Co-expression of citrulline-nitric oxide cycle enzymes and decreased glutamine synthetase expression in different regions of brain in epilepsy rat model

The aim of this study was to determine the mRNA expression of nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL) and glutamine synthetase (GS) in different regions of brain in rats subjected to kainic acid (KA) mediated epilepsy. The short term (acute) group animals were sacrificed after 2 h and the long term (chronic) group animals were sacrificed after 5 days of single injection of KA. After decapitation of rats, cerebral cortex (CC), cerebellum (CB) and brain stem (BS) were separated and in their homogenates, the relative amount of nNOS, iNOS, AS, AL and GS mRNA was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results showed an increased expression of iNOS in all brain regions tested in chronic group as compared to either control or acute group, and it indicate a favorable condition of nitric oxide production. AL expression was significantly increased only in CB in acute group whereas in chronic group it is increased in CC and CB and decreased in BS as compared to control. The aforementioned increased expression of AL may contribute effective recycling of citrulline to arginine. No change in expression of nNOS and AS in both acute and chronic groups of epilepsy. GS expression was significantly decreased only in chronic group of epilepsy in all brain regions tested when compared with control group. The decreased GS may be contributing prolonged availability of glutamate in chronic epilepsy.


INTRODUCTION
Generation of nitric oxide (NO), a versatile molecule in signaling processes and unspecific immune defense, is intertwined with synthesis, catabolism and transport of arginine which thus ultimately participates in the regulation of a fine-tuned balance between normal and pathophysiological consequences of NO production (Wiesinger, 2001).NO is synthesized from arginine by nitric oxide synthase (NOS; EC 1.14.13.39), and the citrulline generated as a by-product can be recycled to arginine by successive actions of argininosuccinate synthetase (AS; EC 6.3.4.5) and argininosuccinate lyase *Corresponding author.E-mail: mswamy@kb.usm.my or mummedys@yahoo.co.in.Fax: +609-765 3370.
(AL; EC 4.3.2.1) via the citrulline-NO cycle (Zhang et al., 2000).In the mammalian tissue, NO is synthesized by a family of three isoenzymes, namely neuronal NOS (nNOS), inducible NOS (iNOS), endothelial NOS (eNOS) and their functioning neuronal signaling process, immune defense and vascular relaxation, respectively.Coinduction of AS, Cationic amino acid transporter-2 and NOS in activated murine microglial cells (Kawahara et al., 2001) and co-induction of inducible NOS and arginine recycling enzymes in cytokine-stimulated PC12 cells and high output production of NO were reported (Zhang et al., 2000).Kainic acid (KA), a glutamate analogue is widely used to induce exitotoxicity in experimental animals, that is, a model for temporal lobe epilepsy and a model for neurodegenerative disorders (Sperk, 1994).KA induced status epilepticus was associated with both apoptotic and necrotic cell death (White, 2002) and induction of heat sensitive proteins in hippocampus and cortical regions of rodent brain (Kato et al., 1999;Akbar et al., 2001).The exact mechanisms contributing to increased concentration of NO in epilepsy are not well established.Earlier studies reported that NOS knockout mice were more severely affected by epileptic activity than controls and the response to NO during epilepsy depends on its concentration (Itoh and Watanabe, 2009).It was also indicated that NO may be regarded as an anticonvulsant, as NOS knockout mice were more severely affected by epileptic activity than controls and proconvulsant, because high NO is formed in normal rats subjected to convulsions by pentylenetetrazole (PTZ) (Itoh and Watanabe, 2009).Earlier studies observed that the inhibition of PTZ induced convulsion in L-argininepretreated animals have suggested that NO mediates the anticonvulsant action of L-arginine (Tsuda et al., 1998).Others have proposed a proconvulsant action for NO, because in their studies L-arginine potentiated N-methyl-D-aspartate-induced convulsions in rodents (Mollacce et al., 1991;De Sarro et al., 1993).Neuronal excitation involving the excitatory glutamate receptors is recognized as an important underlying mechanism in neurodegenerative disorders (Dong et al., 2009).In the central nervous system (CNS), the conversion of glutamate to glutamine by glutamine synthetase (GS), that takes place within the astrocytes, represents a key mechanism in the regulation of excitatory neurotransmission under normal conditions as well as in injured brain (Szatkowski and Attwell, 1994).In our earlier study, we reported the increased activities of NOS, AS and AL and decreased activity of GS in acute and chronic groups of KA mediated epilepsy (Swamy et al., 2011).Therefore the present study was conducted to assess the expression of nNOS, iNOS, AS, AL and GS in cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of rats in acute and chronic groups of KA mediated epilepsy and thought, to provide additional information in understanding the increased generation of NO and its effects on GS modulation in KA mediated epilepsy.

Animals and epilepsy induction
Male Sprague Dawley rats weighing 200 to 250 g were used for the study.The animals had free access to food and water.Animal Ethics Committee of Universiti Sains Malaysia, Health Campus, Kubang Kerian, Malaysia, approved the experimental design [USM/Animal Ethics Approval/2007/(34) (105)].The animals were divided into control, acute group and chronic groups (n = 6 rats/group).In the acute group, epilepsy was produced by subcutaneous administration of KA (15 mg/kg body weight, dissolved in normal saline) and control group received normal saline subcutaneously (Milatovic et al., 2002).The animals showed convulsions after 40 to 45 min of KA injection for 2 to 3 min and afterwards became drowsy.The animals were sacrificed after 2 h of injection using the guillotine and the brains were quickly removed, Swamy et al. 1523 placed in ice cold saline and blotted with filter paper to remove blood, and the different regions (CC, CB and BS) were separated as described (Sadasivudu and Lajtha,1970).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from three different regions of brain separately by using RNeasy Mini Kit # 74104 (Qiagen, Germany).RNA concentration was determined by measuring the absorbance at 260 nm. 2 µg of RNA was transcribed to cDNA by using RevertAid TM H Minus First Strand cDNA Synthesis Kit # K1632 (Fermentas, USA) by following manufacturer protocols.Briefly, reaction mixture of total RNA and oligo(dT)18 primer were prepared in an eppendorf tube.Reaction mixture was then made up until 12 µl by adding DEPC-treated water.The mixture was spin down and incubated for 3 to 5 s at 70°C.Later, 5x reaction buffer, Ribolock Ribonuclease Inhibitor and 10 mM dNTP mix was added into the reaction mixture.Incubation at 37°C was conducted for 5 min and then RevertAid H Minus M-MuLV was quickly added.The mixture was incubated for 60 min at 42°C.Finally, the reaction was stopped by heating process at 70°C for 10 min.PCR reaction was carried out in MJ research machine and performed using 2 µg of cDNA for each amplification reaction.The 2 µg of cDNA was added into PCR buffer.The sequence for oligonucleotides primers were designed based on GeneBank accession numbers as described in Table 1 by using Primer3Plus software and blast using BLAST GeneBank.Reaction mixture of PCR was prepared by mixing 1X Taq Buffer, 200 µM dNTP, 1 µM forward primer, 1 µM reverse primer, 0.5 µg of sample, 2 mM of MgCl2 and 1.25 U/µl of Taq Polymerase.The mixture was made up until 20 µl by adding nuclease free water.Initial denaturation step was performed at 94°C for 5 min, followed by denaturation step at 94°C for 1 min, annealing step at 55°C for 1 min , extension step at 72°C for 1 min and finally the final extension at 72°C for 5 min.
PCR products were separated on a 1.8% of agarose gel by using Biorad Electrophoresis Set with the setting of 70 V in 45 min.Each band was analysed under UV light by using Image Analyzer Biorad.The relative amount of nNOS, iNOS, AS, AL and GS mRNA was calculated as a relative ratio by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.

Statistical analysis
Results were reported as mean ± standard error of mean (SEM) from 6 animals for each parameter calculated.Statistical analysis of results was done by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test, using the SPSS software (version 12.0.1).P value of < 0.05 was taken as statistically significant at 95% confidence interval.

RESULTS
The relative expression of nNOS mRNA to glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA are represented in Figure 1.The nNOS expression was not changed significantly in all the brain regions tested in acute and chronic groups as compared to control.The iNOS expression (Figure 2) was significantly increased (P < 0.001) in chronic group as compared to control or acute group in all the brain regions tested.The expression of iNOS in acute group was not significant when compared with control group.Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant.

Gene
The increased expression of iNOS in chronic group was uniform in all the three brain regions tested.The expression of AS (Figure 3) was not changed significantly in all the brain regions tested in acute and chronic groups Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant; ***P < 0.001.were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant.

AS/GAPDH density ratio
as compared to control.The AL expression (Figure 4) was significantly increased (P < 0.001) only in cerebellum in acute group whereas in chronic group, it was increased (P < 0.001) in cerebral cortex and cerebellum and decreased (P < 0.001) in brain stem as compared to control.The GS expression (Figure 5) was significantly decreased in all the brain regions tested, only in chronic group of epilepsy as compared to control group (in CC P < 0.001, CB and BS P < 0.05).A representative gel showing the RT-PCR result for one of the enzyme is given in Figure 6.

DISCUSSION
NO is postulated to be involved in the pathophysiology of many epilepsy models resulting from increased action of excitatory neurotransmitter namely glutamate (Rundfeldt et al., 1995;Lapouble et al., 2002).In neurons, NO synthesis is stimulated by Ca 2+ -influx, which is induced by the activation of glutamate receptors, preferentially Nmethyl D-aspartate (NMDA) receptor (Radenovic and Selakovic, 2005).The literature findings implicate neuronal NO generation in the pathogenesis of both direct and secondary excitotoxic neuronal injuries in vivo.Although, NMDA receptors likely contribute critically to neuronal injury in various acute conditions, several observations support the hypothesis that AMPA/KA receptors may be of greater importance to the neurodegenerative process (Carriedo et al., 1998).Though, there are most kainate receptors in the hippocampus, earlier studies showed a high production of NO in cortex than amygdala and hippocampus (Milatovic et al., 2002).
The increased concentration of NO in brain in epilepsy supports the involvement in pathophysiology of NO in excitotoxicity (Milatovic et al., 2002;Guix et al., 2005;Swamy et al., 2009Swamy et al., , 2011)).The sustained increase of NOx in chronic group indicate the continuous increased production of NO and its deleterious effects in CNS in chronic conditions of epilepsy (Swamy et al., 2011).The increased iNOS expression observed in all the regions in chronic group in this study support the increased activity of NOS reported (Swamy et al., 2011).The increased expression of AL in CB in acute, CC and CB in chronic group indicate the co-expression of iNOS and AL in these regions.The co-expression of iNOS and AL may favor the effective recycling of citrulline to arginine.Such a coinduction of iNOS, AS and AL were reported earlier in cytokine-stimulated PC12 cells (Zhang et al., 2000).
Neuronal excitation involving the excitatory glutamate receptors is recognized as an important underlying mechanism in neurodegenerative disorders (Dong et al., 2009;Messripour and Mesripour, 2011).In the brain, the conversion of glutamate to glutamine by GS represents a key mechanism in the regulation of excitatory neurotransmission (Szatkowski and Attwell, 1994).The glutamine synthetase is present in all parts of brain and it is equally high in cerebral cortex, cerebellum and hippocampus (Girard et al., 1993;Rose and Felipo, 2005).The modulation of GS activity in brain, therefore, is important and its impairment or saturation may have pathological consequences (Rodrigo and Felipo, 2007).The decreased  activity of GS was reported in acute and chronic groups of epilepsy (Swamy et al., 2011).The exact mechanism of inhibition of GS by NO is not known, but it is thought to be as a covalent modification as a result of nitrosylation or nitration of tyrosine in GS (Kosenko et al., 2003;Rose and Felipo, 2005).It was reported earlier that GS becomes nitrated and inhibited during PTZ induced seizure model at repeated PTZ seizure induction, but there was no decrease in GS protein level (Bidmon et al., 2008), however, there were gene expression studies indicating decreased GS expression in chronic phase of epilepsy induced by KA (Hammer et al., 2008).The decreased expression of GS observed in all the brain regions of chronic group in this study support the earlier report of decreased GS expression (Hammer et al., 2008).The results of GS expression in acute group did not show any change in expression and the reported decrease of GS activity (Swamy et al., 2009) may be due to its nitration in this condition.It is also reported that haploinsufficiency of GS increases susceptibility to experimental febrile seizures (Van Gassen et al., 2009).The decreased GS expression observed in this study and decreased activity reported earlier (Swamy et al., 2009(Swamy et al., , 2011) ) may contribute for prolonged availability of glutamate for excitotoxicity.The increased formation of NO along with increased activities of NOS, AS and AL reported earlier (Swamy et al., 2009(Swamy et al., , 2011) ) and the mRNA expression results presented in this study are in agreement with increased formation of NO in KA mediated epilepsy and NO involvement in modulation of GS.It is proposed that the inhibition of GS may provide prolonged availability of glutamate, which is causing excitotoxicity in chronic epilepsy.

Conclusions
This study clearly demonstrated the increased expression of iNOS in all the brain regions studied and indicate favorable condition for high NO synthesis in chronic epilepsy.The co-expression of AL along with iNOS in CB in acute, CC and CB in chronic group may partly be responsible for increased recycling of citrulline to arginine which may further increase NO generation.Decreased GS expression observed in this study provides a reason for the decreased GS activity in addition to the nitration of GS which was reported earlier.The decreased GS expression observed in this study and decreased activity (reported earlier) may contribute for prolonged availability of glutamate for excitotoxicity.

Figure 1 .
Figure 1.Relative ratio of nNOS mRNA expression level to glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA level in different regions of rat brain in acute and chronic groups of epilepsy.Band intensities were quantified by densitometer and reported as relative value to the GAPDH band.Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant.

Figure 2 .
Figure 2.Relative ratio of iNOS mRNA expression level to glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA level in different regions of rat brain in acute and chronic groups of epilepsy.Band intensities were quantified by densitometer and reported as relative value to the GAPDH band.Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant; ***P < 0.001.

Figure 3 .
Figure 3.Relative ratio of AS mRNA expression level to glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA level in different regions of rat brain in acute and chronic groups of epilepsy.Band intensities were quantified by densitometer and reported as relative value to the GAPDH band.Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant.

Figure 4 .
Figure 4. Relative ratio of AL mRNA expression level to glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA level in different regions of rat brain in acute and chronic groups of epilepsy.Band intensities were quantified by densitometer and reported as relative value to the GAPDH band.Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant; **P < 0.01, ***P < 0.001.

Figure 5 .
Figure 5.Relative ratio of GS mRNA expression level to glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA level in different regions of rat brain in acute and chronic groups of epilepsy.Band intensities were quantified by densitometer and reported as relative value to the GAPDH band.Results were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using Bonferroni's test and expressed as mean value ± standard error of mean for six animals in each group.NS = statistically not significant; *P < 0.05, *** P < 0.001.

Figure 6 .
Figure 6.Representative autoradiography of AS and glyceraldehyde-3-phosphate dehydrogenage (GAPDH) mRNA expression in different regions of rat brain in acute and chronic groups of epilepsy.Lane 1: control, Lane 2: acute, Lane 3: chronic.