Carbamazepine inhibits angiotensin I-converting enzyme, linking it to the pathogenesis of temporal lobe epilepsy

Abstract

We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems.

Citation: Transl Psychiatry (2012) 2, e93, doi:10.1038/tp.2012.21

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Carbamazepine inhibits angiotensin I-converting enzyme, linking it to the pathogenesis of temporal lobe epilepsy

SS Almeida1, MG Naffah-Mazzacoratti2, PB Guimares1, F Wasinski1, FEG Pereira1, M Canzian3, RS Centeno3, H Carrete3, EM Yacubian3, AK Carmona1, RFF Vieira1, CR Nakaie1, RA Sabatini1, SR Perosa2, RFP Bacurau4, TLF Gouveia2, G Gallo5, M Wrtele5, EA Cavalheiro2, JA Silva Jr6, JB Pesquero1 and RC Araujo1

We nd that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly nd that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the reninangiotensin and kallikreinkinin systems.

Translational Psychiatry (2012) 2, e93; doi:http://dx.doi.org/10.1038/tp.2012.21

Web End =10.1038/tp.2012.21 ; published online 13 March 2012

Introduction

Epilepsy is a chronic neurological disease characterized by abnormal neural activity leading to epileptic seizures aficting

B1% of the population worldwide. We have recently found that temporal lobe epilepsy (TLE) patients show increased expression of the Angiotensin II (Ang II) AT1 and AT2 receptors1 as well as the kinin B1 and B2 receptors in their brain,2 thus providing a link between one of the most common forms of epilepsy, TLE associated to mesial sclerosis, to the so-called reninangiotensin (RAS) and kallikreinkinin systems (KKS).1,2 These systems, associated with regulation of blood pressure and several functions in the central nervous system,1 consist of a series of regulatory proteolytic steps, including conversion of angiotensinogen by renin to angiotensin I (Ang I), which is further degraded by angiotensin I-converting enzyme (ACE) to Ang II and conversion of kininogen by kallikrein to the peptide bradykinin, which in turn can be degraded by ACE.3

Because of its dual function as an angiotensin processing and kinin degrading enzyme, we therefore undertook the task to examine patients suffering from epilepsy for the occurrence of the common ACE insertion (I)/deletion (D) polymorphism, where the introduction of a 287-bp Alu-repeat sequence into intron 16 of the ACE gene has been shown to induce lower ACE plasma activities in the carriers of this mutation.4,5

Our surprising nding in this report that a majority of patients suffering from severe forms of epilepsy belong to the DD genotype, led us to postulate that distinct activities of this enzyme (levels of angiotensin/kinins) might be involved in TLE. Aiming to corroborate these results, we then tested directly the hypothesis that polypharmacological

effects of current available drugs could have effects mediated by ACE inhibition, leading to the nding that carbamazepine, one of the most commonly used drugs in the treatment of epilepsy and psychiatric disorders is a direct inhibitor of ACE.

Methods

Genotyping. The ACE I/D polymorphism was genotyped as described6 in 72 patients with epilepsy who underwent surgery and 368 controls. All subjects abstained from alcohol and caffeine intake for at least 24 h before the blood tests. Smokers and subjects who had received antihypertensive drugs were excluded from the control group. Each subject gave informed consent before collection of DNA at the beginning of the experimental procedure. The genotypes of all experimental groups are shown in Supplementary Table 1.

Surgical procedures. Surgical specimens from patients with intractable epilepsy were submitted to standard corticoamygdalohippocampectomy at the So Paulo Hospital (Epilepsy Research and Treatment Unit, UNIPETE, So Paulo, Brazil) for seizure control. All cases showing neoplasm, vascular malformations, post-traumatic and ischemic lesion in preoperative magnetic resonance imaging (MRI) were excluded. Selected patients (n 9) had detailed anamnesis, video electroencephalo

gram recordings, and MRI studies. Antiepileptic drugs used by these patients for seizure control included: carbamazepine, phenobarbital, valproic acid and phenytoin. Surgery was performed at least 48 h after the last seizure. The outcome of seizures after epilepsy surgery was classied according to

1Department of Biophysics, Federal University of So Paulo, So Paulo, Brazil; 2Experimental Neurology and Department of Pathology, Federal University of So Paulo, So Paulo, Brazil; 3Department of Neurology and Neurosurgery, Federal University of So Paulo, So Paulo, Brazil; 4School of Arts, Sciences and Humanities of University of So Paulo, So Paulo, Brazil; 5Department of Science and Technology, Federal University of So Paulo, So Jos dos Campos, So Paulo, Brazil and

6Departament of Rehabilitation Sciences, Nove de Julho University (UNINOVE), So Paulo, BrazilCorrespondence: Professor RC Araujo, Department of Biophysics, Federal University of So Paulo, Rua Pedro de Toledo 669, 9o andar, So Paulo, SP 04023-032, Brazil.

E-mail: mailto:[email protected]

Web End [email protected] Keywords: angiotensin I-converting enzyme; carbamazepine; epilepsy; TLE

Received 23 December 2011; revised 10 February 2012; accepted 11 February 2012

Carbamazepine inhibits ACE SS Almeida et al

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Engels four categories.7 Control tissues were obtained from autopsies (less than 5 h post mortem) (n 9) from brains

showing no evidence of pathology based on a gross classication after routine histological examination, and were obtained from autopsies carried out by a pathologist trained especially for this purpose from the Anatomical Pathology Department (INCOR, FMUSP). By using this procedure, similar hippocampal areas from epileptic patients and autopsied subjects could be compared. Hippocampi were dissected, frozen in liquid nitrogen and stored at 80 1C. All

experiments were performed under approval from the Institutional Ethics Committee of the Federal University of So Paulo (UNIFESP).

ACE activity assays. Somatic ACE activity from human hippocampi was assayed using a uorescence resonance energy transfer substrate containing ortho-aminobenzoic (Abz) and dinitrophenyl (Dnp) as uorescence donor/ acceptor pair, namely Abz-FRK(Dnp)P-OH, as described.8 ACE specic activity for each tissue was calculated by division of velocity values in mM min 1 by the respective protein concentrations, as determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Activity of puried, or plasma ACE (control n 127; TLE

n 43) was measured in the same way but normalized

either by the enzyme amount or plasma volume, respectively. In the cellular assays, Chinese hamster ovary cells (CHO) were transfected with human ACE and its activity evaluated either with the uorescence resonance energy transfer substrate AbzFRK(Dnp)POH, as described,9 or

with the endogenous substrate Ang I as assayed by high-performance liquid chromatography (HPLC). Measurements were done in triplicate and ACE activity was reported as mM

of Abz-FRK(Dnp)P-OH hydrolyzed per minute of substrate hydrolyzed after 6 min of incubation in the presence of different concentrations of carbamazepine. For the determination of the inhibitory effect of carbamazepine on ACE hydrolytic activity with Ang I, samples were preincubated for 2 h with different concentrations of carbamazepine. Samples were then incubated in the same buffer with 10 mM of Ang I (Sigma-Aldrich, St Louis, MO, USA), in the presence or absence of 2.5 mM of lisinopril. Supernatant aliquots (400 ml)

were collected after 0, 2, 5, 15 and 30 min, and the reaction was stopped by the addition of 2 ml 10% triuoroacetic acid (TFA). The conversion of Ang I to Ang II was determined by

HPLC analysis using a Vydac C18 (4.6 150 mm) column

equilibrated with solvent A (0.1% TFA/H2O) and eluted with solvent B (0.1% TFA; 60% acetonitrile/H2O) with a 3045% acetonitrile gradient of solvent B for 15 min, with a ow of1.5 ml min 1 and detection at 220 nm wavelength. Some peaks were collected and analyzed in an automatic amino-acid sequencer to conrm their sequence (data not shown). Measurements were made in duplicate and the values of ACE activity were shown as percentage of basal Ang I degradation per minute per milligram of total protein (%min 1mg 1).

Kallikrein assay. Assays were performed using puried enzyme (0.57 mg ml 1; Enzyme Research Laboratories,

South Bend, IN, USA) in 50 mM Tris-HCL buffer (pH 7.4)

using the uorogenic substrate Z-Phe-Arg-AMC as described.6

Animal model. C57Bl/6 mice were obtained from the Center for the Development of Experimental Models for Medicine and Biology (CEDEME) at the Federal University of So Paulo and were kept at 22 1C in a lightdark cycle (12 in 12 h), with ad libitum supply of water and standard rodent chow. Carbamazepine was administered in mice by gavage, at a dose of 150 mg kg 1. After 60 min, animals were killed by cervical dislocation for blood collection; plasma was extracted by centrifugation and stored at 801C. All experiments were conducted in accordance with the NIH guide for care of laboratory animals and their uses (Institute of Laboratory Animal Resources, National Academy Press, Washington DC, 1996) after approval by the Ethics Committee in Research of UNIFESP.

Statistics. All data are given as means.e.m. Statistical evaluations were performed using Students t-test for unpaired data or one-way analysis of variance followed by the Tukeys post-hoc test to identify signicant data points. Genotype frequencies observed in our cohort were in HardyWeinberg equilibrium. Results were considered signicant with Po0.05.

Results and Discussion

Owing to the association between ACE and epilepsy, we examined male patients suffering from refractory TLE for the occurrence of the common ACE insertion (I)/deletion (D) polymorphism, which is known to induce lower ACE plasma activities in II subjects.4 Surprisingly, we found a signicant higher number of patients suffering from TLE with the DD genotype (46%) when compared with the normal subjects (27%; Figures 1ad). Concomitantly, TLE patients of both sexes with the II genotype, that express less ACE, showed a signicantly better recovery rate after corticoamygdalohippocampectomies (Figure 1e). As the D form leads to higher expression levels of ACE, we expected to be able to measure higher ACE activities in the plasma of male patients suffering from TLE. ACE activity was indeed signicantly elevated in subjects with the DD genotype of the control group. However, we could only detect a non-signicant trend to higher ACE activities in patients suffering from TLE with the DD genotype (Figures 1f and g). Furthermore, when we examined ACE activity in the hippocampus from seizure-control corticoamygdalohippocampectomies, we detected a twofold lower ACE activity in these patients (Figure 1h).

As these results seemed contradictory, we therefore hypothesized that if higher ACE expression is casually involved in the pathogenesis of TLE, perhaps its treatment could be linked to a reduction of ACE activity. Consequently, we decided to screen biochemically for the effects on ACE activity of different drugs commonly used to treat epilepsy. All the drugs tested showed insignicant changes in ACE activity, except carbamazepine, which signicantly inhibited ACE activity in a dose-dependent manner (Figure 2a). Carbamazepine is normally used to treat TLE in doses of 4001200 mg per day,9 where the minimal dose (400 mg per day) is

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a

b

**

c

d

**

e f

g h

Figure 1 ACE activity and TLE. (a, b) Distributions of the I/D ACE polymorphism in the control and TLE group (control n 368; TLE n 72). (c, d) Allele frequencies.

(e) Percentage of class 1A (best) outcome (Engels classication7) depending on genotype. (f, g) Mean (s.e.m.) soluble ACE activity according to I/D distribution in

the control group (n 127) and in patients with TLE (n 43). (h) Hippocampal tissue ACE activity. *Po 0.05, **Po0.01, ***Po0.001.

equivalent to a concentration of 0.34 mM in the blood. At this level, carbamazepine was able to inhibit B25% the plasma activity of human ACE (Figure 2a).

To exclude artifacts from other proteases present in blood plasma, we repeated this experiment using puried ACE corroborating our results (Figure 2b). Inhibition of ACE by carbamazepine was additionally conrmed in a dose-dependent manner using HPLC to identify direct degradation of the native Ang I peptide by the enzyme produced in ACE-transfected CHO. Carbamazepine was able to inhibit B75% of ACE at the cellular level (Figure 2c, Supplementary

Figure 1). Taken together all these independent experiments indicate in vitro direct inhibition of ACE by carbamazepine at concentrations similar to those used by TLE patients. To analyze the action of carbamazepine in vivo, we fed mice with a dose of 150 mg per kg of carbamazepine. The mice were killed after 60 min, and their serum analyzed for ACE activity. Again in this experiment we could conrm B25%

inhibition of ACE activity by carbamazepine (Figure 2d). Finally, direct inhibition of ACE by carbamazepine was additionally conrmed by comparing treated with noncarbamazepine-treated TLE patients. In this experiment,

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Carbamazepine inhibits ACE

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a

125

100

75

50

Percentage of

ACE inhibition

25

0

-25

-50

-75

0,25mM 0,5mM 1,25mM 2,50mM

-100

vehicle valproic acid

phenobarbital

phenytoin carbamazepine

vehicle valproic acid

phenobarbital

phenytoin carbamazepine

vehicle valproic acid

phenobarbital

phenytoin carbamazepine

vehicle valproic acid

phenobarbital

phenytoin carbamazepine

b c

d e

Figure 2 Inhibition of ACE by carbamazepine. (a) Inhibitory prole of different drugs used in the treatment of epilepsy. Carbamazepine was the only antiepileptic drug able to inhibit plasmatic ACE activity. (b) Enzymatic activity of puried ACE. Mean (s.e.m.) (*Po0.05, ***Po0.001). As a non-specicity control, carbamazepine had no

inhibitory effect on either human plasmatic or puried kallikrein (Supplementary Figure 2). (c) HPLC assay demonstrating carbamazepine inhibition of Ang I degradation by

ACE (***Po 0.001). (d) Mean (s.e.m.) plasmatic ACE activity is decreased 60 min after carbamazepine administration by gavage (150 mg kg 1) in mice (**Po0.01).

(e) Mean (s.e.m.) plasmatic ACE activity of TLE patients treated with carbamazepine (n 35) resembles that of the control group (n 55), whereas non-carbamazepine-

treated TLE patients (n 15) have signicantly higher ACE activities (*Po0.05).

treatment with carbamazepine led to a similar ACE activity as in the control group, whereas non-carbamazepine-treated TLE patients had a higher ACE activity (Figure 2e). However, we did not observe blood pressure differences between the

treated TLE and non-treated TLE groups (Supplementary Table 2).

All components of the RAS and KKS have been found in the brain10 modulating functions such as stress, anxiety, learning

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Carbamazepine inhibits ACE SS Almeida et al

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and memory acquisition.11 Under pathological conditions the RAS promotes inammatory responses increasing neural excitability,12 glial scar formation and transformation of a normal neural network into a seizure-generating network, thus providing a basis for a linkage between angiotensin signaling and epilepsy.13 Our results provide further evidence for this hypothesis, as we nd a higher proportion of TLE patients with the DD ACE polymorphism phenotype, which leads to higher ACE activity levels of the enzyme. Furthermore we surprisingly nd that carbamazepine is an inhibitor of ACE in the concentration range used to treat patients.

It has been recently reported that TLE patients show increased expression of the Ang II AT1 and AT2 receptors1 as

well as the kinin B1 and B2 receptors in the hippocampus2 and that captopril, a classical ACE inhibitor, enhances the anti-convulsant activity of carbamazepine.14 These results indeed strengthen the case for a link between the RAS and KKS and the pathogenesis of epilepsy. Furthermore, as carbamaze-pine is thought to display anticonvulsant activity due to its inhibition of sodium channels,9 our results enrich once again the list of known drugs with polypharmacological effects.

Conict of interest

The authors declare no conict of interest.

Acknowledgements. We acknowledge nancial support from INNT, CNPq, CAPES CInAPCe and FAPESP.

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