ATIVIDADE DA CREATINA QUINASE CEREBRAL É AUMENTADA PELA

ADMINISTRAÇÃO CRÔNICA DE PAROXETINA

Patrícia M. Santos, Emilio L. Streck

Laboratório de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense,

UNESC, Criciúma, SC, Brasil.

Correspondência: Prof. Dr. Emilio L. Streck, Laboratório de Fisiopatologia

Experimental, Universidade do Extremo Sul Catarinense, 88806-000, Criciúma, SC,

Brasil. Fax: #55 48 3431-2644. E-mail: emiliostreck@gmail.com

Resumo

A depressão maior é um transtorno grave e recorrente manifestado muitas vezes com

sintomas de níveis psicológicos, comportamentais e fisiológicos. A paroxetina é um

inibidor seletivo de recaptação de serotonina, a nortriptilina é um dos antidepressivos

tricíclicos e a venlafaxina é um inibidor seletivo da recaptação de serotonina e

noradrenalina. Além disso, vários trabalhos também sugerem o metabolismo energético

cerebral como um mecanismo subjacente da depressão. A creatina quinase (CK)

desempenha um papel central no metabolismo energético de tecidos que consomem

muita energia, tais como cérebro, onde ele funciona como um eficiente sistema de

tampão celular nos níveis de adenosina trifosfato. No presente trabalho, foi avaliada a

atividade da CK em cérebro de ratos após administração crônica de paroxetina,

nortriptilina e venlafaxina. Ratos adultos Wistar receberam injeções diárias de

paroxetina (10 mg/kg), nortriptilina (15 mg/kg), venlafaxina (10 mg/kg) ou salina

durante 15 dias. Doze horas após a última injeção, os ratos foram decapitados, o

hipocampo, estriado e córtex pré-frontal foram imediatamente removidos. A atividade

de CK foi então medida. Os resultados demonstraram que a administração crônica de

paroxetina aumentou a atividade da CK no córtex pré-frontal, hipocampo e estriado de

ratos adultos. Por outro lado, a administração crônica de nortriptilina e venlafaxina não

afetou a atividade da CK em nenhuma das estruturas cerebrais. Considerando que o

metabolismo energético está provavelmente envolvido na fisiopatologia dos transtornos

depressivos, um aumento na atividade da CK através dos antidepressivos pode ser um

importante mecanismo de ação destes fármacos.

Palavras-chave: Creatina quinase; cérebro; paroxetina; venlafaxina; nortriptilina.

1. Introdução

A depressão maior é um transtorno grave e recorrente manifestado muitas vezes

com sintomas de níveis psicológicos, comportamentais e fisiológicos, afetando 17-20%

da população do mundo, podendo resultar em morte prematura, sendo uma das

principais consequências econômicas e sociais [20, 35]. Pensa-se que um em cada cinco

indivíduos sofra um transtorno do humor na vida. A Organização Mundial de Saúde

estima que a depressão seja a quarta causa mais importante a nível mundial de

incapacidade e perda da qualidade de vida [22, 24, 30].

Fármacos antidepressivos são amplamente utilizados no tratamento de

transtornos de humor e outras condições. Vários antidepressivos eficazes estão

disponíveis, mas não funcionam para todos os pacientes, sendo incerta a eficiência do

tratamento. É digno de nota que a farmacoterapia da depressão é cara e amplamente

prescrita pelos médicos, entretanto menos da metade dos pacientes tratados atinja

remissão completa após a terapia com um único antidepressivo e outros se mostram

parcialmente imunes ou intolerantes ao tratamento farmacológico [27]. Além disso, uma

resposta terapêutica pode levar várias semanas, ou ainda não ser observada. Durante

este período, o paciente sofre morbidade continuada, além de aumento do risco de

suicídio. Portanto, a escolha do antidepressivo adequado para cada paciente é um

problema significativo em psiquiatria [17].

Os antidepressivos têm contribuído para a melhora da depressão do paciente, e

qualidade de vida, fazendo o tratamento da depressão mais satisfatório [7, 13, 16, 29].

Vários trabalhos sugerem o metabolismo cerebral como um mecanismo subjacente da

depressão. Neste contexto, foi demonstrado que alguns antidepressivos podem melhorar

o metabolismo energético [18, 28, 36, 40].

A creatina quinase (CK) desempenha um papel central no metabolismo de

tecidos que consomem alta demanda de energia tais como cérebro, onde ele funciona

como um eficiente sistema de tampão celular dos níveis de adenosina trifosfato (ATP).

A enzima catalisa a transferência reversível do grupo N-fosforil da fosfocreatina para

adenosina difosfato (ADP) regenerando o ATP. Também é conhecido que uma

diminuição da atividade da CK pode potencialmente prejudicar a homeostase

energética, contribuindo para a morte celular [1, 6, 11, 12, 39]. Neste contexto, tem sido

amplamente demonstrado que uma diminuição da atividade da CK está associada a um

percurso neurodegenerativo que resulta em perda neuronal após isquemia cerebral [39],

doenças neurodegenerativas [1, 6] e em outros estados patológicos [11, 12]. Foi

recentemente demonstrado que a CK está inibida em modelos animais com transtornos

neuropsiquiátricos, como transtorno bipolar [37] e após a eletroconvulsoterapia [4].

Portanto, considerando que a CK desempenha um papel importante na

homeostase energética cerebral e que alguns antidepressivos podem modular o

metabolismo energético, decidimos investigar a atividade cerebral da CK de ratos após

administração crônica de paroxetina (inibidor seletivo, da recaptação da serotonina),

nortriptilina (antidepressivo tricíclico) e venlafaxina (inibidor seletivo da recaptação de

serotonina e noradrenalina).

2. Metodologia

2.1. Animais: ratos machos adultos Wistar (250-300 g) foram obtidos do Biotério da

Universidade do Extremo Sul Catarinense. Os animais foram mantidos em ambiente

climatizado (23 ± 1°C) com ciclo claro-escuro de 12 horas (luzes acesas 07h00min),

com água e alimentação padrão livre. Este estudo foi executado conforme as

recomendações para o cuidado com o animal de acordo com a Sociedade Brasileira de

Neurociências e Comportamento (SBNeC) e com a aprovação do Comitê de Ética da

Universidade do Extremo Sul Catarinense.

2.2. Fármacos: Os animais receberam diariamente injeções intraperitoneais de

paroxetina (10 mg/kg), nortriptilina (15 mg/kg) ou venlafaxina 10 (mg/kg) durante 15

dias. Todos os fármacos foram dissolvidos em solução salina (veículo). Controle os

animais receberam o veículo.

2.3. Preparação dos tecidos e homogenato: Doze horas após a última injeção, os ratos

foram mortos por decapitação, o cérebro foi rapidamente removido e o hipocampo,

estriado e córtex pré-frontal separados. As estruturas cerebrais foram homogeneizadas

em tampão SETH (1:10, p/v) pH 7,4 (sacarose 250 mM, EDTA 2 mM, Trizma Base 10

mM e heparina 50 UI/mL). Os homogeneizados foram centrifugados a 800 × g durante

10 minutos e os sobrenadantes mantidos a -70 °C até a determinação da atividade das

enzimas utilizadas. O período máximo entre a preparação do homogenato e a análise

enzimática foi sempre inferior a 5 dias. O teor de proteínas foi determinado pelo método

descrito por Lowry e colaboradores [25] usando albumina bovina como padrão.

2.4. Atividade da creatina quinase: A atividade da CK foi avaliada em

homogeneizados cerebrais pré-tratados com lauril maltosídeo 0,625 mM.

Resumidamente, a reação consistiu numa mistura contendo Tris-HCl 60 mM, pH 7,5,

fosfocreatina 7,0 mM, MgSO4 9,0 mM e cerca de 0,4-1,2 mg de proteína em um

volume final de 100 µL. Após 15 min de pré-incubação a 37°C, a reação foi então

iniciada pela adição de ADP 4,0 mM e parou após 10 minutos por meio da adição de 20

µL de ácido p-hidroximercuribenzóico (p-HMB) 50 mM. A creatina formada foi

estimada de acordo com o método colorimétrico de Hughes [15]. A cor foi desenvolvida

por meio da adição de 100 µL de �-naftol 2%, 100 µL diacetil 0,05% em um volume

final de 1,0 mL e lido espectrofotometricamente após 20 min a � = 540 nm. Os

resultados foram expressos em unidades / min x mg de proteína.

2.5. Análise estatística: Os dados foram analisados pela análise de variância de uma

via, seguido pelo teste de Tukey quando o F foi significativo, e são expressos como

média ± desvio padrão. Todas as análises foram realizadas utilizando o programa SPSS

(Statistical Package for the Social Sciences).

3. Resultados

No presente trabalho foi avaliada a atividade cerebral da CK em ratos após

administração crônica de paroxetina, nortriptilina e venlafaxina. Nossos resultados

demonstraram que a administração crônica de paroxetina aumentou a atividade da CK

no córtex pré-frontal, hipocampo e estriado de ratos adultos. Por outro lado, a

administração crônica de nortriptilina e venlafaxina não afetou a atividade da CK nessas

áreas cerebrais (Figura 1).

4. Discussão

No presente estudo foi verificada a atividade da CK em cérebro de rato após a

administração crônica de paroxetina, nortriptilina e venlafaxina. O sistema

creatina/CK/fosfocreatina é importante para manutenção da homeostase energética

normal [21, 33], exercendo várias funções integradas, como proteção de energia

temporária, capacidade metabólica, transferência de energia e controle metabólico [32].

O cérebro de ratos adultos, tal como outros tecidos com altas e variáveis taxas de

metabolismo de ATP, apresentam uma alta concentração de fosfocreatina e atividade da

CK [21, 33].

Existe um forte corpo de evidências sugerindo que a disfunção no metabolismo

cerebral está relacionada com distúrbios neuropsiquiátricos, como depressão e

transtorno bipolar [2, 19, 23]. Neste contexto, o estudo de Streck e colaboradores [37]

mostraram que a administração de anfetamina inibiu a atividade da CK no cérebro de

ratos e os estabilizadores de humor não afetaram a inibição desta enzima.

Recentemente, tem sido demonstrado que os estabilizadores de humor impedem e

revertem o efeito inibitório sobre a atividade da citrato sintase causada pela anfetamina

em um modelo animal de mania [5]. Danos a cadeia de transportes de elétrons tem sido

sugerida como um fator importante na patogênese de uma série de transtornos

psiquiátricos [8, 26], incluindo depressão maior. Gardner e colegas [10] mostraram uma

diminuição significativa das taxas de produção de ATP e da enzima mitocondrial

muscular em relação aos controles em pacientes com transtorno depressivo maior.

Madrigal e colegas [26] também relataram que complexos I-III e II-III da cadeia

respiratória mitocondrial foram inibidos em cérebros de ratos após o estresse crônico

(imobilização por seis horas durante 21 dias). Recentemente, também foi relatada a

inibição da cadeia respiratória mitocondrial, atividades dos complexos I, III e IV após o

estresse crônico por 40 dias, sugerindo que o prejuízo no metabolismo energético pode

ocorrer nos transtornos depressivos [31].

A deficiência de energia tem sido associada à morte neuronal e

neurodegeneração [3, 14, 34]. Considerando que o metabolismo energético está

provavelmente envolvido na fisiopatologia dos transtornos depressivos, um aumento na

atividade da CK por antidepressivos pode ser um importante mecanismo de ação destes

medicamentos. O presente trabalho mostrou que a atividade da CK aumentou após

administração crônica de paroxetina. Por outro lado, a administração crônica de

nortriptilina e venlafaxina não afetou a atividade da CK no cérebro de ratos. Alguns

outros estudos também apontam para a possibilidade de que as drogas utilizadas no

tratamento de tais desordens modulam o metabolismo energético [4, 9, 38].

Alguns achados apóiam esta hipótese. Já foi demonstrado que a atividade da

Na+,K+-ATPase foi afetada pela fluoxetina e eletroconvulsoterapia, mostrando

importantes mudanças metabólicas que ocorrem após a terapia antidepressiva. Algumas

evidências também apontam para a possibilidade de que as drogas utilizadas no

tratamento de tais desordens modulam o metabolismo energético [9, 38]. Além disso,

temos recentemente revelado que a CK é inibida após eletroconvulsoterapia [4].

Em conclusão, nossos dados corroboram com outros estudos, sugerindo que

alguns medicamentos antidepressivos modulam o metabolismo energético cerebral.

Mais estudos são importantes para avaliar se outras enzimas envolvidas no metabolismo

são também afetadas por estes antidepressivos.

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Figura 1. Efeitos da administração crônica da paroxetina, venlafaxina e nortriptilina na

atividade da creatina quinase no córtex pré-frontal, hipocampo e estriado de ratos.

* P <0,01 vs. grupo salina, de acordo com a ANOVA seguida pelo teste de Tukey.

Brain creatine kinase activity is increased by paroxetine chronic

administration

Patrícia M. Santos, Emilio L. Streck

Laboratório de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense,

UNESC, Criciúma, SC, Brasil.

Correspondence: Prof. Dr. Emilio L. Streck, Laboratório de Fisiopatologia

Experimental, Universidade do Extremo Sul Catarinense, 88806-000, Criciúma, SC,

Brasil. Fax: #55 48 3431-2644. E-mail: emiliostreck@gmail.com

Abstract

Major depression is a serious and recurrent disorder often manifested with symptoms at

the psychological, behavioral, and physiological levels. There is a growing body of

evidence, which extends our understanding of depression and antidepressant function

involved the monoaminergic synapse. The paroxetine is a selective serotonin reuptake

inhibitor, the nortriptiline is an antidepressive tricyclic and the venlafaxine is selective

serotonin-norepinephrine reuptake inhibitor. In addition, several works also suggest

brain metabolism impairment as a mechanism underlying depression. Creatine kinase

(CK) plays a central role in metabolism of high-energy consuming tissues such as brain,

where it functions as an effective buffering system of cellular adenosine triphosphate

levels. In the present work, we decided to investigate CK activity from rat brain after

chronic administration of paroxetine, nortriptiline and venlafaxine. Adult male Wistar

rats received daily injections of paroxetine (10 mg/kg), nortriptiline (15 mg/kg),

venlafaxine (10 mg/kg) or saline in 1.0 ml/kg volume for 15 days. Twelve hours after

the last injection, the rats were killed by decapitation, the hippocampus, striatum and

prefrontal cortex were immediately removed. The activity of CK was then measured.

Our results demonstrated that paroxetine chronic administration increased the creatine

kinase activity in prefrontal cortex, hippocampus and striatum of adults rats. On the

other hand, nortriptiline and venlafaxine chronic administration did not affect CK

activity in the structures brain. Considering that metabolism impairment is probably

involved in the pathophysiology of depressive disorders, an increase in CK activity by

antidepressants may be an important mechanism of action of these drugs.

Key words: creatine kinase; brain; paroxetine; venlafaxine; nortriptiline.

1. Introduction

Major depression is a serious and recurrent disorder often manifested with

symptoms at the psychological, behavioral, and physiological levels affecting 17-20%

of the population of the world and may result in premature death, and major social and

economic consequences [20, 35]. It is thought that every fifth individual suffers by a

mood disorder in the lifetime. The World Health Organization estimates that major

depression is the fourth most important cause worldwide of loss in disability-adjusted

life years [22, 24, 30].

Antidepressant drugs are widely used in the treatment of mood disorders and

other conditions. Several effective antidepressants are available but do not work for all

patients, and it is not possible to predict which patient will respond to which drug. It is

worthy of note that the pharmacotherapy of depression is costly and widely prescribed

by physicians, although less than half of treated patients attain complete remission after

therapy with a single antidepressant and others exhibit partial, refractory or intolerant

responses to the pharmacological treatment [27]. Furthermore, it can take several weeks

before a therapeutic response, or lack of response, becomes apparent. During this

period, the patient suffers ongoing morbidity and increased risk of suicide. Therefore,

choosing the appropriate antidepressant for each patient is a significant problem in

psychiatry [17].

The antidepressants have contributed to the improvement of the depressive

patient's quality of life and to make the treatment of depression more satisfactory [7, 13,

16, 29]. Several works suggest brain metabolism impairment as a mechanism

underlying depression. In this context, it has been demonstrated that some

antidepressants may improve energy metabolism [18, 28, 36, 40].

Creatine kinase (CK) plays a central role in metabolism of high-energy

consuming tissues such as brain, where it functions as an effective buffering system of

cellular adenosine triphosphate (ATP) levels. The enzyme catalyzes the reversible

transfer of the phosphoryl group from phosphocreatine to adenosine diphosphate,

regenerating ATP. It is also known that a diminution of CK activity may potentially

impair energy homeostasis, contributing to cell death [1, 6, 11, 12, 39]. In this context,

it has been widely shown that a decrease in CK activity is associated with a

neurodegenerative pathway that results in neuronal loss following brain ischemia [39]

and with neurodegenerative diseases [1, 6] and other pathological states [11, 12]. We

have recently showed that CK is inhibited in animal models of neuropsychiatry

disorders, such as bipolar disorder [37] and after electroconvulsive shock [4].

Therefore, considering that CK plays an important role in brain energy

homeostasis and that some antidepressants may modulate energy metabolism, we

decided to investigate CK activity from rat brain after chronic administration of

paroxetine (selective serotonin reuptake inhibitor), nortriptiline (tricyclic antidepressant)

and venlafaxine (selective serotonin-norepinephrine reuptake inhibitor).

2. Methods

2.1. Animals: Adult and male Wistar rats (250–300 g) were obtained from Central

Animal House of Universidade do Extremo Sul Catarinense. They were caged in group

of five with free access to food and water and were maintained on a 12-h light-dark

cycle (lights on 7:00 am), at a temperature of 23 ± 1oC. All experimental procedures

were carried out in accordance with the National Institutes of Health Guide for the Care

and Use of Laboratory Animals and the Brazilian Society for Neuroscience and

Behavior recommendations for animal care, with the approval of the Ethics Committee

from Universidade do Extremo Sul Catarinense.

2.2. Drugs: Animals received daily intraperitoneal injections of paroxetine (10 mg/kg),

nortriptiline (15 mg/kg) or venlafaxine 10 mg/kg) in 1.0 ml/kg volume for 15 days. All

the drugs were dissolved in saline solution (vehicle). Control animals received the

vehicle (1.0 mL/kg).

2.3. Tissue and homogenate preparation: Twelve hours after the last injection, the

rats were killed by decapitation, the brain was removed and the hippocampus, striatum

and prefrontal cortex were homogenized (1:10, w/v) in SETH buffer, pH 7.4 (250 mM

sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/ml heparin). The homogenates were

centrifuged at 800 × g for 10 min and the supernatants kept at −70oC until used for

enzymes activity determination. The maximal period between homogenate preparation

and enzyme analysis was always less than 5 days. Protein content was determined by

the method described by Lowry and colleagues [25] using bovine serum albumin as

standard.

2.4. Creatine kinase activity: CK activity was measured in brain homogenates pre-

treated with 0.625 mM lauryl maltoside. Briefly, the reaction mixture consisted of 60

mM Tris-HCl, pH 7.5, containing 7.0 mM phosphocreatine, 9.0 mM MgSO4 and

approximately 0.4–1.2 mg protein in a final volume of 100 mL. After 15 min of

preincubation at 37oC, the reaction was then started by the addition of 4.0 mM of ADP

and stopped after 10 min by the addition of 20 mL of 50 mM phydroxy-mercuribenzoic

acid. The creatine formed was estimated according to the colorimetric method of

Hughes [15]. The color was developed by the addition of 100 mL 2% a-naphtol and 100

mL 0.05% diacetyl in a final volume of 1.0 mL and read spectrophotometrically after 20

min at � = 540 nm. Results were expressed as units/min x mg protein.

2.5. Statistical analysis: Data were analyzed by Student’s t test and are expressed as

mean ± standard deviation. All analyses were performed using the Statistical Package

for the Social Science (SPSS) software.

3. Results

In the present work, we evaluated CK activity in rat brain after chronic

administration of paroxetine, nortriptiline and venlafaxine. Our results demonstrated

that paroxetine chronic administration increased CK activity in the prefrontal cortex,

hippocampus and striatum of adults rats. On the other hand, chronic administration of

nortriptiline and venlafaxine did not affect CK activity in these brain areas (Figure 1).

4. Discussion

In the present study, we verified CK activity from rat brain after chronic

administration of paroxetine, nortriptiline and venlafaxine. The

creatine/phosphocreatine/CK system is important for normal energy homeostasis [21,

33] by exerting several integrated functions, such as temporary energy buffering,

metabolic capacity, energy transfer and metabolic control [32]. The brain of adult rats,

like other tissues with high and variable rates of ATP metabolism, presents high

phosphocreatine concentration and CK activity [21, 33].

There is a strong body of evidence suggesting that dysfunction in brain

metabolism is related to neuropsychiatry disorders, such as, depression and bipolar

disorder [2, 19, 23]. In this context, the study of Streck and collaborators [37] showed

that administration of amphetamine inhibited CK activity in brain of rats and mood

stabilizers did not affect the inhibition this enzyme. We have recently showed that mood

stabilizers prevent and reverse inhibitory effect on cytrate synthase activity caused by

amphetamine in an animal model of mania [5]. Damage to the mitochondrial electron

transport chain has been suggested to be an important factor in the pathogenesis of a

range of psychiatric disorders [8, 26], including major depression. Gardner and

colleagues [10] showed a significant decrease of mitochondrial ATP production rates

and mitochondrial enzyme ratios in muscle compared to controls in major depressive

disorder patients. Madrigal and colleagues [26] also reported that complexes I-III and

II-III of mitochondrial respiratory chain were inhibited in rat brain after chronic stress

(immobilization for six hours during 21 days). It has also been recently reported

inhibition of mitochondrial respiratory chain complexes I, III and IV activities after

chronic variety stress for 40 days, suggesting that energy metabolism impairment may

occur in depressive disorders [31].

Energy impairment has been linked to neuronal death and neurodegeneration [3,

14, 34]. Considering that metabolism impairment is probably involved in the

pathophysiology of depressive disorders, an increase in CK activity by antidepressants

may be an important mechanism of action of these drugs. In the present work we

showed that CK activity was increased after chronic administration of paroxetine. On

the other hand, chronic administration of nortriptiline and venlafaxine did not affect CK

activity in the brain of rats. Some other studies also point to the possibility that drugs

used in the treatment of such disorders modulate energy metabolism [4, 9, 38].

Some findings support this hypothesis. It has been demonstrated that Na+,K+-

ATPase activity was affected by fluoxetine and electroconvulsive shock, showing that

important metabolic changes occur after antidepressant therapy. Some evidence also

point to the possibility that drugs used in the treatment of such disorders modulate

energy metabolism [9, 38]. In addition, we have recently showed that CK is inhibited

after electroconvulsive shock [4].

In conclusion, our data corroborate with other studies, suggesting that some

antidepressants drugs modulate brain energy metabolism. Further studies are important

to evaluate whether other enzymes involved in metabolism are also affected by these

antidepressants.

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Figures captions

Figure 1. Effects of paroxetine, venlafaxine, and nortriptiline chronic administration on

creatine kinase activity in the prefrontal cortex, hippocampus, and striatum of rats.

*P<0.01 vs. saline group, according to ANOVA followed by the Tukey test.