NMDA receptors by an associated phos- phatase kinase signaling complex. Science 285, 93–96.
Whitcomb, K., Lupica, C.R., Rosen, J.B., Berman, R.F., 1990. Adenosine involvement in postictal events in amygdala-kindled rats. Epilepsy Res. 6, 171–179.