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Effects of Progesterone on Total Brain Tissue Adenosine Deaminase Activity inExperimental EpilepsySadrettin Pençe a; brahim Erkutlu b; Naciye Kurtul c; Mehmet Alptekin b Üner Tan det al.a Department of Physiology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey b Department ofNeurosurgery, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey c Department of Chemistry,Faculty of Science, Sutcu Imam University, Kahramanmaras, Turkey d Faculty of Sciences Department ofPhysics, Çukurova University, Adana, Turkey

Online Publication Date: 01 February 2009

To cite this Article Pençe, Sadrettin, Erkutlu, brahim, Kurtul, Naciye, Alptekin, Mehmet Tan, Üneret al.(2009)'Effects of Progesteroneon Total Brain Tissue Adenosine Deaminase Activity in Experimental Epilepsy',International Journal of Neuroscience,119:2,204 —213

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International Journal of Neuroscience, 119:204–213, 2009Copyright C© 2009 Informa Healthcare USA, Inc.ISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450802055374

EFFECTS OF PROGESTERONE ON TOTAL BRAINTISSUE ADENOSINE DEAMINASE ACTIVITY INEXPERIMENTAL EPILEPSY

SADRETTIN PENCE

Department of PhysiologyFaculty of Medicine University of GaziantepGaziantep, Turkey

IBRAHIM ERKUTLU

Department of NeurosurgeryFaculty of Medicine University of GaziantepGaziantep, Turkey

NACIYE KURTUL

Department of ChemistryFaculty of Science Sutcu Imam UniversityKahramanmaras, Turkey

MEHMET ALPTEKIN

Department of NeurosurgeryFaculty of Medicine University of GaziantepGaziantep, Turkey

UNER TAN

Cukurova UniversityFaculty of Sciences Department of PhysicsAdana, Turkey

Address correspondence to Dr. Sadrettin Pence, Gaziantep University, Medical School,Department of Physiology 27310 Gaziantep, Turkey. E-mail: pence@gantep.edu.tr

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PROGESTERONE AND ADA IN EXPERIMENTAL EPILEPSY 205

Single seizure and epilepsy is one of the most commonly encountered neurologicdisorders in elderly individuals, arising as a result of complex and often multipleacquired underlying pathologies. Adenosine, acting at A1 receptors, exhibitsanticonvulsant effects in experimental epilepsy and inhibits progression to statusepilepticus. Adenosine deaminase is the enzyme for the regulation of adenosinelevels. Therefore any change in adenosine deaminase levels will reflect to adenosinelevels. Adenosine deaminase levels were decreased in the groups that were givenprogesterone. Progesterone may have an antiseizure effect with the additionalfinding decreased levels of adenosine deaminase that would have resulted inincreased adenosine levels that exerts anticonvulsant effect via GABA-A receptors.Further studies are needed to evaluate the role of progesterone effects on adenosinedeaminase levels and its mechanism(s) in the pathogenesis.

Keywords epilepsy, adenosine deaminase, progesterone, brain, mice

INTRODUCTION

Epilepsy may be affected by sex hormones, complicating the reproductivestate in men and women, exhibiting changes in seizure frequency, and severityfollowing many hormonal cycles (Beyenburg et al., 2001; Dagci, Koylu, Tan,Yan, & Pogun, 2002; Tan & Tan, 2001; Tan, Kalyoncu, & Tan, 2002). Animalmodels have been used to understand the interrelation between hormones andepilepsy in humans. The results have supported the clinical observations thatsteroid hormones may significantly affect epilepsy in humans.

Progesterone has been shown to depress spontaneous interictal spikes,protect against pentylenetetrazol-induced seizures, and raise the seizurethreshold in female but not in male rats (Wheless & Kim, 2002). Ovariansteroids have been reported to change neuronal excitability at the membrane andin the genome. Progestins and metabolites have antiseizure effects. However,the mechanisms and sites of action of these effects are not well understood(Rhodes & Frye, 2005). Intravenous infusion of progesterone led to a significantdecrease in spike frequency in epileptic women (Backstrom, Blom, Zetterlund,& Romano, 1984). In brain, 3α, 5α-THP acts like a sedative, decreasing anxietyand reducing seizure activity by enhancing the function of GABA-A receptorswithin the brain (Bitran, Hilvers, & Kellog, 1991).

Adenosine regulates many neural processes such as the seizure sus-ceptibility, neuroprotection, pain perception, sleep induction, respiration,cardioprotection, heart rate, blood pressure, and body temperature (Dunwiddie& Masino, 2001). The metabolism of adenosine is generated mainly byintracellularly or extracellularly localized 5′-nucleotidases and cytoplasmicS-adenosylhomocysteine hydrolase. The processes of adenosine elimination

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206 S. PENCE ET AL.

involves reactions catalyzed by adenosine deaminase (ADA) and adenosinekinase (AK) yielding inosine or 5′-AMP, respectively (Shryock & Belardinelli,1997). Inhibitory action of adenosine is exerted through A1 receptors (Ribeiro,Sebastiao, & Mendonca, 2003). GABA is the main inhibitory neurotransmitterin the central nervous system. Therefore, the knowledge of how GABAand adenosine interact is relevant to understand how neuronal excitability iscontrolled. Adenosine modulates GABAergic responses in the hippocampusby several mechanisms, involving modulation of GABA release through A2A

receptors (Cunha & Ribeiro, 2000) and regulation of GABA-A receptor activityby A1 receptors (Akhondzadeh & Stone, 1994; Isabel, Joaquim, & Ana, 2006).In light of these studies, we studied the effects of progesterone on total braintissue ADA levels in experimental epilepsy.

MATERIALS AND METHODS

Animals

Experiments were carried out on 80 mice, in eight groups consisting of 10 mice(5 males and 5 females in each group). Mice were weighing between 25–35g. Before the experiment, animals were kept on a 12-h light–dark cycle andallowed free access to food and water. All experiments were performed at atemperature of 22–24◦C, in a silent room, between 10:00 a.m. and 12:00 a.m.The experiments were performed in accordance with “Guide for the Care andUse of Laboratory Animals, DHEW, publication no. (NIH) 85–23, 1985” andapproved by local ethical committee at Faculty of Medicine, University ofGaziantep.

The animals were randomly divided into eight experimental groups.Group 1 (sham group) included mice decapitated in artificial cerebrospinal

fluid solution and their brains were extracted.Group 2 (control group): NaCl (% 0.9–60 mg/kg) was given via intra-

peritoneal route (ip). These mice were decapitated in brain solution and thentheir brains were extracted.

Group 3 (PTZ group) (convulsive dose of 60 mg/kg) included mice thatreceived single Pentylenetetrazole (PTZ) injection in a convulsive dose of60 mg/kg ip. They had a generalized epilepsy about 5 min later. One hour latermice were decapitated in brain solution and their brains were extracted.

Group 4 (convulsive PTZ plus progesterone group) included mice thatreceived single PTZ injection in convulsive dose of 60 mg/kg ip. They had a gen-eralized epilepsy in about 5 min. One hour later they had received 39.5 mg/kg

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progesterone. One hour after those mice were decapitated in brain solution andtheir brains were extracted.

Group 5 (progesterone group): Five doses of progesterone (39.5 mg/kg)has been administered to the mice at the 1st, 3rd, 5th, 7th, and 10th days. At theend of the 10th day, mice were decapitated in brain solution and their brainswere extracted.

Group 6 (single-dose progesterone group): Single-dose progesterone(39.5 mg/kg) has been administered to the mice. One hour later mice weredecapitated in brain solution and their brains were extracted.

Group 7 (kindling group): Five doses of PTZ (40 mg/kg) has beenadministered to the mice at the 1st, 3rd, 5th, 7th, and 10th days. At the endof the 10th day, mice were decapitated in brain solution and their brains wereextracted.

Group 8 (kindling plus progesterone group): Subconvulsive dose, fivedoses of PTZ (40 mg/kg) and 1 hr later progesterone (39.5 mg/kg) have beenadministered to the mice at the 1st, 3rd, 5th, 7th, and 10th days. At the endof the 10th day, mice were decapitated in brain solution and their brains wereextracted.

In all groups brains were divided into five parts using preparationmicroscope: the right hemisphere, left hemisphere, right brain stem, leftbrain stem, and cerebellum. These brain regions have been homogenisatedin phosphate buffer with a homogenisator. The resulting solutions were kept at−80◦C in deep freeze.

Chemicals

PTZ (Sigma) was dissolved in sterile isotonic saline and administeredintraperitoneally at a dose of 60 mg/kg, which is between the 50% effectivedose (ED50) of 33 mg/kg (ip) and the median effective lethal dose (LD50) of75 mg/kg (ip) (Loscher & Fiedler, 1996). Progesterone (Sigma) was also used.Both PTZ and progesterone were administered in 1 ml solutions prepared withsterile saline.

Assessment of Seizures

Immediately after injection of PTZ, mice were placed into the plexiglas boxesand observed for a period of 30 min. Generally, following injection of PTZ, micedisplayed walking and sniffing types of behavior and periods of immobility. Atthe same instance the animal displayed myoclonic jerks followed by a seizure.

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Table 1. Brain tissue ADA levels (U/g tissue) in eight groups

Brain regions n Mean ± SD df F (72–7) p

Right hemisphereGroup 1 10 0.2029 ± 0.03132 7 45.076 0.000Group 2 10 0.2059 ± 0.02918Group 3 10 0.4490 ± 0.12688Group 4 10 0.3860 ± 0.02565Group 5 10 0.2126 ± 0.02006Group 6 10 0.2218 ± 0.01726Group 7 10 0.6651 ± 0.16805Group 8 10 0.1993 ± 0.03633

Left hemisphereGroup 1 10 0.2250 ± 0.03598 7 47.359 0.000Group 2 10 0.2160 ± 0.02716Group 3 10 0.4107 ± 0.06090Group 4 10 0.3829 ± 0.04700Group 5 10 0.2158 ± 0.04096Group 6 10 0.2218 ± 0.03534Group 7 10 0.5016 ± 0.09083Group 8 10 0.2087 ± 0.04022

Right brain stemGroup 1 10 0.7870 ± 0.10045 7 31.014 0.000Group 2 10 0.7780 ± 0.09295Group 3 10 1.4879 ± 0.47271Group 4 10 1.1730 ± 0.11392Group 5 10 0.7300 ± 0.06018Group 6 10 0.7350 ± 0.05563Group 7 10 1.4191 ± 0.17536Group 8 10 0.6338 ± 0.06950

Left brain stemGroup 1 10 0.7269 ± 0.06156 7 21.015 0.000Group 2 10 0.7149 ± 0.05157Group 3 10 1.1622 ± 0.51228Group 4 10 1.0690 ± 0.08569Group 5 10 0.7071 ± 0.02832Group 6 10 0.7141 ± 0.03203Group 7 10 1.4411 ± 0.22006Group 8 10 0.6174 ± 0.05407

CerebellumGroup 1 10 0.3435 ± 0.03387 7 39.575 0.000Group 2 10 0.3353 ± 0.02832Group 3 10 0.5492 ± 0.28558Group 4 10 0.5759 ± 0.09078Group 5 10 0.3030 ± 0.03561Group 6 10 0.3150 ± 0.03240Group 7 10 1.0098 ± 0.13590Group 8 10 0.4053 ± 0.05009

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Table 2. Results of Tukey test and Post-hoc comparisons of the groupsa

Groups

Righthemisphere

(Groups)

Lefthemisphere

(Groups)

Right brainstem

(Groups)Left brain stem

(Groups)Cerebellum

(Groups)

1 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 72 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 73 1, 2, 5, 6, 7, 8 1, 2, 5, 6, 7, 8 1, 2, 4, 5, 6, 8 1, 2, 5, 6, 8 1, 2, 5, 6, 74 1, 2, 5, 6, 7, 8 1, 2, 5, 6, 7, 8 1, 2, 3, 5, 6, 8 1, 2, 5, 6, 7, 8 1, 2, 5, 6, 7, 85 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 76 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 77 1, 2, 3, 4, 5, 6, 8 1, 2, 3, 4, 5, 6, 8 1, 2, 5, 6, 8 1, 2, 4, 5, 6, 8 1, 2, 3, 4, 5, 6, 88 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 7 3, 4, 7

aPost hoc comparison results for the Tukey test for the brain tissue ADA levels (U/g tissue).Only results with statistical significance have been presented. P < .05 has been interpreted assignificant.

Latency was defined as the average length of time in minutes between drugadministration and seizure onset. The generalized seizure was characterizedby symmetric forelimb and hind limb tonus, and then hind limb clonus andflipping activity. There was concomitant high-pitched vocalization in somecases. Since some animals died due to effects of the seizure, after this periodthey were monitored for another 30 min only to assess mortality. Since ananimal occasionally had another fit either while the first one was going onor somewhat later than the first one, the seizure duration was calculated asthe sum of these multiple seizures for each animal to be assessed as onecombined fit. The researcher injecting the mice and observing the seizure wasblind to the exposure condition (PTZ or PTZ + progesterone) of each mouse.Subsequently, latency to first seizure onset, total seizure duration, the numberof seizure episodes and mortality were recorded for each subject.

Biochemical Evaluation

Serum AD activities were estimated spectrophotometrically by the method ofGiusti (1974), which is based on the direct measurements of the formationof ammonia produced when AD acts in excess of adenosine. Results wereexpressed as units per liter of serum (U/L). One enzyme unit was the amountof enzyme necessary to convert 1 µM of adenosine to inosine and ammoniaper min at 37◦C.

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Statistical Analysis

One-way ANOVA test was applied to evaluate differences between the groups.Tukey test was applied to evaluate the differences between the parts of thegroups. Paired samples T test was applied to evaluate the differences betweenparts of each group.

RESULTS

The results of ADA levels in different groups are presented in Table 1. One-wayANOVA showed that the ADA levels were significantly different betweengroups. This analysis also revealed significant differences in total ADA levelsof mice given PTZ at convulsive and subconvulsive doses, progesterone. Tukeytest was applied to find out the significance of these differences betweenthe groups (see Table 2). ADA levels in PTZ-induced epilepsy groups withor without administration of progesterone were higher than those in controlgroup and progesterone only non-epileptic group. ADA levels of all groupsfrom high to low levels were cerebral hemispheres, cerebellum, and brainstem,respectively. In the right and left cerebral hemisphere, right and left brainstemand cerebellum, ADA levels were different between groups (see Table 1).

In all the brain regions ADA levels were higher in kindling groupscompared to other groups. After progesterone treatment, ADA levels of groupsfrom high to low were Group 3 (single dose PTZ), Group 4 (single dose PTZ+ progesterone), and Group 8 (kindling + progesterone), respectively. ADAlevels in Group 6 (single dose progesterone) and Group 5 (5 dose progesterone)were similar in those of sham and control groups and there was no significantdifference between these groups. In cerebral hemispheres, although there wasfound significant difference between Group 3 (single dose PTZ) and Group 4(single dose PTZ + progesterone) there was no significant difference in otherbrain regions.

In all the brain regions we found significant differences in ADA levelsbetween Group 7 (kindling) and Group 8 (kindling + progesterone). ADAlevels in Group 8 (kindling + progesterone) were similar in the sham andcontrol groups. In all the brain regions we have found significant differencesin ADA levels in Group 3 (single dose PTZ) and Group 7 (kindling) whencompared with sham and control groups.

In subgroup analysis results of the paired samples t test for the braintissue ADA levels (U/g tissue) of the groups showed that there weresignificant differences between left and right cerebral hemispheres, left andright brainstems, and cerebellum in all groups (P < .05).

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DISCUSSION

In this study, we have found significant differences in ADA levels of righthemisphere, left hemisphere, right-brain stem, left-brain stem, and cerebellumbetween groups. Differences of ADA levels of all groups reflect lateralization.Our results demonstrated that the highest ADA levels are measured in brainstem regions when compared to the other parts of the brain, probably due toautonomic centers and nuclei in the brain stem (Geiger & Nagy, 1996). Wehave also found increased ADA levels in all brain parts of the study groups 3(convulsive dose) and 7 (subconvulsive dose) when compared to control (shamand NaCl-treated) groups.

The interesting point that attracted our attention was that while ADA levelsin Group 4 (PTZ + progesterone) were significantly lower than those observedin Group 3 (single dose PTZ) in cerebral hemispheres, there was no changein ADA levels in brainstem and cerebellum. These results were consistentwith animal studies concerning GABA-A receptor subtypes (Sieghart & Sperk,2002). The ADA levels showed regional specificity with differences among thecerebral hemispheres, cerebellum, and brainstems structures. We have foundhigh ADA activity in the PTZ kindling group in comparison with the control andsham groups. High ADA activity may implicate increased purine catabolismduring PTZ kindling seizure in mice brain tissue. On the basis of the presentfindings, progesterone treatment may prevent epileptic activity by decreasingADA levels. In view of the distinct main locations of adenosine A1 receptors andGABA-A receptors, we can propose that the antiseizure effect of progesteroneon adenosine A1-mediated responses may have occurred via binding to GABA-A receptors could influence presynaptic adenosine A1 receptors through NO, aretrograde messenger (Isabel, Joaquim, & Ana, 2006).

Previous studies suggested that a potentiation of GABA-A mediatedresponses by adenosine may occur through the activation of postsynapticchloride channels (Akhondzadeh & Stone, 1994). Although much has beendone about adenosine modulation of GABAergic responses, the mechanisms bywhich steroid hormones modulate epileptic activity is not yet fully understood.

Conclusions

We have found that ADA activity is not homogeneous throughout the brainand suggested that this enzyme might exert a more prominent modulatoryaction in specific brain areas. Furthermore, progesterone decreased ADA levels,suggesting an anticonvulsive effect, consistent with the scientific literature. The

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results showed that progesterone, through GABA-A receptors, may exert aninhibitory effect through a predominant adenosine-mediated action in the brain.The final question remaining is what is the relevance of hormone actions onthe brain to human and animal neurophysiology and, especially, to epilepticphenomenon?

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