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Purpose: As reported by several authors, angiotensin II (AngII) is a proinflammatory molecule that stimulates the release of inflammatory cytokines and activates nuclear factor κB (NFκB), being also associated with the increase of cellular oxidative stress. Its production depends on the activity of the angiotensin converting enzyme (ACE) that hydrolyzes the inactive precursor angiotensin I (AngI) into AngII. It has been suggested that AngII underlies the physiopathological mechanisms of several brain disorders such as stroke, bipolar disorder, schizophrenia, and disease. The aim of the present work was to localize and quantify AngII AT1 and AT2 receptors in the cortex and hippocampus of patients with temporal lobe epilepsy related to mesial temporal sclerosis (MTS) submitted to corticoamygdalohippocampectomy for seizure control.
Method: Immunohistochemistry, Western blot, and real-time PCR techniques were employed to analyze the expression of these receptors.
Results: The results showed an upregulation of AngII AT1 receptor as well as its messenger ribonucleic acid (mRNA) expression in the cortex and hippocampus of patients with MTS. In addition, an increased immunoexpression of AngII AT2 receptors was found only in the hippocampus of these patients with no changes in its mRNA levels.
Discussion: These data show, for the first time, changes in components of renin-angiotensin system (RAS) that could be implicated in the physiopathology of MTS.
The renin-angiotensin system (RAS) participates in the regulation of several physiological processes including body water balance, maintenance of blood pressure, fluctuation of reproductive hormones, sexual behavior, and regulation of pituitary gland hormones. In this system, angiotensinogen is the inactive precursor and after its cleavage by the protease renin, the angiotensin I (AngI) is released. AngI, which has very low biological activity, is the main substrate for angiotensin converting enzyme (ACE), which generates angiotensin II (AngII), the main active peptide. After that, a cascade of events could be triggered, leading to the release of diverse angiotensin-related peptides with a variety of biological effects (for review, see Wright & Harding, 2004).
Although widely distributed in peripheral systems, all RAS components have also been found in the brain where they actively modulate several functions, including exploratory behavior, stress, anxiety, learning, and memory acquisition (Chen et al., 1993; Meffert et al., 1996; Yamada et al., 1996; Wright & Harding, 2004; Saavedra et al., 2005). Interestingly, AngII has been considered to be the major active peptide of the RAS, acting in the periphery as a paracrine and endocrine hormone. This polypeptide could also be considered as a neurotransmitter/neuromodulator through its action on AT1 and AT2 receptors present in several brain regions (Wright et al., 2002). In addition, growing evidence has emerged indicating the RAS participation's in some neurodegenerative disorders such as Alzheimer's (Barnes et al., 1991; Tian et al., 2004), Parkinson's (Grammatopoulos et al., 2005), and Huntington's (Bird, 1980) diseases.
Recent works of Tchekalarova (Tchekalarova et al., 2005; Tchekalarova & Georgiev, 2005, 2006) indicate that angiotensin-related peptides may have anticonvulsant activity in pentylenetetrazol (PTZ), bicuculline- and picrotoxin-induced seizures. Although the mechanisms by which RAS peptides could exert the anticonvulsant activity are not completely understood, these authors suggested that the underlying process could involve the participation of other neurotransmitters (Wright et al., 2002). In this sense, AngII was shown to be able to increase the threshold for PTZ-induced seizures through a dopaminergic-related mechanism as dopamine (DA) receptor (D1/D2) agonists or DA uptake inhibitors increase while DA antagonists attenuate the above-mentioned AngII action (Georgiev et al., 1985). Furthermore, an involvement of GABAergic interneurons mediating the inhibitory effect of AngII was also suggested in the basolateral amygdala, where GABAA receptor antagonist effectively blocked the discharge rate decrease normally induced by AngII (Tchekalarova et al., 2005). In addition, results in mice have shown that the increased threshold of PTZ-induced seizures exerted by adenosine analogues was attenuated by AngII receptor antagonists (for review, see Tchekalarova & Georgiev, 2005). Interestingly, Das (2005) has shown that AngII stimulates the release of proinflammatory cytokines, activates NFκB, and increases oxidative stress—a group of activities that has been considered to facilitate neuronal loss. These findings would justify the association of AngII with the physiopathology of stroke, bipolar disorder, schizophrenia, and Alzheimer's disease. Thus, ACE inhibitors and AngII receptor antagonists should be considered important coadjutants in the treatment of chronic inflammatory brain diseases.
Temporal lobe epilepsy related to mesial temporal lobe sclerosis (MTS) is characterized by hippocampal sclerosis, which includes atrophy, induration of shrunken tissue, granule cell loss in the dentate gyrus and extensive pyramidal cell loss in Ammon's horn, and astroglial proliferation in the hippocampus (Babb et al., 1991). The predominant cell loss occurs in pyramidal neurons of the stratum pyramidale of Ammons horn. Neuronal population of the stratum oriens survives in most sclerotic hippocampi, along with interneurons scattered throughout the neuron-depleted pyramidal layer. In the dentate gyrus, many of the granule cells are lost and neurons in the granule cell layer appear to be more dispersed. Thus, several lines of evidence point to sclerotic hippocampus as the major structure involved in chronic seizures observed in MTS (for review, see Lanerolle & Lee, 2005).
Based on these data, the present work was designed to study the expression of AngII receptors (AT1 and AT2) in the cortex and hippocampus obtained from patients with MTS, submitted to surgery for seizure control. With this purpose, double staining procedure associated to Western blot was employed to localize and quantify these alterations, respectively. In addition, AngII AT1 and AT2 receptors mRNA levels were also analyzed using real-time PCR to study the synthesis of these two receptors in both cortex and hippocampus tissues. All patients presented hippocampal sclerosis visualized by image analysis and by pathological studies. The expression of AngII receptors and their respective mRNA were also studied in hippocampal and cortical areas removed during autopsy of subjects without neurological disease, which were used as control tissues.
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All experiments were performed under approval from the Institutional Ethics Committee of the Universidade Federal de São Paulo (UNIFESP). Surgical specimens from patients with intractable epilepsy were submitted to standard corticoamygdalohippocampectomy at the Hospital São Paulo (UNIPETE, Brazil). All cases showing neoplasm, vascular malformations, post-traumatic and ischemic lesions on preoperative MRI were excluded. Selected patients (n = 12) had detailed anamnesis, video-EEG recordings, and MRI studies. The age of patients with epilepsy varied from 19 to 64 years (38.8 ± 17 years). Control tissues were obtained from autopsies (less than 5 h of postmortem) and the age of the subjects at death varied from 28 to 64 years (47.25 ± 18 years) (n = 12). The antiepileptic drugs used by these patients for seizure control included: carbamazepine, phenobarbital, diphenylhydantoin, clobazam, valproic acid, and topiramate. Surgery was performed at least 48 h after the last seizure. Table 1 summarizes detailed clinical features concerning all the patients with epilepsy included in this study. Control hippocampi and cortices were obtained from brains showing no evidence of pathology on the basis of gross and routine histological examination. These tissues were obtained from autopsies carried out by a pathologist from the Anatomical Pathology Department, (INCOR, FMUSP) who had been trained especially for this purpose by neurosurgeons responsible for the surgery of patients with epilepsy. Using this procedure, similar hippocampal and cortical areas from epileptic patients and autopsied subjects could be compared.
Table 1. Clinical evaluation of patients with TLE
|Patients||First seizure (age)||Duration of epilepsy/years||Seizure frequency/month||Engel's outcome scale|
| 1.||1 year ||30||2||IA|
| 2.||5 years||34||4||IA|
| 3.||7 years||32||12 ||IA|
| 4.||3 years||31||12–20||IA|
| 5.||1 year ||31||2–3||IA|
| 6..||4 years||27||8–12||III|
| 7.|| 10 months||40||2–4||IA|
| 8.||5 years||30||2–3||IA|
| 9.||5 years||34||1–3||IA|
|11.||12 years ||18||16 ||III|
Brain tissues removed during surgery or autopsy were rapidly immersed in buffered 1% paraformaldehyde (pH = 7.4) for 24 h at 4°C. Collected hippocampi and cortices were sliced into sections of 0.5 cm through longitudinal axis. All tissues were then dehydrated in a solution containing increased alcohol concentration, followed by xylol (100%) before inclusion in paraffin. The paraffin-embedded tissue was sliced (3 μm) in microtome (Jung-Leica, Munich, Germany) and maintained in covered silane slides. Slices from the hippocampus and cortex were submitted to classical Nissl staining (NS), after paraffin removal. The tissue from each patient was analyzed using at least three different sections for Nissl or double staining procedures. Thus, cresyl-violet staining was done to assess the specimen orientation and to check the localization and the extent of lesion. Adjacent sections were selected for immunofluorescence (three sections of three different patients were used).
Paraffin was removed from slices using xylol solution (100%) and endogenous peroxidases were blocked using H2O2 (3%) for 15 min. After this procedure, slices were heated for 10 min in a 700 W microwave oven.
In order to verify whether Ang II AT1 and AT2 receptors' immunoreactivity were localized in hippocampal and cortical neurons, we employed a double-label immuno-fluorescence protocol to colocalize the AngII AT1 and AT2 receptors containing cells and the neuronal marker (Neu-N). The anti-AT1 receptor antibody employed was a rabbit polyclonal antibody and the anti-AT2 receptor was a goat polyclonal antibody, followed by the mouse anti-NeuN (monoclonal antibody from Chemicom International, Temecula, CA, U.S.A.). The tissues were removed and placed in 5% formal saline fixative. Forty-four hours later, the hippocampus and the cortex were dehydrated and embedded in paraffin wax. Three micrometer sections adhered onto poly-l-lysine (Sigma, St. Louis, MO, U.S.A.) were dewaxed in xylene, rehydrated by sequential immersion in 100% ethanol, 95% ethanol and distilled water for 5 min each, permeabilized with 0.1% trypsin for 3 min, and nonspecific binding sites were blocked with 1% bovine serum albumin for 1 h. Incubation with 100 μl of the primary antibodies, Neu-N diluted 1:300, anti-AT1 receptor, and anti-AT2 receptor diluted 1:50 was performed at 4°C for overnight. Binding of the first antibody was detected with an antimouse antibody coupled to Alexa Fluor 488 (green) 1:300 (Molecular probe, Eugene, OR, U.S.A.) for 1 h. The second antibody was detected with an antirabbit antibody coupled to Alexa Fluor 594 (red) 1:300 for 1 h. Binding of the anti-AT2 receptor was detected with an antigoat antibody coupled to Alexa Fluor 594 (Red) 1:300 for 1 h. The sections were then washed in PBS and cover-slipped with Flouromount-G (Electronic Microscopy Science, Fort Washington, PA, U.S.A.). Magnified images were obtained by dual channel confocal argon/krypton laser scanning and these images were collected using appropriated emission filters. During colocalization of AngII AT1 receptors and Neu-N, AT1 receptors were stained in red, while Neu-N positive cells were stained in green. A similar procedure was employed to covisualized AT2 receptors and Neu-N. To analyze the antibody specificity both primary antibodies were excluded during double staining and no labeling was found, indicating a good specificity for the employed antibodies (data not shown).
Western blot protocol for AngII AT1 and AT2 receptors
Protein quantification for AngII AT1 and AT2 receptors was performed in the hippocampi and cortices (n = 4 for each group) by Western blot. Tissues were dissected and stored at –80°C until assay. Samples were homogenized in lysis buffer with protease inhibitor cocktails (0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 0.001 M EDTA pH 8.0, 1% NP-40, 10% Glycerol, 10μM PMSF, 1 mM sodium metavanadate, 0.05 M NaF, 2 nM okadaic acid). Protein content was determined using the Lowry method (1951). Samples were diluted in Laemmli buffer and boiled for 5 min. Two standard curves were performed using several protein concentrations. A linear range was obtained using 40 μg of protein for AngII AT1 receptor and 80 μg for AngII AT2 receptor. Thus, an equivalent amount of protein was electrophored on 10% polyacrylamide mini-gels and transferred to nitrocellulose membrane by electroblotting. Blocking was performed in 5% nonfat milk for 2 h at room temperature. Thereafter, blots were then probed with an affinity-purified goat polyclonal antibody raised against a peptide mapping near the carboxy terminus of rabbit AT1 receptor (1:150, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.) or with a goat polyclonal antibody raised against a peptide mapping the C-terminus of AT2 receptor (1:150, Santa Cruz Biotechnology, Inc) in TBS-T (50 mM Tris-HCl, 154 mM NaCl, pH7.5 + 0.1% Tween 20) plus 2% nonfat milk. After overnight incubation, blots were washed in TBS-T (3 × 10 min) and incubated for 2 h in biotinylated antigoat or antirabbit immunoglobulin (Calbiochem, Darmstadt, Germany), diluted 1:150 in TBS-T plus 2% nonfat milk. Blots were washed in TBS-T (3 × 10 min) and incubated for 1 h in streptavidin-horseradish peroxidase (Vector Laboratories, Inc., Burlingame, CA, U.S.A.). After washing, enhanced chemiluminescence reagents (ECL, Amershan Pharmacia Biotech, Cardiff, UK) were applied to the blots. The membranes were then exposed to an x-ray film (Amersham) and the bands were quantified by densitometry. Equal protein loading was shown by stripping and reprobing the membrane with an anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000, Proteimax Biotecnologia Ltda., São Paulo, Brazil).
Western blot data analysis
The ratio between optical density of AngII AT1 and AT2 receptors and the internal standard GAPDH bands are presented as means ± standard deviation (SD). Significance of differences was assessed by Student's t-test and p-value <0.05 was considered significant.
Quantitative real-time TaqMan PCR for angiotensin receptors
Dissected hippocampi and cortices were frozen in liquid nitrogen and stored at –80°C. Thawed tissue was homogenized in 1 ml TRIzol reagent (Gibco BRL, Gaithersburg, MD, U.S.A.) and total RNA was isolated according to the manufacturer's instructions. Samples were submitted to a 20 μl reaction using TaqMan Amplification System with a Mastercycler Realplex (Eppendorf, Wesseling-Berzdorf, Germany). Real-time PCR was performed with 900 ng of cDNA for AT1 and AT2 receptors and 100 ng of cDNA for GAPDH, used as an internal standard (GAPDH mRNA accession number NM_002046). Beta2-microglobulin was used as a second internal standard (β2 microglobulin mRNA accession number AF072097). Oligonucleotide primer and fluorogenic probe sets for TaqMan real-time PCR were designed for AT1 and AT2 receptors and GAPDH or beta2-microglobulin using Assays-by-Design Service (Applied Biosystems, Foster City, CA, U.S.A.) to meet all TaqMan design guidelines. Probes were synthesized with a reporter dye 6-carboxyfluorescein (6-FAM) covalently linked at the 5′ end and a quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA) was linked to the 3′ end of the probe. AT1 and AT2 probes used were: 5′-FAM-CGACGCACAATGCTTGTAGCCAAAGTCA-TAMRA-3′ and 5′-FAM-TTGTTCTGGCCTTCATCATTTGCTGGC-TAMRA-3′, respectively. The utilized primers were:
|AT1||5′AGA TGA CGG CTG||5′ AGG ACA AAA GCA|
|receptor|| CTC GAA GA 3′|| TAG GGA G 3′|
|AT2||5′ CAA AGC ATT CTG||5′ TCC TTA TGC CTT TGG|
|receptor|| CAG CCT GA 3′|| TTG TTG TTG AAG T 3′|
Each reaction was carried out with 10 μl of RealMasterMix Probe (Eppendorf) and 1 μl of a mix containing two primers (18 μM each) and a probe (5 μM), specific to mRNA of angiotensin AT1 receptor or angiotensin AT2 receptor. The cycle conditions were: 50°C for 2 min, then 95°C for 10 min, followed by 50 cycles of 95°C for 15 s (melting step), 60°C for 1 min (anneal/extend step). Each group was composed of five subjects.
Gene expression data analysis
Increased amount of reporter dye fluorescence during the 50 cycles of amplification were monitored using Eppendorf Cycle Manager. The PCR cycle when a given fluorescence threshold is crossed by the amplification curve is considered the first parameter to analyze mRNA expression and named Ct. The bigger the initial copies amount, the smaller the Ct number. A normalized value is obtained by subtracting Ct of GAPDH or β2 microglobulin from Ct of AT1 and AT2 receptors, resulting in ΔCt. As it is uncommon to use ΔCt as a relative expression data due to this logarithmic characteristic, the 2−ΔCt parameter was used to express the relative expression data, which were analyzed as mean ± SD. Hippocampi and cortices from patients with MTS and control tissue were compared using Student's t-test. A value of p < 0.05 was considered to be statistically significant.
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Nissl-stained cortices from patients with epilepsy presented no difference with regards to cellular distribution in all layers of temporal cortex when compared to control tissues (data not shown). However, this staining revealed hippocampal sclerosis in all patients with epilepsy, confirming data obtained during MRI analysis, which previously demonstrated atrophy, signal changes, and abnormal shape of the hippocampus. The hippocampi of patients with epilepsy presented intense cell loss mainly in the hilus as well as in the CA1 and CA3 regions, when compared with the control area. The hippocampi of the control group showed a normal pattern of cell distribution (Fig. 1).
Figure 1. Nissl staining of control and sclerotic hippocampus: (A) control, (B) sclerotic hippocampus (scale bars 400 um).
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The double staining showed intense AngII AT1 labeling of pyramidal neurons, colocalizing with Neu-N protein, in control and epileptic tissues. As shown (Fig. 2, top panel) in the cortex of patients with epilepsy the density of AngII AT1 receptor is increased in each neuron, mainly in apical regions as clusters, near the plasma membrane (Fig. 2, top panel). This type of labeling was less visualized in control cortex.
Figure 2. Double staining: colocalization of NeuN and AngII AT1 or AT2 receptor in the temporal cortex and hippocampus (n = 3). NeuN is stained in green while AngII AT1 and AT2 receptors are stained in red. The top panel shows immunostaining of AngII AT1 receptors of control tissues on right panel and epileptic tissue on left panel. The lower panel shows immunostaining of AngII AT2 receptors of control tissue on right panel and epileptic tissue on left panel.
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The double staining of AngII AT1 receptor and Neu-N also showed increased expression of AngII AT1 receptor in CA3 regions of sclerotic hippocampus and apical regions of neurons were more stained (Fig. 2, top panel). In contrast, granule cells from patients showed no immunoreactivity against AngII AT1 receptors (Fig. 3), demonstrating that more vulnerable cells are preferentially stained. Only hilar neurons were double-labeled.
Figure 3. Double staining: colocalization of NeuN and AngII AT1 receptor in the dentate gyrus of patient with hippocampal sclerosis. Note that the granular cells were not stained by anti-AT1 receptor while hilar cells are immunolabeled.
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Western blot analysis indicated a 55 kDa band, identified as Ang II AT1 receptor. Optical density analysis, after normalization with GAPDH (35–40 kDa) confirmed an increased expression of AT1 receptor in the hippocampus (0.425 ± 0.04, p = 0.038) and cortex (0.386 ± 0.03, p = 0.012) of patients with epilepsy, when compared to control hippocampus (0.331 ± 0.03) and cortex (0.277 ± 0.03), respectively (Fig. 4, top and lower panels). In addition, quantification of mRNA for AngII AT1 receptor by real-time PCR demonstrated an increased level of this mRNA in the hippocampus (0.55 ± 0.105, p = 0.00057) and cortex (2.5 ± 0.179, p = 5.49 × 10−9) of patients with epilepsy, when compared with the control hippocampus (0.33 ± 0.034) and control cortex (0.86 ± 0.089) (Fig. 6, top panel). These data demonstrated an upregulation of AngII AT1 receptors in the cortex and hippocampus of patients with MTS.
Figure 4. Western blot analysis of AngII AT1 receptor in the cortex (top panel) and hippocampus (lower panel) of control and epileptic tissues. Representative Western blot analysis: rows: 1, 2, 3, 4—control tissues; 5, 6, 7, 8—epileptic tissues. *p = 0.012 (top panel); *p = 0.038 (lower panel), control versus epileptic tissues (n = 4 per group).
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Figure 6. Top panel: quantification of AngII receptors mRNA expression in the cortex and hippocampus. Top: level of AngII AT1 receptors mRNA in the human cortex measured by real-time PCR assay. All data have been normalized for levels of β2 microtubulin expression within the same sample and are expressed as relative levels detected in each group sample as 2 –ΔCt. Data are expressed as mean + SD (n = 5 per group). Lower: level of AngII AT1 receptors mRNA in the human hippocampus measured by real-time PCR assay. All data have been normalized for levels of β2 microtubulin expression within the same sample and are expressed relative to levels detected in each group sample as 2–ΔCt. Data are expressed as mean + SD (n = 5 per group). Lower panel: quantification of AngII receptors mRNA expression in the cortex and hippocampus. Top: level of AngII AT2 receptors mRNA in the human cortex measured by real-time PCR assay. All data have been normalized for levels of β2 microtubulin expression, within the same sample and are expressed as relative levels detected in each group sample as 2 –ΔCt. Data are expressed as mean + SD (n = 5 per group). Lower: level of AngII AT2 receptors mRNA in the human hippocampus, measured by real-time PCR assay. All data have been normalized for the levels of β2 microtubulin, within the same sample and are expressed relative to levels detected in each group sample as 2 – ΔCt. Data are expressed as mean + SD (n = 5 per group).
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The double staining for AngII AT2 receptors and NeuN showed that in the temporal cortex no visible difference was found concerning AT2 receptor expression in the control and epileptic tissue. This procedure allows us to visualize few neurons stained in control and epileptic cortex, showing no difference between them (Fig. 2, lower panel).Vessels and glial-processes were also found in both tissues.
In contrast, an increased immunoreactivity against AngII AT2 receptors was found in sclerotic hippocampus. Magnifications of this figure revealed few cells in CA3 region immunostained against AngII AT2 receptors in the hippocampus of these patients. In addition, the double staining of AngII AT2 receptor and Neu-N also revealed that besides pyramidal cells, several fibers, glial processes, and vessels were visualized in the sclerotic hippocampus (Fig. 2, lower panel). However, granular cells were not labeled in sclerotic hippocampus (data not shown).
Western blot analysis indicated a 50-kDa band, identified as AngII AT2 receptor. Optical density determination, after normalization with GAPDH (35–40 Kda), showed an increased expression of AngII AT2 receptor in the hippocampus (0.83 ± 0.07) of patients with epilepsy, when compared with control tissue (0.307 ± 0.07, p = 0.0532 × 10−3). No difference was observed in the temporal cortex (2.17 ± 0.14, p = 0.288) of these patients, when compared to controls (2.04 ± 0.13) (Fig. 5, lower panel).
Figure 5. Western blot analysis of AngII AT2 receptor in the cortex and hippocampus of control and epileptic tissues. Representative Western blot analysis: rows 1, 2, 3, 4—control tissues; 5, 6, 7, 8—epileptic tissues. p = 0.2885 (top panel), **p = 5.32.10–5 (lower panel), control versus epileptic tissues (n = 4 per group).
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Although Western blot has shown increased expression of AngII AT2 receptor in the sclerotic hippocampus, the quantification of their mRNA expression by real-time PCR showed similar results in the hippocampus of patients with epilepsy (0.13 ± 0.016) and control subjects (0.12 ± 0.024, p = 0.348) as well as in the cortex of patients (0.22 ± 0.018) and control subjects (0.20 ± 0.028, p = 0.437) (Fig. 6, top and lower panel). All together, these data showed that although AngII AT2 receptor is upregulated in the hippocampus of epileptic patients, its synthesis is not induced during an interictal period. Concerning the epileptic cortex, no difference was found related to the expression of AngII AT2 receptor. The employment of GAPDH as housekeeping on RT–PCR did not modify the obtained results (data not shown).
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In the present work we showed, for the first time, a relationship between human temporal lobe epilepsy related to mesial temporal sclerosis (MTS) and the RAS. Increased amount of AngII AT1 receptors was found in neurons from cortex and hippocampus of patients with MTS. In addition, mRNA for AngII AT1 receptor was increased in both tissues from these patients showing that the expression as well as the synthesis of this receptor is upregulated in patients with MTS. This study also showed an upregulation of AngII AT2 receptor in the hippocampus of patients with MTS, although its synthesis was not modified during the interictal period of analysis. Altogether, these results strongly suggest the participation of angiotensin systems in the pathophysiology of temporal lobe epilepsy (TLE) associated with mesial sclerosis.
AngII AT1 and AT2 receptors have similar but not identical brain distribution in all mammalian species studied so far. AngII AT1 receptor expression predominates in adult animals while AngII AT2 receptors are highly expressed in developing brain (Tsutsumi & Saavedra, 1991; Saavedra, 2005). Despite the fact that AngII AT1 receptor is almost entirely responsible for the classical effects of AngII, it has been found that AngII AT2 receptor is expressed at a low amount in many healthy adult tissues and is upregulated in a variety of human diseases. AngII AT2 receptors have been related to ionic fluxes, cell differentiation, neuronal tissue regeneration, and programmed cell death in the brain. According to Wilms et al. (2005), AngII AT2 receptor could also be involved in neuronal protection following brain injury, such as stroke or CNS trauma, while AngII AT1 receptor might act as a receptor linked to neuronal injury. As reported by Das (2005), the binding of peptide AngII to the AT1 receptor is also involved in the stimulation of leukotriens, prostaglandins, free radicals generation, activation of the NFκB and increase of C-reactive protein levels, triggering the expression of proinflammatory and pro-oxidant genes. In addition, the association between inflammatory processes and temporal lobe epilepsy is already well known (Vezzani & Granata, 2005). Several inflammatory targets are upregulated in tissues involved in this syndrome (Vezzani et al., 1999; Vezzani et al. 2000) and proinflammatory cytokines have been linked to the pathogenesis of seizures. Jamali et al. (2006) showed a dysregulation of the neurotransmission and complement systems in the entorhinal cortex of humans with temporal lobe epilepsy, showing the participation of immune system with infiltration of C3 positive leucocytes and membrane attack complex deposition on neurons. In addition, Lerner-Natoli et al. (2000) showed that during long-lasting seizures the neuronal firing activates NF-κB and the major histocompatibility complex expression by glial cells of hippocampal formation, which are also related to inflammatory processes of TLE. Wilms et al. (2005) have shown that AngII AT1 receptor, although linked to inflammatory cascade production, is also associated to growth factors induction in neurons and glia. This receptor is also related with the modulation and proliferation in various cell types, showing a dual function for this receptor.
ACE, the main enzyme of RAS, is also related to the kallikrein-kinin system, which is linked to the inflammatory process. In this regard our group recently showed an overexpression of kinin B1 and B2 receptors in the hippocampus of patients with temporal lobe epilepsy, associated with mesial sclerosis (Perosa et al., 2007). Together, these data suggest that both systems could be involved in seizure modulation or in epileptogenic processes, via inflammatory pathways.
Interestingly, ACE gene polymorphism has also been associated with Alzheimer's disease being related to hippocampus and amygdala atrophy, showing that the RAS system is involved with brain pathologies, linked to severe cell loss and brain degeneration (Sleegers et al., 2005). Although the present work did not evaluate ACE expression, the differences reported here concerning the expression of angiotensin-related receptors between control and sclerotic hippocampus add new evidence to the participation of the RAS in brain diseases, associated with atrophy of important structures such as the hippocampus.
In the present work, the expression of AngII AT1 receptors was increased in the cortex and in the hippocampus of patients with temporal lobe epilepsy, while the AT2 receptor was elevated only in the hippocampus of these patients. These results suggest that changes in the release of AngII may occur via not yet known mechanisms and that this polypeptide would have pro- or antiepileptogenic function depending on its receptor activation. In this sense, our idea follows those of Tchekalarova & Georgiev (2005) who demonstrated the involvement of this system in seizure genesis and/or control. According to them, the activation of AngII AT1 receptor elicits neuronal depolarization, via the inhibition of potassium channels or by opening a nonselective sodium–calcium channel, facilitating the discharge firing in several brain regions. Increased c-Fos expression with consequent expression of immediate-early genes was also found, showing neuronal activation. In this context, our findings suggest that the upregulation of AngII AT1 receptor is related to increased tissue excitability, in the hippocampus and cortical areas.
In addition, dopaminergic system mobilization has been also reported. In vivo and in vitro studies revealed that AngII AT1 receptor activation potentates the depolarization-induced release of DA as well as the susceptibility to seizures. In this sense, a previous paper from our group (Naffah-Mazzacoratti et al., 1996) has shown increased utilization rate of DA in the cortical tissue of patients with temporal lobe epilepsy, a finding that now could be attributed to the present results on the upregulation of angiotensin system. Tchekalarova & Georgiev (2006) reported the modulation of PTZ-induced seizures by AngII AT1 receptor associated with a noradrenergic mechanism, mediated by the peptides AngII and AngIII.
Recently, Stragier et al. (2006) have also shown that AngIV is able to inhibit pilocarpine-induced seizures via the activation of somatostatin receptor 2. In this work, the authors observed that the administration of AngIV increased the hippocampal release of DA and serotonin, and that this effect is blocked by somatostatin receptor 2 antagonist, a finding that confirms the participation of these monoamines in the modulation of seizures induced by angiotensin-related peptides.
The anticonvulsant effect of AngII has also been attributed to the observation that this compound may have a direct action on the GABA-benzodiazepine receptor–ionophore complex, probably through allosteric mechanisms or by an interaction of AngII receptor with the GABAA receptor (Tchekalarova & Georgiev, 2005). In addition, Schelman et al. (2004) and Jing et. al. (2004) found that AngII acting on AT2 receptors attenuates the N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity, via antiapoptotic mechanisms, altering the expression level of Bcl-2 protein and rescuing cells from death. In the present work, we found an increased expression of AngII AT2 receptor in neurons from the hippocampus of patients with MTS, a region with severe cell loss. These data could be related with an AngII AT2 receptor mediated effect of neuroprotection or regenerative process. In agreement with this idea, Grammatopoulos et al. (2004) reported that AngII attenuates chemical hypoxia-induced caspase-3 activation in primary cortical neuronal cultures via the AT2 receptor and stimulates the p21 ras/mitogen activated protein kinase pathway, which is known to induce neuronal survival signals. In addition, oxidative stress induced apoptosis via upregulation of Jun-N-terminal kinases (JNK) could also be inhibited by AT2 receptor stimulation. Furthermore, Lucius et al. (1998) showed using the optic nerve of adult rats that AngII, acting through the AT2 receptor induces neurite outgrowth. Thus, the increased expression of these receptors in the hippocampus may be related to an attempt to control tissue degeneration.
According to Wilms et al. (2005), a cross-talking between AngII AT1 and AT2 receptors may occur. In this sense, the understanding of how distinct signal transduction elements are targeted by AT1 and AT2 receptors in different types of neurons will pave the way for a deeper insight into the role of these receptors during health and diseases of the nervous system.
In conclusion, the present work shows for the first time changes in the RAS in temporocortical areas of patients with MTS and suggests its participation in the physiopathologic mechanisms underlying epileptogenesis.