UNIVERSIDADE FEDERAL FLUMINENSE
CENTRO DE ESTUDOS GERAIS
INSTITUTO DE BIOLOGIA
PROGRAMA DE NEUROIMUNOLOGIA
ALEXANDRE DOS SANTOS RODRIGUES
ESTUDO DO SISTEMA PURINÉRGICO GLIAL:
MODULAÇÃO DA MAP CINASE E DO
TRANSPORTE DE ADENOSINA EM
CULTURAS DE GLIA DA RETINA
E ASTRÓCITOS CEREBRAIS
TESE SUBMETIDA À UNIVERSIDADE
FEDERAL FLUMINENSE VISANDO A
OBTENÇÃO DO GRAU DE DOUTOR EM
NEUROIMUNOLOGIA
Orientador: Roberto Paes de Carvalho
NITERÓI
2011
INSTITUTO DE BIOLOGIA
PROGRAMA DE NEUROIMUNOLOGIA
Trabalho desenvolvido no laboratório de Neurobiologia Celular do
Departamento de Neurobiologia, Instituto de Biologia, UFF.
Tese de Doutorado submetida à
Universidade Federal Fluminense como
requisito parcial para obtenção de grau
de Doutor em Neuroimunologia.
Orientador: Roberto Paes de Carvalho
Niterói
ii
2011
ALEXANDRE DOS SANTOS RODRIGUES
ESTUDO DO SISTEMA PURINÉRGICO GLIAL:
MODULAÇÃO DA MAP CINASE E DO
TRANSPORTE DE ADENOSINA EM
CULTURAS DE GLIA DA RETINA
E ASTRÓCITOS CEREBRAIS
Tese de Doutorado submetida à
Universidade Federal Fluminense como
requisito parcial para obtenção de grau
de Doutor em Neuroimunologia.
BANCA EXAMINADORA
__________________________________________________________________
Dr. José Luiz Martins do Nascimento – UFPA
__________________________________________________________________
Dra. Olga Maria Martins Silva de Almeida – UERJ
__________________________________________________________________
Dra. Regina Célia Cussa Kubrusly – UFF
__________________________________________________________________
Dra. Elizabeth Giestal de Araujo – UFF (Revisora)
__________________________________________________________________
Dr. Roberto Paes de Carvalho – UFF (Orientador)
__________________________________________________________________
Dra. Mariana Rodrigues Pereira – UFF (Suplente)
Niterói
2011
iii
cod. aa
Rodrigues, Alexandre dos Santos
Estudo do sistema purinérgico glial: Modulação
da MAP cinase e do transporte de adenosina em
culturas de glia da retina e astrócitos cerebrais
/Alexandre dos Santos Rodrigues. – Niterói: [s.n.],
2011. xxp
Tese de Doutorado do Programa de Pós-graduação em
Neuroimunologia – Universidade Federal Fluminense,
2011.
1. Adenosina . 2. MAPK. 3. Células Gliais
4. Retina de Galinha
CDD.: 000.000
iv
Este trabalho foi desenvolvido no Laboratório de Neurobiologia Celular, do Programa de
Neurociências do Instituto de Biologia da Universidade Federal Fluminense sob orientação do Prof.
Roberto Paes de Carvalho e na vigência de auxílios concedidos pelo Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal
de Ensino Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do
Rio de Janeiro (FAPERJ) e pelo Programa de Núcleos de Excelência (PRONEX-MCT).
Parte deste trabalho também foi desenvolvido no Centro de Neurociências e Biologia Celular de
Coimbra, Instituto de Bioquímica da Faculdade de Medicina da Universidade de Coimbra, sob
orientação do Prof. Rodrigo A. Cunha, durante a realização do Doutorado-Sanduíche, no período de
Setembro/2008 à Outubro de 2009.
v
Se
Se és capaz de manter a tua calma quando
Todo o mundo ao teu redor já a perdeu e te culpa;
De crer em ti quando estão todos duvidando,
E para esses no entanto achar uma desculpa;
Se és capaz de esperar sem te desesperares,
Ou, enganado, não mentir ao mentiroso,
Ou, sendo odiado, sempre ao ódio te esquivares,
E não parecer bom demais, nem pretensioso;
Se és capaz de pensar --sem que a isso só te atires,
De sonhar --sem fazer dos sonhos teus senhores.
Se encontrando a desgraça e o triunfo conseguires
Tratar da mesma forma a esses dois impostores;
Se és capaz de sofrer a dor de ver mudadas
Em armadilhas as verdades que disseste,
E as coisas, por que deste a vida, estraçalhadas,
E refazê-las com o bem pouco que te reste;
Se és capaz de arriscar numa única parada
Tudo quanto ganhaste em toda a tua vida,
E perder e, ao perder, sem nunca dizer nada,
Resignado, tornar ao ponto de partida;
De forçar coração, nervos, músculos, tudo
A dar seja o que for que neles ainda existe,
E a persistir assim quando, exaustos, contudo
Resta a vontade em ti que ainda ordena: "Persiste!";
Se és capaz de, entre a plebe, não te corromperes
E, entre reis, não perder a naturalidade,
E de amigos, quer bons, quer maus, te defenderes,
Se a todos podes ser de alguma utilidade,
E se és capaz de dar, segundo por segundo,
Ao minuto fatal todo o valor e brilho,
Tua é a terra com tudo o que existe no mundo
E o que mais --tu serás um homem, ó meu filho!
[Poema “If”, de Rudyard Kipling (1865-1936),
tradução por Guilherme de Almeida]
vi
AGRADECIMENTOS
Dedico esta tese aos meus pais e aos meus familiares, especialmente ao meu tio João Carlos, que sempre me
incentivaram, ajudaram e sugeriram o melhor caminho que eu deveria seguir.
Agradeço ao Roberto Paes de Carvalho, meu orientador aqui na UFF já há alguns anos ... rs, pela pessoa que
é e, pelo exemplo de amor à ciência e ao trabalho, pela dedicação, atenção e incentivo que acredito que seja
não apenas para mim, mas aos muitos alunos que estão aqui e aos que já passaram pelo laboratório.
Ao Rodrigo Cunha, meu obrigado por ter me dado a oportunidade de realizar um doutorado-sanduíche no
seu laboratório, pela oportunidade de crescimento técnico e científico e por tudo o que aprendi sob diversos
pontos de vista.
Agradeço à Prof. Karin Calaza e aos karinérgicos: Raquel, Vivian, Elisa, Daniel, Raul “algo mais?” rs,
Havana pelos bons momentos, pelas discussões nos seminários e pela ótima integração entre todos os alunos
dos dois laboratórios, com amizade e também colaboração científica. Este é o caminho!
Agradeço ao Rafael (“Presida da EPA”), a metamorfose ambulante rs, a figura do lab!, que nestes últimos
anos elevou o nome da adenosina a níveis estratosféricos!!! E a campanha segue ... Só espero que não queira
tornar-se um Hugo Chávez e ficar no trono eternamente! rs.
Agradeço aos mais novos do lab de Neurobiologia Celular e do lab do Marcelo (ou nem já tão novos...):
Nádia e William, pela boa convivência no lab; Felipe, Ivan e Thaísa: ótimas pessoas, animadas e engajadas
no laboratório, já às portas do Mestrado ... sucesso pra vcs!; Marcelo Cossenza, sempre muito gestual e
ilustrativo, que sempre contribuiu e muito com as discussões, e principalmente na resolução de dúvidas
técnicas! Bom prosseguimento e paciência, que em breve os trabalhos começam a sair!
A Camila e Renato, pelos bons momentos vividos no lab, as comemorações, ao bom desenvolvimento
científico e muito aprendizado no período em Coimbra.
A Mariana e Eliza, obrigado pelo convívio, pela colaboração, por terem lidado com o “pepino da glia” rs que
foi pra vocês conseguirem as culturas em 2009! Sucesso na caminhada!
A “multifuncional” Sarah (estudante da UFRJ, técnica de lab da UFF, IC do lab, que mais? rs), que mesmo
com tudo isso ainda conseguiu me ajudar na parte experimental em 2010. Sucesso pra você, muito obrigado
e vamos manter a caminhada!
A Luzeli, grande pessoa e importante no lab, uma tremenda figura, sempre animada e querendo trabalhar
contra o chefe! “Não suja nada aí, hein!” rs. Brincadeiras a parte, obrigado pelo companheirismo, pela
amizade e pela admiração e vou sempre tentar retribuir a altura.
vii
Aqueles que passaram pelo lab e não puderam ou resolveram seguir outros caminhos: Rodrigo, Rochele,
Igor, Daniel, Cristiane, Telmo, Ana, Octavia; obrigado pelos bons momentos. Quem sabe um dia seja
possível um grande encontro?
A Jainne, que me ajudou muito na minha fase inicial no laboratório e pela grande pessoa que é. Apesar da
distância, torço pra que esteja bem e siga na luta!
Aos professores e estudantes do depto. de Neurobiologia pelas boas relações e pela integração,
principalmente nos churrascos! rs. Um abraço especial para o Pablo (sempre resolvendo os bugs de
informática!), Paulo Emílio (colega de trabalho e de apartamento, o homem das frases e gestos simples! rs),
Liana (os diálogos de longa duração ... rs), Aninha e Ísis, a dupla que vem de longe!
A profa. Beth, por ter aceitado ser a revisora da tese com o cenário que apresentei pra ela em Novembro rs!
Aos colegas de moradia em Coimbra (Pablo, Britta, Maria e Cindy)! Vocês foram ótimos, obrigado pela
atmosfera de respeito e os momentos de integração que todos tiveram um para com o outro. Até um dia
destes...!
A todos os integrantes do LEF ou Purines at CNC, enfim! (Ângelo, Attila, Carla, Henrique, Manu, Nuno,
Paula Canas, Pedro, Rui, Samira, Paula Agostinho, Rodrigo), sempre existirão diferenças, é lógico, mas
muito obrigado pelo bom espírito de grupo, de ajuda que encontrei aí. Isso foi essencial e com certeza, não é
tudo, mas é importantíssimo para o sucesso pessoal e profissional de do grupo e de todos. Quem sabe um dia,
venhamos a nos encontrar por aí!
Ao Marco, colega direto de bancada e grande companheiro, sempre de bom humor (ok, nem sempre rs) e de
frases clássicas, do tipo: “tá tudo...” censurado! rs. Muito obrigado pela maneira como fui recebido e pela
integração que recebi até o último dia aí em Coimbra. A gente se vê! “Falou?”;
Ao Pablo Pandolfo, seguidor de “Vig An", grande pessoa, bem-humorado, super tranqüilo de convivência e
que sabe tudo de comportamento animal.
À Betty, Carla e Patrícia, que trio! Vocês são incansáveis e possuem muita força interior. Desejo tudo de
bom pra vocês! Muito obrigado pelos papos, cafés, jantares e por toda força durante todo este tempo.
À todo o corpo técnico e estudantes do Centro de Neurociências de Coimbra (CNC), da Universidade de
Coimbra, que contribuíram sob distintas formas.
viii
SUMÁRIO
LISTA DE ABREVIATURAS
x
RESUMO
xviii
ABSTRACT
xviv
1. INTRODUÇÃO
01
1.1 Adenosina, seus receptores e seu metabolismo
01
1.2 Transportadores de nucleosídeos
03
1.3 Importância terapêutica dos Transportadores de Nucleosídeos
06
1.4 MAPKs
07
1.4.1 ERKs
08
1.5 Retina, um modelo biológico de estudo
09
1.6 Regulação das ERKs por adenosina
12
1.7 Adenosina na retina
13
1.8 Transporte de Adenosina e Regulação por vias de sinalização
17
1.9 Modulação dos níveis de adenosina pela Adenosina Cinase
21
1.10 Astrócitos corticais
24
2. OBJETIVOS
26
3. MANUSCRITOS
27
3.1 “Modulation of ERK phosphorylation by A1 adenosine receptor
27
in cultures of avian retinal glial cells: Involvement of PKC and Src
cinase.”
3.2 “Modulation of adenosine transport by MAP kinase cascade in
62
cultured avian retinal cells.”
3.3 “Equilibrative Nucleoside Transporter 1 (ENT1)-mediated
90
adenosine transport in cultures of rat cortical astrocytes:
Modulation by activation of adenosine A1 receptors.”
4. DISCUSSÃO
117
5. PERSPECTIVAS
126
6. REFERÊNCIAS BIBLIOGRÁFICAS
127
ix
LISTA DE ABREVIATURAS
ADA – adenosina deaminase
ADP - adenosina difosfato
AMP - adenosina 3’-5’ monofosfato
AMPc - adenosina 3´-5´monofosfato cíclico
ATP - adenosina 5´-trifosfato
BDNF - fator neurotrófico derivado do cérebro
Bmax – ligação máxima
BSA - albumina de soro bovino
C3 - terceiro dia de cultura
C4- quarto dia de cultura
C21- vigésimo primeiro dia de cultura
CAMKII - Proteína cinase II dependente de cálcio e calmodulina
CKII- caseína cinase II
CMF - solução sem cálcio e magnésio
CNT- tranportador concentrativo de nucleosídeos
CPM - contagens por minuto
CREB – proteína ligante ao elemento de resposta ao AMPc
DAT – transportador ativo de dopamina
DIV – dias in vitro
EHNA - eritro-9-(2-hydroxi-3-nonil) adenina
ENT- transportador equilibrativo de nucleosídeos
EPSC – corrente excitatória pós-sináptica
ERK – proteína cinase ativada por sinais extracelulares
GABA - ácido gama amino butírico
GLUT – transportador de glicose
x
Gi - proteína G inibitória
Gs - proteína G estimulatória
GSK3β - cinase 3β da glicogênio sintase
GTP- guanosina trifosfato
IFN-γ – interferon gama
IP3 – inositol trifosfato
JNK – proteína cinase c-Jun NH2-terminal
Kd – constante de dissociação
Km – constante de Michaelis
LPS - lipopolissacarídeo
LTP – potenciação de longa duração
MAP – proteína associada ao microtúbulo
MAPK – proteína cinase ativada por mitógeno
MEK – MAP cinase cinase
MEM - meio mínimo essencial
mRNA - ácido ribonucléico mensageiro
NBTI - S-(Nitrobenzil)-6-tioinosina
NMDA – N-metil-D-aspartato
NO- óxido nítrico
NOS- óxido nítrico sintase
PI3K – cinase do fosfatidil inositol na posição 3
PLC – fosfolipase C
PKA – proteína cinase dependente de AMPc
PKC – proteína cinase dependente de Ca+2
Raf – MAP cinase cinase cinase
Ras – proteína G monomérica
xi
SAH - S-adenosilhomocisteína
SAPK – proteína cinase associada a sinais de estresse
SDS - dodecil sulfato de sódio
SFB – soro fetal bovino
SNC – sistema nervoso central
SOS – fator de troca de nucleotídeo
Src- proteína tirosina cinase citoplasmática purificada do sarcoma da retina de pinto
TBS - solução tamponada de Tris
TBS-T - solução tamponada de Tris acrescida de tween-20
xii
RESUMO
Adenosina (Ado) é um importante neuromodulador no SNC com funções na transmissão sináptica e
em processos de neuroproteção. Atua através dos receptores A1, A2A, A2B e A3, que estimulam
diferentes vias de sinalização incluindo a cascata das MAPKs, a qual tem importantes papéis em
várias funções neurais e gliais. Células gliais desempenham importante papel na regulação dos
níveis intra e extracelulares de adenosina. O objetivo deste trabalho foi caracterizar os sistemas de
captação de Ado em culturas mistas e purificadas de glia de retina de galinha e culturas de
astrócitos corticais. Investigamos também os mecanismos de modulação da captação de Ado pela
ERK e a regulação da ERK por receptores A1 em culturas purificadas de glia. NBTI, um inibidor
dos transportadores equilibrativos de nucleosídeos, promoveu uma forte redução da captação nos
três tipos de culturas utilizadas neste estudo. Pré-incubação de [3H] Ado com adenosina deaminase
e inibição da adenosina cinase com iodotubercidina também promoveram forte inibição da captação
nas diferentes culturas. Em culturas mistas e gliais retinianas, inibição da ERK com PD98059 ou
U0126, diferentes inibidores da MEK, reduziu fortemente a captação de Ado, mas U0124, um
análogo inativo do U0126, não teve efeito. Glutamato induziu a liberação de purinas das culturas
mistas, mas este efeito não foi significativamente inibido por PD98059. Em culturas de astrócitos
corticais, ativação dos receptores A1 com o agonista seletivo CPA diminuiu a captação de [3H] Ado
e aumentou o Kd de ligação do [3H] NBTI. Em conclusão, nossos resultados identificam ENT1
como o principal transportador de adenosina em culturas de retina e de astrócitos cerebrais e, tendo
em vista que a ERK é regulada pela estimulação dos receptores A1 na retina, nossos resultados
sugerem que a adenosina regule a atividade do próprio transportador via ativação de receptores A1
e da via da ERK.
xiii
ABSTRACT
Adenosine (Ado) is an important neuromodulator in the CNS, regulating synaptic transmission and
processes of neuroprotection. Ado acts through A1, A2A, A2B and A3 receptors, and regulate
different signaling pathways including MAPKs cascade which has important roles in several neural
and glial functions. Glial cells are well known regulators of intra or extracellular adenosine levels.
The aim of this work was to characterize the Ado uptake systems in chicken retinal cultures and
cortical astrocytes cultures. We also investigated the mechanisms of transport modulation by ERK
and the regulation of ERK by A1 receptors in purified glial cultures. NBTI, an inhibitor of
equilibrative nucleoside transporters, promoted a strong reduction of uptake in all three types of
cultures used in this study. Preincubation of [3H] Ado with adenosine deaminase and adenosine
kinase inhibition with iodotubercidin also promoted a strong inhibition of uptake in different
cultures. In retinal glial and mixed cultures, ERK inhibition with the different MEK inhibitors
PD98059 and U0126 strongly reduced Ado uptake whereas U0124, a UO126 inactive analog, had
no effect. Glutamate induced the release of purines in mixed cultures, but this effect was not
significantly inhibited by PD98059. In astrocytes cortical cultures, activation of A1 receptor with its
selective agonist CPA decreased [3H] Ado uptake and increased the Kd of 3H-NBTI binding. In
conclusion, these data identify ENT1 as the main adenosine transporter in retinal and astrocytes
cultures and based on the fact that ERK is regulated by stimulation of A1 receptors in the retina, our
results suggest that adenosine regulates its own transporter activity via activation of A1 receptors
and the ERK pathway.
xiv
1. INTRODUÇÃO
1.1 Adenosina, seus receptores e seu metabolismo
A adenosina é um nucleosídeo composto de uma base purínica, a adenina, e de uma
pentose, a ribose. A adenosina é referida classicamente como uma substância
neuromoduladora e não como um neurotransmissor clássico. Essa classificação se deve ao
fato de um grande número de evidências indicarem que a adenosina não é armazenada e nem
liberada pela fusão de vesículas sinápticas às membranas dos terminais pré-sinápticos.
Contudo, é importante ressaltar que recentemente alguns relatos tem sugerido que a
adenosina possa ser liberada por uma
dinâmica similar em algumas características a
liberação de neurotransmissores clássicos (Wall & Dale, 2007; Klyuch et al., 2010).
A adenosina, presente no meio extracelular, vai atuar através da interação com seus
receptores metabotrópicos, que foram subdivididos em quatro subtipos: A1, A2A, A2B e A3
(Schulte & Fredholm, 2003; Fredholm et al., 2005). Classicamente, os receptores A1 e A3
estão acoplados à proteína Gi, e por meio desta inibem a enzima adenilil ciclase, o que leva à
diminuição da formação do segundo mensageiro AMPc. Já os receptores A2A e A2B
geralmente estão acoplados à proteína GS, e com isso aumentam a atividade da adenilil
ciclase, induzindo aumento nos níveis de AMPc (Schulte & Fredholm, 2003; Fredholm et
al., 2005). Cabe salientar que, em sistemas celulares onde foi feita a transfecção dos
receptores de adenosina, foi possível observar o acoplamento destes receptores a outras
proteínas G, o que sugere existir a possibilidade de sinalização, que não a clássica, para os
receptores de adenosina (para revisão, ver (Schulte & Fredholm, 2003)).
De um modo geral, a ativação de receptores A1 induz neuroproteção (Johansson et
al., 2001). Dados da literatura correlacionam o efeito neuroprotetor da adenosina à
1
diminuição da liberação de aminoácidos excitatórios, à hiperpolarização da membrana
neuronal, à queda no influxo de cálcio e ao decréscimo da formação de radicais livres (de
Mendonca et al., 2000; Boison, 2008).
Em eventos isquêmicos, acredita-se que o principal efeito neuroprotetor da adenosina
seja devido à ativação dos receptores pré-sinápticos A1 que leva à diminuição da liberação de
glutamato (Ribeiro et al., 2002). No entanto, os receptores A1 de adenosina também podem
ter efeitos relacionados à excitotoxicidade. Já foi demonstrado que a ativação dos receptores
A1, na presença de adenosina desaminase, participa da neurotoxicidade induzida pelo kainato
(Rebola et al., 2005).
Quanto aos receptores A2A, também existem relatos desta dualidade de efeitos. Além
de efeitos neuroprotetores (Ferreira & Paes-de-Carvalho, 2001), também já foi visto que estes
receptores podem estar envolvidos em modificações sinápticas induzidas por stress no
hipocampo (Cunha et al., 2006). Desta maneira, podemos sugerir que o contexto celular seja
determinante para os efeitos mediados tanto pelo receptor A1 como pelo receptor A2A.
Os níveis intracelulares de adenosina são regulados pela: captação deste nucleosídeos
feita pelos seus transportadores ou pela síntese desta purina que é feita pela enzima 5´nucleotidase a partir do AMP. Já a concentração extracelular de adenosina está diretamente
correlacionada à atividade de seus transportadores, à degradação de nucleotídeos de adenina
liberados pelos transportadores de nucleotídeos, à atividade das ecto-nucleotidades e à
conversão do ATP liberado na fenda sináptica (Dunwiddie et al., 1997).
As principais enzimas responsáveis pela metabolização da adenosina são a adenosina
cinase, que gera o AMP e a adenosina deaminase, que metaboliza adenosina em inosina
(Latini & Pedata, 2001).
2
1.2 Transportadores de nucleosídeos
Os transportadores bidirecionais de nucleosídeos desempenham importante papel no
controle dos níveis intra e extracelulares de adenosina. De uma maneira geral, eles são
divididos em transportadores equilibrativos e concentrativos e se constituem em proteínas
integrais de membrana responsáveis pelo transporte de adenosina, pelo transporte de outros
nucleosídeos, de nucleobases e de drogas que sejam análogos de nucleosídeos (Kong et al.,
2004).
Os transportadores equilibrativos realizam suas funções de acordo com os níveis de
nucleosídeos nos meios intra e extracelulares. Já os transportadores concentrativos, como o
nome já sugere, promovem o influxo de nucleosídeos contra o gradiente de concentração,
usando para tal a energia derivada do gradiente de concentração de sódio existente nas
membranas celulares (Podgorska et al., 2005).
Os transportadores equilibrativos dividem-se em sensíveis e insensíveis ao inibidor
nitrobenziltioinosina (NBTI), sendo que os sensíveis são inibidos nas concentrações
nanomolares desta droga, enquanto que os insensíveis são resistentes a concentrações de até 1
µM (Podgorska et al., 2005). Atualmente existem descritos 4 subtipos de transportadores
equilibrativos, que são denominados ENT1, ENT2, ENT3 e ENT4, sendo que apenas o ENT1
e o ENT3 são sensíveis ao NBTI, sendo este último bem menos susceptível à inibição
(Podgorska et al., 2005). Estes transportadores podem se apresentar na forma glicosilada e
possuem 11 domínios transmembrana. Sua localização no plano da membrana se dá da
seguinte maneira: a cauda N-terminal está no citoplasma e a cauda C-terminal se localiza no
espaço extracelular e possui uma grande alça extracelular entre os domínios transmembrana 1
e 2 e uma grande alça intracelular que interliga os domínios transmembrana 6 e 7 (Figura 1)
(Sundaram et al., 1998)(para revisão, (Baldwin et al., 2004)).
3
Os transportadores concentrativos são divididos em 3 subtipos (CNT1, CNT2 e
CNT3). Eles apresentam 13 domínios transmembrana dispostos com a cauda N-terminal
citoplasmática e a cauda C-terminal localizada no espaço extracelular e podem apresentar
glicosilação (Kong et al., 2004).
Figura 1: Representação esquemática do ENT1 de camundongos (Retirado de Kiss et al., 2000)
A ligação do NBTI ao ENT1 se dá com alta afinidade (Kd 1-10 nM) através de uma
interação não-covalente, no sítio de ligação de alta afinidade para a adenosina nele presente.
Por outro lado, o ENT2 não é afetado pelo NBTI em concentrações nanomolares e somente
torna-se inibido com altas concentrações de NBTI (> 10 µM) (Kong et al., 2004). Acredita-se
que o NBTI interaja na região compreendida entre a 3a e a 6a região transmembrana em um
sítio de reconhecimento ao substrato no lado extracelular (Baldwin et al., 2004).
Também já foi demonstrado que o ENT1 é incapaz de transportar nucleobases, como
por exemplo, adenina, guanina e hipoxantina. No entanto, o ENT2 também transporta essas
nucleobases. As regiões transmembrana 5 e 6 do ENT2 parecem ser importantes para o
reconhecimento das nucleobases, pois a inserção dessa região de uma isoforma de ENT2 de
4
rato para uma isoforma de ENT1 de rato fez com que este transportador passasse a ter
habilidade para o transporte de nucleobases (Kong et al., 2004).
O ENT1 é um transportador amplamente expresso na maioria dos tipos celulares,
encontrado principalmente na membrana plasmática, sendo considerado o principal regulador
para a manutenção dos níveis de adenosina dentro da faixa fisiológica (Bone et al., 2007).
Existem relatos de clonagem deste transportador a partir, pelo menos, de tecidos de ratos,
camundongos, humanos e de cães (Hammond et al., 2004). Dados da literatura demonstram
que a inibição do ENT1 pode potenciar os efeitos neuroprotetores e cardioprotetores
provocados pela adenosina quando em situações de injúria (Bone & Hammond, 2007).
Existem descritas duas isoformas de ENT1 em camundongos, denominadas de
mENT1a e mENT1b (Kiss et al., 2000; Bone et al., 2007). As únicas diferenças entre essas
isoformas estão localizadas na alça intracelular central que conecta as regiões transmembrana
6 e 7, sendo que o mENT1b tem uma serina na posição 254 seguida por uma seqüência lisinaglicina, enquanto que o mENT1a possui uma arginina na posição 254 e a seqüência lisinaglicina está deletada nesta isoforma. A Ser254 é parte de uma potencial seqüência consenso
para fosforilação pela caseína cinase II (CK II). Esta característica diferencial entre essas
isoformas de transportadores, sugere a possibilidade de uma modulação diferenciada em
relação à CK II (Bone et al., 2007).
Animais que não expressam os transportadores de nucleosídeos equilibrativos (ENT1)
demonstraram uma tendência a um maior consumo de álcool. Este efeito parece ser devido à
redução da inibição das correntes excitatórias de glutamato mediadas pelo receptor A1 no
núcleo accumbens, região importante na regulação da auto-administração e no sistema de
recompensa no uso de drogas (Choi et al., 2004). (Jennings et al., 2001) demonstraram que
existe uma co-localização entre ENT1 humano e receptores A1 em diversas estruturas do
5
cérebro, o que sugere que esse transportador possua um papel importante no controle da ação
neuromoduladora dos receptores A1 de adenosina.
1.3 Importância terapêutica dos transportadores de nucleosídeos
Sendo os nucleosídeos necessários para a síntese de nucleotídeos, células em processo
de divisão celular e ou com alta taxa metabólica apresentam uma grande demanda por essas
moléculas (Abdulla & Coe, 2007). A importância fisiológica dos nucleosídeos como
substratos para a síntese de ácidos nucléicos tem levado ao desenvolvimento de compostos
análogos de nucleosídeos, que são úteis para o tratamento de alguns tipos de cânceres e
infecções virais. Isso se deve ao fato dos análogos de nucleosídeos serem anti-metabólitos,
pois uma vez fosforilados interferem com a síntese de novos nucleosídeos e na biossíntese de
nucleotídeos levando à indução da apoptose as células tratadas com estas drogas (para
revisão, (Kong et al., 2004)). O mecanismo geral de ação destes compostos é mostrado
abaixo (Figura 2). Estes análogos são tipicamente hidrofílicos e requerem proteínas
transportadoras para poder entrar nas células. Muito pouco ainda é conhecido sobre a
estrutura, função e regulação dos transportadores, e por isso são necessários mais estudos que
venham a permitir uma melhor otimização dos tratamentos quimioterapêuticos que são
baseados nos análogos citados acima (Abdulla & Coe, 2007).
6
Figura 2: Mecanismos de ação dos análogos de nucleosídeos (NA) anti-virais e anti-cânceres (Retirado de
Kong et al., 2004) Legendas: NAMP, NADP e NATP são análogos de nucleosídeos mono, di e trifosforilados, respectivamente. dCK:deoxicitidina cinase
1.4 MAPKs
As MAPKs (Figura 3) constituem uma família de proteínas cinases que atuam
promovendo a fosforilação de proteínas em resíduos de serina e de treonina. Estas enzimas
são efetores finais de módulos de cinases seqüenciais bem conservados, que podem ser
ativados por receptores acoplados às proteínas G ou por receptores tirosina cinases. As
MAPKs podem ser divididas em três sub-famílias principais: ERKs (proteínas cinases
reguladas por sinais extracelulares), p38 e JNK. Esta última é conhecida também como
proteína cinase ativada por sinais de estresse (SAPKs) (Schulte & Fredholm, 2003), embora à
ativação da p38 também tenha sido relacionada ao estresse.
7
Figura 3: Vias de sinalização simplificadas das MAP cinases (Modificado a partir de um esquema obtido da
Cia. Cell Signaling).
1.4.1 ERKs
A sub-família das ERKs é constituída por seis diferentes isoformas: ERK1, ERK2,
ERK3α, ERK3β, ERK5 e ERK7. As ERKs mais bem estudadas são ERK1, ERK2 e ERK5.
No sistema nervoso, as ERKs 1/2, também conhecidas como p44 MAPK e p42 MAPK,
respectivamente, estão geralmente relacionadas com a regulação de diversos processos
celulares, tais como proliferação, diferenciação e plasticidade sináptica (Sweatt, 2001;
Schulte & Fredholm, 2003; Thomas & Huganir, 2004).
As ERKs 1/2 podem fosforilar alvos extra-nucleares tais como as proteínas cinases
ribossomais S6 (RSKs), que então translocam-se para o núcleo. As próprias ERKs também
podem translocar-se para o núcleo e possuem como alvos principais: fatores de transcrição
como Elk1 e AP-1 e as cinases ativadas por sinais de stress e mitógenos (MSKs), que estão
constitutivamente presentes no núcleo. Além destes substratos, estas enzimas também podem
catalisar a fosforilação de diferentes substratos citoplasmáticos e proteínas do citoesqueleto
8
(Para revisão sobre alvos das ERKs, ver (Yoon & Seger, 2006)). Já é conhecido que as ERKs
podem fosforilar os canais de potássio dependentes de voltagem (KV4.2) localizados nos
dendritos (Adams et al., 2000; Morozov et al., 2003). A fosforilação deste canal provoca
uma redução da condutância, que está relacionada à formação da memória de longa duração
(Morozov et al., 2003).
Também já foi visto que a despolarização neuronal induz um aumento da formação
dos dendritos em neurônios simpáticos, por um mecanismo dependente da ativação das ERKs
1/2 (Vaillant et al., 2002). Neste mesmo trabalho, eles observaram
que estas enzimas
aumentavam à fosforilação da proteína associada ao microtúbulo (MAP2) (Vaillant et al.,
2002). Outro trabalho já relatou que a ERK fosforilada pode associar-se com a vimentina e
isto mostrou-se importante para a translocação retrógrada da ERK para uma região de lesão
no nervo ciático (Perlson et al., 2005). (Chuderland et al., 2008) demonstraram que em
células quiescentes as ERKs 1/2 estariam ancoradas a diferentes proteínas citoplasmáticas e
na presença de um estímulo, estas cinases seriam liberadas destes ancoradores e poderiam
exercer os diferentes eventos de sinalização celular, como por exemplo, a translocação
nuclear, que mostrou-se ser regulada pelos níveis citosólicos de cálcio.
1.5 Retina, um modelo biológico de estudo
A retina (Figura 4) de pinto é um excelente modelo para estudos biológicos e
bioquímicos do desenvolvimento neuronal (Coulombre, 1955). A retina possui a mesma
origem embrionária das estruturas do SNC sendo um tecido de fácil acesso cujas células
apresentam em cultura muitas propriedades neuroquímicas encontradas no tecido in vivo
(Paes de Carvalho & de Mello, 1982; Paes de Carvalho et al., 1990; Kubrusly et al., 2005).
9
A retina madura de pinto é composta de seis tipos celulares e também já é bem
caracterizado o padrão de gênese, a nível temporal destas células retinianas, a saber: células
ganglionares, células horizontais, fotorreceptores do tipo cone, células amácrinas, e por
último as células bipolares e a glia de Müller, levando à formação das camadas celulares da
retina madura (Prada et al., 1991)(para revisão, (Martins & Pearson, 2008)).
Figura 4: Representação dos tipos e camadas celulares distintas de uma típica retina de vertebrados. PRS:
segmentos externos dos fotorreceptores; ONL: camada nuclear externa; OPL: camada plexiforme externa;
INL: camada nuclear interna; IPL: camada plexiforme interna; GCL: camada de células ganglionares; BV:
vasos sanguíneos; P: pericitos; G: ganglionares; AS: astrócitos; E: pés terminais da glia de Muller; A:
amácrinas; H: horizontais; B: bipolares; M: células de Muller; R: bastonetes; C: cones; RPE: epitélio
pigmentado da retina (Retirado de Bringmann et al., 2009).
O tipo majoritário de célula glial encontrada na retina de pinto é a glia de Müller. Os
oligodendrócitos e os astrócitos não são encontrados neste tecido (Newman & Reichenbach,
1996; Bringmann et al., 2009). A glia de Müller executa muitas funções similares aquelas
10
realizadas pelos astrócitos, oligodendrócitos e células ependimais em outras regiões do
sistema nervoso central (SNC). Sua localização percorre desde a membrana limitante interna
até a membrana limitante externa, estando os corpos celulares localizados na camada nuclear
interna. Erroneamente, estas células foram inicialmente consideradas apenas como células de
suporte neuronal, no entanto, nos últimos 15 anos, foi demonstrado que a glia de Müller é
importante em diferentes contextos, tais como processos de migração celular, modulação da
transmissão sináptica, remoção de debris celulares e como uma fonte geradora de novos
neurônios em processos degenerativos (Fischer & Reh, 2003; Newman, 2003b).
A glia de Müller expressa diversos receptores e transportadores de neurotranmissores,
receptores purinérgicos e canais iônicos dependentes de voltagem (Kubrusly et al.,
2005)(para revisão, (de Melo Reis et al., 2008)). As células gliais estão envolvidas na
modulação da transmissão sináptica. Neurotransmissores podem ativar receptores em células
gliais; bem como podem induzir a liberação de neurotransmissores por células da glia, que
irão ativar receptores presentes nos neurônios (Newman, 2003b, a).
Também foi demonstrado que a glia de Müller em cultura, na ausência de neurônios, é
capaz de expressar marcadores dopaminérgicos, o que sugeriu que a comunicação neurônioglia na retina intacta seja uma característica essencial para a glia de Müller manter seu
fenótipo fisiológico (Kubrusly et al., 2008).
Na literatura, existem alguns relatos que demonstram a presença das ERKs 1/2
fosforilada na glia de Müller in vivo. A injeção intravítrea de fatores tróficos, tais como
BDNF, CNTF ou FGF2, induz um aumento temporal na fosforilação de ERK em retinas de
camundongos C57BL/6J (Wahlin et al., 2000). Em um modelo de inflamação ocular,
induzida por LPS, também já foi demonstrado que ocorre um aumento da fosforilação de
ERK na glia de Müller (Takeda et al., 2002). Em processos isquêmicos induzidos pelo
aumento na pressão intraocular, observa-se um aumento nos níveis de p-ERK na glia de
11
Müller. Neste mesmo trabalho foi igualmente observado que o bloqueio da ERK conferia
uma proteção significativa em relação ao dano induzido pelo evento isquêmico (Roth et al.,
2003).
No entanto, que nosso grupo tenha conhecimento, não há nenhum trabalho que
correlacione ativação de ERKs por receptores de Ado na glia de Müller de retina de galinha,
o que nos motivou a analisar se isso também ocorria.
1.6) Regulação das ERKs por adenosina
Na literatura existem diversos relatos que correlacionam a fosforilação de MAPKs
mediada por ativação de receptores de adenosina. A ativação das ERKs ½, mediada pelo
receptor A1, em células CHO se dá de maneira dose e tempo dependentes e envolve a enzima
MEK, pois o inibidor desta enzima, PD 98059, bloqueou completamente o efeito
estimulatório induzido pela ativação do receptor A1. O bloqueio da proteína Gi pela toxina
pertussis, o bloqueio das tirosinas cinases com genisteína e a inibição da PI3-cinase por LY
294002 ou Wortmannina também impediram o efeito da adenosina, indicando também o
envolvimento destas vias (Dickenson et al., 1998). Em outro estudo, (Robinson & Dickenson,
2001) identificaram que, na linhagem de células de músculo liso DDT1MF-2, a regulação das
ERKs 1/2 é semelhante à observada em células CHO, mas a fosforilação é independente de
tirosina cinases. Também já foi visto que a ativação das ERKs 1/2 induzida pelos receptores
A1 é importante para a maturação dos espermatozóides murinos. Este efeito é mediado pelo
influxo de cálcio, pela PKC e pelas proteínas Gαi2 and Gq/11 (Minelli et al., 2008). Outro
trabalho relacionado à ativação de ERK pelos receptores A1 também observou a dependência
de PKC e da fosfolipase C em uma linhagem celular de músculo liso CASMCs (Ansari et al.,
12
2009). Assim, mais uma vez podemos observar que a ativação das ERKs após estimulação
dos receptores A1 depende do contexto celular.
(Schulte & Fredholm, 2000) transfectaram todos os subtipos de receptores de
adenosina em células CHO e através do agonista não seletivo de receptores de adenosina
NECA demonstraram que a ativação de qualquer subtipo do receptor é capaz de aumentar a
fosforilação de ERK nestas células. (Germack & Dickenson, 2004) caracterizaram as cascatas
de sinalização de ERKs induzidas por ativação de receptores de adenosina em cardiomiócitos
de ratos neonatos, e demonstraram que as ERKs podem ser ativadas por qualquer um dos
subtipos de receptores de adenosina. Além disso, mostraram que a fosforilação das ERKs
mediada por receptores A3 parece ser dependente de PLC e PKC, que mediariam a ativação
da adenilil ciclase que então seria responsável pelo disparo da via de sinalização das MAPKs.
(Schulte & Fredholm, 2002) caracterizaram que a fosforilação das ERKs em células CHO
transfectadas com o receptor A3 depende da proteína Gi , da subunidade βγ, Ras, PI3-kinase
e MEK.
(Arslan & Fredholm, 2000) demonstraram, em células PC12, que a ativação do
receptor A2a induz aumento da fosforilação das ERKs 1/2 de maneira dose dependente.
Outros estudos também identificaram a ativação das MAPKs por receptores A2a e
caracterizaram os alvos intermediários da sinalização. Um exemplo é o estudo que
demonstrou que em células CHO a via de sinalização envolve pelo menos a participação da
Gαs, adenilil ciclase, PKA, Rap1, B-Raf e MEK, enquanto que em células HEK 293 a via de
sinalização está associada com ativação da Ras (Seidel et al., 1999).
1.7 Adenosina na retina
13
Diversos trabalhos na literatura relatam a presença de adenosina, seus receptores e
transportadores em retinas de diferentes espécies (Schaeffer & Anderson, 1981; Braas et al.,
1987; Blazynski, 1991).
A presença de um sistema de captação de adenosina em retinas de peixe-dourado
(Studholme & Yazulla, 1997), em retinas de coelhos (Perez et al., 1986), em retinas de ratos
(Schaeffer & Anderson, 1981) e em retinas de galinha (Perez & Bruun, 1987; Paes de
Carvalho et al., 1990), dentre outros, já foi demonstrada.
Na retina de pinto, foi demonstrada a presença de transportadores de nucleosídeos,
através da técnica de binding com o [3H] NBTI, a partir do 80 dia embrionário até animais
pós-eclosão (de Carvalho et al., 1992). Neste mesmo trabalho foi demonstrado que nos
estágios mais tardios do desenvolvimento, os sítios de transportadores estavam localizados
predominantemente nas camadas plexiformes, tendo uma localização similar à encontrada
para os receptores A1 (Paes de Carvalho, 1990). Essa co-localização também já havia sido
demonstrada em outras estruturas do SNC (Jennings et al., 2001), que propuseram estar esta
co-localização associada a uma atividade modulatória do transportador sobre a ativação dos
receptores A1.
Em retinas embrionárias de pinto foi demonstrado que adenosina eleva a produção de
AMPc, sendo o maior efeito observado no 17o dia embrionário. É importante enfatizar que o
mesmo efeito fora também observado em culturas mistas de células de retina de embrião de
pinto (Paes de Carvalho & de Mello, 1982). Em um outro estudo, foi caracterizado que entre
o 10o dia e o 20o dia de desenvolvimento da retina de pinto, a ativação do receptor A1 é capaz
de inibir o acúmulo de AMPc induzido pela dopamina (Paes de Carvalho & de Mello, 1985).
A presença do receptor A1 na retina em desenvolvimento foi estudada através do ensaio de
ligação de [3H]CHA, um agonista seletivo do receptor A1, demonstrando a presença de
14
receptores em diversos estágios de desenvolvimento, sendo que no 17o dia foram encontrados
os níveis mais elevados deste receptor (Paes de Carvalho, 1990).
(Perez et al., 1986) demonstraram que a maior parte da [3H]–adenosina captada pelas
células da retina de coelhos é convertida em nucleotídeos de adenina. Após um estímulo
despolarizante ocorre um aumento da liberação de purinas, na forma majoritária de
hipoxantina, xantina e inosina. Este efeito foi inibido pelo dipiridamol (inibidor de
transportador equilibrativo de nucleosídeos) demonstrando que em parte esta liberação se dá
pelo transportador na forma de nucleosídeos.
Em culturas purificadas de neurônios e fotorreceptores da retina de pinto,
demonstrou-se a existência de um sistema de captação específico de alta afinidade para
adenosina. Sob um estímulo despolarizante ocorre aumento da liberação de purinas, sendo
que a maior parte da radioatividade liberada é encontrada na forma de inosina (Paes de
Carvalho et al., 1990). Neste mesmo trabalho, a incubação das culturas com um inibidor do
ENT1, NBTI (10 nM), provocou uma inibição da captação de mais de 80%, o que indica que
o ENT1 é o principal, senão o único, transportador presentes nestas células. Na concentração
utilizada de NBTI, há apenas o bloqueio do ENT1 e não do ENT2. Foi também demonstrado
que a captação era independente de íons sódio e que a captação de adenosina era bloqueada
por uma prévia incubação com adenosina deaminase, enzima que converte adenosina em
inosina, o que indica que este transportador não carreia inosina. Também nessas culturas
purificadas de neurônios, foi observado que o neurotransmissor dopamina é capaz de
aumentar a liberação de purinas (Figura 5).
15
Figura 5: Liberação de purinas estimulada por dopamina em culturas purificadas de neurônios de retina de
pinto (Retirado de Paes-de-Carvalho, 2002)
Em culturas mistas (neurônios + células gliais) de retina de pinto, já foi demonstrada a
presença do transportador ENT1, através da técnica de binding com a utilização do [3H]NBTI, o ligante de alta afinidade deste transportador (Paes-De-Carvalho, 2002). Também foi
visto, nas mesmas culturas mistas, que a incubação crônica de adenosina com o EHNA, um
inibidor de adenosina deaminase, provocou uma redução significativa do número de
transportadores, pois houve uma grande redução dos valores de Bmax, (Paes-De-Carvalho,
2002) o que sugere uma modulação do transportador pela ativação de receptores de
adenosina.
Em culturas mistas de retina de pinto (Paes-de-Carvalho et al., 2005), também foi
observado que mais de 90% da [3H]adenosina captada pelas células é convertida em
nucleotídeos de adenina, enquanto que cerca de 80% da liberação de purinas estimulada pela
ativação de receptores ionotrópicos de glutamato é encontrada na forma de inosina e
hipoxantina, de modo semelhante ao observado em culturas purificadas de neurônios (Paes de
16
Carvalho et al., 1990). Esse efeito estimulatório da liberação de purinas estimulada por
glutamato é bloqueado pelo NBTI, inibidor do transportador de nucleosídeos equilibrativo do
tipo 1 (Paes-de-Carvalho et al., 2005).
1.8 Transporte de adenosina e sua regulação por vias de sinalização
A regulação da expressão dos transportadores de nucleosídeos pode ser dada tanto ao
nível transcricional, onde a taxa de síntese de mRNA é modificada por indução ou supressão
transcricional, ou no nível pós-transcricional, onde a proteína em si é regulada por diversos
mecanismos (Kong et al., 2004).
Com relação aos transportadores concentrativos, não há muita informação sobre os
mecanismos de regulação destes, um dos poucos trabalhos existentes demonstrou que o alltrans- ácido retinóico aumentou a taxa de inserção do CNT3 na membrana plasmática por um
mecanismo que envolve a ativação de p38, TGF-β1 e ERK 1/2 (Fernandez-Calotti & PastorAnglada, 2010) e um outro trabalho também identificou que uma isoforma selvagem do
CNT3 pode ser encontrada tanto em lipid rafts quanto em domínios não-lipid rafts, porém
uma isoforma deste CNT3, conhecida como CNT3C602R, foi encontrada em menores
quantidades nos lipid rafts quando comparado aos níveis da isoforma selvagem do CNT3.
Com respeito aos transportadores equilibrativos, diversos trabalhos na literatura
demonstram que estes podem ser regulados por ação de diversos agonistas ou por proteínas
cinases, mas, no entanto ainda não está estabelecido se a modulação desses transportadores
ocorre por fosforilação direta ou via interações com proteínas secundárias. Já foram
demonstrados potenciais sítios de fosforilação para PKC em ENT1 de camundongos e cães
(Kiss et al., 2000; Hammond et al., 2004) nas alças intracelulares, um sítio próximo ao fim da
17
região carboxi-terminal que interliga o 2o e 3o domínios transmembrana e outro na maior alça
intracelular que interliga o 6o e 7o domínios transmembrana.
Além da PKC, também já foram demonstrados potenciais sítios de fosforilação para
CK II em ENT1 (Kiss et al., 2000; Bone et al., 2007). Apesar de não ter sido demonstrada
fosforilação direta dos transportadores por nenhuma dessas cinases, existem diversos relatos
de modulação dos transportadores e dos níveis de adenosina, como os citados a seguir.
A exposição aguda ao etanol de células de linfoma S49 provocou uma severa inibição
da captação de adenosina e este efeito parece ocorrer através da diminuição de
transportadores sensíveis ao NBTI na membrana celular (Nagy et al., 1990). Posteriormente
este mesmo grupo mostrou que este efeito inibitório do etanol na captação de adenosina é
dependente da ativação de PKA (Nagy et al., 1991). Um outro trabalho deste mesmo grupo,
também em células cromafins da medula adrenal, demonstrou que a ativação da PKC provoca
uma redução da captação de adenosina e uma diminuição da presença de sítios ligantes dos
transportadores sensíveis ao NBTI (Delicado et al., 1991). Estes efeitos inibitórios na
captação por ação da PKA e PKC também foram observados em uma linhagem de
neuroblastoma (Sen et al., 1999). No entanto, também já foi demonstrado que a ativação da
PKC pode regular positivamente a captação de adenosina em linhagens celulares humanas
(Coe et al., 2002) e em uma linhagem celular de cardiomiócitos de camundongos (Chaudary
et al., 2004).
(Sweeney, 1996) mostrou que a inibição da subunidade α da Gi ou Go com toxina
pertussis leva a uma diminuição da liberação de adenosina em culturas de neurônios
granulares do cerebelo evocada por um estímulo despolarizante com potássio, enquanto que a
ativação da subunidade α da Gs com toxina de cólera provoca um estímulo da liberação de
adenosina nas mesmas condições descritas acima. ATP foi capaz de aumentar
significativamente o transporte de uridina em vesículas de membranas plasmáticas de células
18
chromafins, um efeito que pode ser mediado por aumento da inserção de transportadores na
membrana (Delicado et al., 1994).
Também já foi visto que, de acordo com o tipo de estímulo elétrico, de alta ou baixa
frequência, especificamente em fatias hipocampais, podem ser encontradas variações no local
de formação de adenosina, favorecendo o metabolismo intracelular por ação das
nucleotidases ou a liberação da adenosina per se através dos transportadores equilibrativos,
respectivamente (Cunha et al., 1996). Em sinaptossomas hipocampais, já se demonstrou
liberação de adenosina induzida por ATP e esta adenosina no meio extracelular ativa os
receptores A2A que aumentam a LTP induzida por estimulação elétrica de maneira dependente
de PKC (Almeida et al., 2003). Em sinaptossomas hipocampais, demonstrou-se que a
ativação dos receptores A2a provoca um aumento da captação de adenosina e este efeito é
revertido pela inibição da PKC (Pinto-Duarte et al., 2005).
Alguns estudos correlacionam a ativação das MAPKs e a regulação de
transportadores diversos. Por exemplo, (Caivano, 1998) demonstrou que a estimulação
conjunta com LPS e com IFN-γ aumenta a captação de arginina em uma linhagem celular de
macrófagos, um efeito que é bloqueado na presença de um inibidor da via das MAP cinases.
Outro grupo demonstrou que a glicose induz uma translocação do transportador GLUT4 para
a membrana plasmática em adipócitos de ratos, por um mecanismo dependente de ERKs
(Bandyopadhyay et al., 2001). Um trabalho recente demonstrou que a ativação de receptores
D2 de dopamina pode regular a atividade do DAT, transportador de dopamina, por uma
maneira dependente de MAPKs (Bolan et al., 2007).
Nos últimos anos, também tem surgido alguns trabalhos correlacionando a modulação
dos transportadores de nucleosídeos por MAPKs. Em células endoteliais fetais humanas
demonstrou-se que a inibição do transporte de adenosina por glicose ocorre de modo dose e
tempo-dependente e tem a participação da PKC, óxido nítrico e ERKs (Montecinos et al.,
19
2000) e um outro trabalho deste mesmo grupo demonstrou nestas mesmas células que o TGFβ1, através da ativação dos receptores TβRII, modula negativamente os níveis de proteína do
ENT1 por um mecanismo que depende da produção de óxido nítrico (Vega et al., 2009).
Outro grupo demonstrou que altos níveis de glicose induzem uma diminuição dos níveis de
mRNA de ENT1 em culturas primárias de fibroblastos do coração de ratos, por mecanismos
que dependem de PKC e ERK, mas independem de óxido nítrico (Grden et al., 2008). Além
desses, existem outros trabalhos que demonstram que a insulina e a glicose afetam a
expressão dos transportadores de nucleosídeos e, tendo em vista que pacientes diabéticos
podem realizar terapias baseadas em análogos de nucleosídeos por cânceres ou doenças
virais, essa variação na expressão dos transportadores pode ter implicações clínicas na
eficácia e na toxicidade das drogas (Kong et al., 2004).
Este efeito inibitório das ERKs na captação foi observado de maneira similar em
culturas de linfócitos B, onde a inibição da MEK provocou um aumento da expressão do
RNAm do ENT1 que havia sido diminuído pelas altas concentrações de glicose (Sakowicz et
al., 2005). Em uma linhagem de células PC12, demonstrou-se que hipóxia crônica induz um
aumento da liberação de adenosina, por um mecanismo que envolve a down-regulação da
expressão do mENT1 e diminuição da atividade das enzimas adenosina cinase e adenosina
desaminase (Kobayashi et al., 2000).
Como citados anteriormente, diversos estudos demonstram que inibidores de
proteínas cinases podem modular os transportadores ENT1, mas, no entanto, (Huang et al.,
2002; Huang et al., 2003) demonstraram que algumas classes de inibidores podem inibir
diretamente os transportadores, de modo independente da inibição da proteína cinase. Alguns
análogos inativos destes inibidores também tiveram efeitos, por um mecanismo que pode ser
através da interação direta com o sítio de ligação ao NBTI, visto que alguns análogos de
20
inibidores da PKC, p38 e receptores tirosina cinases são análogos estruturais de purinas ou
pirimidinas ou componentes relacionados estruturalmente.
1.9 Modulação dos níveis de adenosina pela adenosina cinase
O metabolismo da adenosina envolve as enzimas adenosina deaminase e adenosina
cinase. A enzima adenosina deaminase promove a deaminação da adenosina à inosina. Esta
via metabólica é majoritariamente citosólica e sua localização correlaciona-se com a dos
transportadores de adenosina (Nagy et al., 1985). No entanto, tem sido sugerido a existência
de uma ecto-adenosina deaminase, que poderia estar ancorada aos receptores A1 catalisando
a degradação de adenosina no meio extracelular, apesar de promover o aumento da ligação de
adenosina ao receptor A1 (Franco et al., 1997; Latini & Pedata, 2001).
A adenosina cinase, presente no meio intracelular, promove a fosforilação da
adenosina, levando à formação de AMP. A inibição da adenosina cinase pode levar ao
aumento da concentração de adenosina extracelular em fatias do hipocampo e consequente
modulação da transmissão sináptica dos neurônios, enquanto que a inibição da adenosina
deaminase não teve qualquer efeito neste modelo celular (Pak et al., 1994). A adenosina
cinase possui um valor de Km menor que o da adenosina deaminase (2µM e 17-45µM,
respectivamente) no cérebro de rato. Em concentrações fisiológicas, a atividade da adenosina
cinase pode ser máxima. No entanto, concentrações elevadas de adenosina podem levar a
uma inibição da enzima, pois é conhecido que a adenosina cinase é uma enzima inibida pelo
próprio substrato. Estudos já demonstraram que esta enzima tem dois sítios ligantes de
adenosina, um de alta afinidade, que corresponde ao sítio catalítico, e outro de menor
afinidade, que serve como sítio regulatório (Arrigoni & Rosenberg, 2006). Deste modo, a
principal via de metabolismo da adenosina em condições fisiológicas deve ser a fosforilação
21
promovida pela adenosina cinase. Em situações de estresse metabólico, quando há aumento
da concentração intra e extracelular de adenosina, a adenosina deaminase pode ser a principal
via de degradação intracelular (ver (Phillips & Newsholme, 1979; Latini & Pedata, 2001)).
De uma maneira geral, o transporte de adenosina em grande parte dos tipos celulares
ocorre por transportadores equilibrativos. A captação contínua de adenosina depende do seu
subseqüente metabolismo, que contribui para manter uma baixa concentração intracelular de
adenosina não-metabolizada e deste modo manter um gradiente de concentração. Nesta
situação, a inibição do metabolismo diminui a captação de adenosina em sistemas que não
expressem os transportadores concentrativos de nucleosídeos. Em culturas primárias de
neurônios corticais, observou-se que a adenosina é metabolizada preferencialmente em
inosina, através da adenosina deaminase, enquanto que em culturas de astrócitos corticais a
rota principal é em direção à formação de nucleotídeos de adenina, através da atividade da
adenosina cinase (Matz & Hertz, 1989). Em culturas mistas de retina de pinto, também foi
demonstrado que há uma alta atividade da adenosina cinase, pois cerca de 95% da adenosina
captada é convertida em nucleotídeos de adenina durante os 15 minutos de captação (Paesde-Carvalho et al., 2005).
A adenosina cinase parece ser o regulador chave e a rota primária na regulação dos
níveis de adenosina. Trabalhos realizados em células do hipocampo têm mostrado que a
expressão da adenosina cinase sofre mudanças durante o desenvolvimento pós-natal,
passando de uma localização neuronal para uma localização astrocítica (Studer et al., 2006).
Também já foi demonstrado que em episódios de isquemia há uma “down-regulação”
transiente da adenosina cinase. Corroborando estes dados, a super-expressão crônica da
adenosina cinase pode causar convulsões e provocar morte celular em eventos epilépticos e
derrames (Boison, 2006).
22
Quanto à modulação da atividade da enzima, (Sahin et al., 2004) demonstraram a
presença de potenciais sítios de fosforilação para CAMKII na sequência de aminoácidos da
adenosina cinase e observaram através de ensaios de fosforilação in vitro que a CAMKII era
capaz de efetivamente fosforilar a ADK, no entanto não muito eficientemente. FK-506, um
imunossupressor que também atua como um inibidor de calcineurina, aumenta a liberação de
adenosina de células endoteliais por um mecanismo que envolve a inibição da atividade da
adenosina cinase associada à membrana plasmática (Hwang et al., 2001). Outro grupo
demonstrou na mesma época que o FK-506 também diminui a captação de adenosina e a
atividade da adenosina cinase em linfócitos T, por um mecanismo que não envolve a inibição
da calcineurina (Spychala & Mitchell, 2002). Também já foi relatado por (Sinclair et al.,
2000) que a ativação de receptores A1 de adenosina em uma linhagem de células musculares
DDT1 MF-2 inibe a atividade da adenosina cinase, por um mecanismo dependente da
ativação de PKC.
A fosforilação da adenosina cinase é um mecanismo potencial, pois a seqüência de
aminoácidos da enzima possui numerosos sítios consenso para fosforilação por PKC, como
demonstrado a partir da clonagem da enzima que já foi feita em tecidos de ratos e humanos
(McNally et al., 1997). (Pawelczyk et al., 2003) demonstraram que esplenócitos de ratos
diabéticos apresentavam uma menor expressão de adenosina cinase, e que os níveis de
expressão da enzima eram restaurados após a administração de insulina. Neste mesmo
trabalho também foi demonstrado que a insulina aumenta a transcrição do mRNA da
adenosina cinase e a atividade em si da enzima em culturas de linfócitos de ratos, por um
mecanismo que envolve a fosforilação da ERK 1/2.
Também já foi demonstrado que o óxido nítrico pode inibir a atividade da adenosina
cinase em culturas de neurônios do prosencéfalo de rato (Rosenberg et al., 2000).
23
1.10 Astrócitos corticais
Os astrócitos corticais são divididos em três subclasses: fibrosos, protoplasmáticos e a
glia radial. Os astrócitos fibrosos (ou fibrilares) são encontrados na substância branca e
possuem longos processos finos que fazem contatos com nodos de Ranvier e vasos
sanguíneos, os astrócitos protoplasmáticos são encontrados na substância cinzenta e possuem
muitos processos ramificados que estão associados com compartimentos pré- e/ou póssinápticos assim como vasos sanguíneos (Barres, 2008). A glia radial, inicialmente descrita
apenas como uma célula mediadora da migração neuronal radial no cortex, atualmente é
conhecida por representar um progenitor comum para neurônios e astrócitos em várias
regiões do SNC em desenvolvimento (Vaccarino et al., 2007). A geração de todos os tipos
celulares no cortex ocorre em fases temporalmente distintas, porém com alguns períodos de
sobreposição, em primeiro lugar são gerados os neurônios, depois os astrócitos e por último
os oligodendrócitos (Sauvageot & Stiles, 2002). Atualmente, é conhecido que os astrócitos,
principalmente os protoplasmáticos, são elementos importantes na regulação da sinalização
sináptica, baseado nisto há alguns anos surgiu a idéia da sinapse tripartite (Araque et al.,
1999).
Atualmente sabe-se que diversas moléculas, tais como glutamato, D-serina, ATP,
adenosina dentre outras, conhecidas como gliotransmissores, também são liberadas pelos
astrócitos e outros tipos gliais (Volterra & Meldolesi, 2005). Com respeito a adenosina,
embora existam diversos trabalhos de caracterização dos mecanismos de captação deste
nucleosídeo em culturas de astrócitos corticais (Hertz, 1978; Bender & Hertz, 1986; Gu et al.,
1996; Peng et al., 2005; Redzic et al., 2010) e também já existam alguns trabalhos
relacionados a expressão dos receptores de Ado em astrócitos corticais (van Calker et al.,
1979; Biber et al., 1997), há muito pouca informação sobre os mecanismos de controle da
24
atividade e/ou expressão dos transportadores de nucleosídeos operados pela ativação de
receptores de Ado.
25
2. Objetivos
2.1 Objetivo Geral
Caracterizar o sistema de captação de adenosina em:
1- Culturas primárias mistas de retina de galinha
2- Culturas purificadas de glia de Müller de retina de galinha
3- Culturas primárias de astrócitos corticais de rato
2.2 Objetivos Específicos
•
Analisar a cinética temporal de captação de Ado nas diferentes culturas descritas
acima
•
Identificar a proporção relativa entre ENTs e CNTs e caracterizar a captação de
inosina nas diferentes culturas descritas acima
•
Identificar a importância relativa da ADK para a captação de Ado nas culturas de
retina de galinha
•
Analisar os efeitos de diferentes inibidores da MEK na captação, metabolismo e
liberação de Ado nas culturas de retina de galinha
•
Caracterizar os efeitos de inibidores da PKC, PLC e CAMK II na captação de Ado
nas culturas de retina de galinha
•
Identificar por western blot, imunocitoquímica e binding de [3H]NBTI a presença do
ENT1 nas culturas de astrócitos corticais
•
Investigar os efeitos da ativação aguda dos receptores A1 na captação de Ado e no
binding de de [3H]NBTI nas culturas de astrócitos corticais
26
Modulation of ERK phosphorylation by A1
adenosine receptor in cultures of avian retinal
glial cells: Involvement of PKC and Src kinase
Alexandre dos Santos-Rodrigues, Mariana R. Pereira, Eliza
Vardiero, Igor L. A. da Silva, Luiz R. Leão-Ferreira and
Roberto Paes-de-Carvalho
Program of Neurosciences, Institute of Biology, Fluminense Federal
University, Niterói, RJ 24001-970, Brazil.
Correspondence to:
Roberto Paes-de-Carvalho
Departamento de Neurobiologia
Instituto de Biologia
Caixa Postal 100180
Centro, Niterói,
RJ 24001-970
Brasil
Tel: (55-21) 2629-2263
Fax: (55-21) 2629-2268
Email: [email protected]
List of Abbreviations:
ADA, adenosine deaminase; CHA, N6-cyclohexyladenosine; CHE, Chelerythrine
chloride; CREB, cyclic AMP response element-binding protein; DAPI, 4′, 6-diamino-2
phenylindole. DPCPX, 8-Cyclopentyl-1,3-dipropylxanthine; ERK, extracellular signalregulated
kinase;
MAPK,
mitogen-activated
protein
kinase;
NECA,
5’-N-
Ethylcarboxamidoadenosine; PD 98059, 2´-amino-3´-methoxyflavone; PKC, protein
kinase
C;
PLC,
phospholipase
C;
PP1,
4-Amino-1-tert-butyl-3-(1′-
naphthyl)pyrazolo[3,4-d]pyrimidine; PVDF, polyvinylidene difluoride;
27
Abstract
Adenosine is an important modulator of neuronal survival and differentiation and also
participates in neuroprotection mechanisms. Besides its classical role in the regulation
of adenylyl cyclase activity, increasing evidence indicates that adenosine receptors
regulate different signaling pathways including MAP kinase cascade which appear to
have important roles in several neural and glial functions. Glial cells play fundamental
roles in the CNS such as regulation of synaptic transmission as well as
neurotransmitter uptake and metabolism. In the present work expression of adenosine
A1 receptors and regulation of extracellular-regulated kinase (ERK) activity of cultured
retinal glial cells by adenosine was evaluated. Expression of A1 receptor in purified
cultures of glial cells obtained from 11 day-old chick embryo retinas was detected by
(3H) DPCPX binding and western blot. Purified cultures were incubated with selective
kinase inhibitors and adenosine receptor selective agonists or antagonists for
determination of phosphorylated ERK (pERK) level by western blotting or
immunocytochemistry. Incubation of cultured glial cells with adenosine deaminase
(0.5 U/ml) or DPCPX (10 µM), an A1 receptor-selective antagonist, reduced basal
pERK level by approximately 50% indicating that endogenous adenosine regulates
ERK phosphorylation through activation of A1 receptor. Incubation with CHA (1 µM),
an A1 receptor-selective agonist, induced an increase of 120% in pERK levels, an
effect completely blocked by DPCPX. Basal pERK level was also reduced 30-50% by
PD98059, a MEK inhibitor, PP1, a Src inhibitor, or Chelerythrine chloride (100 nM), a
PKC inhibitor. Furthermore, these selective inhibitors completely blocked CHAinduced ERK phosphorylation. Immunocytochemistry data revealed that A1 receptorinduced increase in pERK level is mainly localized in the cytosol. These results
28
demonstrate that A1 adenosine receptor is expressed in retinal glial cells and
modulate ERK signaling through a pathway involving PKC and Src kinase.
Keywords: MAP kinase, Muller glial cell, signaling pathway, adenosine deaminase
Running Title: ERK activation by adenosine in retinal glial cells
29
1. Introduction
Adenosine is an ubiquitous nucleoside released in large amounts during
hypoxic and ischemic events (Ribeiro et al., 2002; Schulte and Fredholm, 2003).
Adenosine interacts with G protein-coupled receptors classified into four subtypes:
A1, and A3 receptors, both negatively coupled to adenylyl cyclase, as well as A2A and
A2B receptors, both positively coupled to the same enzyme (Cunha, 2005; Paes-deCarvalho, 2002; Schulte and Fredholm, 2003).
Adenosine, which is not stored or released from synaptic vesicles, is taken up
or released from cells through nucleoside transporters (Jennings et al. 1998; Latini
and Pedata, 2001; Podgorska et al., 2005). Adenosine is synthesized inside as well
as outside neuron and glial cells by intracellular or extracellular conversion from AMP
through 5´-nucleotidase-catalyzed reaction (Franco et al., 1986; Latini and Pedata,
2001). In addition, adenosine can also be deaminated to inosine by adenosine
deaminase or phosphorylated to AMP by adenosine kinase (Latini and Pedata, 2001;
Matz and Hertz, 1989). Altogether, intracellular and extracellular adenosine level is
determined by concerted action of adenosine-dependent enzymes and membrane
transporters.
Adenosine neuromodulatory activity is believed to occur by inhibition or
potentiation of neuronal activity. In retina, the inhibitory action of adenosine involves
an increase of potassium channels conductance mediated by A1 receptors present in
ganglion cells. In this case, adenosine is produced by extracellular dephosphorylation
of ATP released from glial cells (Newman, 2003). On the other hand, long-term
activation of A2A receptor in cultures of chicken retinal neurons induces
neuroprotection from glutamate excitoxicity (Ferreira and Paes-de-Carvalho, 2001) or
from re-feeding-induced death of cultured cells (Paes-de-Carvalho et al., 2003).
30
Recent data also show that this long-term treatment of chicken retinal cells in culture
promotes an increase in A1 receptor expression (Pereira et al., 2010).
Adenosine modulates cell proliferation and differentiation and these processes
seem to be under control of mitogen-activated protein kinases (MAPKs) (Stevens et
al., 2002). The MAPKs constitute a family of serine/threonine protein kinases
activated by receptor tyrosine kinases or G protein-coupled receptors (May and Hill,
2008; Rozengurt, 2007; Werry et al., 2005). MAPKs are classified into three families:
ERKs 1 and 2, P38 and JNK (Schulte and Fredholm, 2003). The ERKs are also
involved in long-term potentiation (LTP) as well as in other types of synaptic plasticity
(Schulte and Fredholm, 2003; Sweatt, 2001). Phosphorylated ERK may translocate
to the nucleus where it catalyzes the phosphorylation of different transcription factors
such as Elk-1 and p90rsk (Schulte and Fredholm, 2003; Sweatt, 2001). Nevertheless,
ERKs can also catalyze the phosphorylation of non-nuclear substrates such as
potassium channels (Morozov et al., 2003) and microtubule-associated protein MAP2
(Vaillant et al., 2002).
Several reports have demonstrated that ERK phosphorylation can be
stimulated by adenosine receptors (Schulte and Fredholm, 2000; Seidel et al., 1999;
for review, Schulte and Fredholm, 2003). Dickenson et al., (1998) and Robinson and
Dickenson, (2001) have shown that stimulation of ERKs can be mediated by A1
receptor activation in CHO and DDT(1)MF-2 cells, respectively. Increased ERK
phosphorylation was also demonstrated in CHO-transfected cells expressing different
adenosine receptors stimulated by the non-selective agonist NECA (Schulte and
Fredholm, 2000). Stimulation of
ERK phosphorylation by different adenosine
receptor subtypes was also demonstrated in cardiomyocytes from neonatal rats
(Germack and Dickenson, 2004).
31
The retina shares the same embryonic origin with other CNS structures and
for that reason it has been considered as a model for studying neuronal interactions
in the developing CNS (Coulombre, 1955). Chicken retinal cells in culture preserves
some properties common to the retina such as modulation of cAMP level by A1 and
A2a receptors as well as nucleoside uptake and release by the high affinity
transporters (Paes-de-Carvalho and de Mello, 1982, 1985; Paes-de-Carvalho et al.,
1990a). The Müller cell is the predominant glial cell type in retina and besides its
classical effects in providing structural and metabolic support to neurons (reviewed
by Bringmann et al., 2006 and de Melo Reis et al., 2008), it also plays an important
role in neuronal activity by regulating release of neurotransmitters such as ATP
(Newman, 2003) or glutamate (Fellin et al., 2004). Some recent data demonstrate
that cultured Muller glial cells from chicken retina express several neuronal markers
such as GAT-1 and GAT-3, tyrosine hydroxylase and β2-nicotinic receptor subunit
(Kubrusly et al., 2005, 2008; de Sampaio Schitine et al., 2007; for review, de Melo
Reis et al., 2008).
In the present work we have investigated the expression of A1 receptors in
purified-cultures of chicken retina glial cells as well as its role in the modulation of
ERK phosphorylation level. Our data show that endogenous adenosine modulates
basal ERK phosphorylation, an effect mediated by A1 receptor through a signaling
pathway involving Src, PKC and MEK. In addition, immunocytochemistry data show a
strong staining for A1 receptor-induced phospho-ERK in the cytosol of glial cells.
32
2. Experimental procedures
2.1. Materials
Fertilized White Leghorn eggs were obtained from a local hatchery and
incubated at 38 °C in a humidified atmosphere. Bovi ne serum albumine (BSA), N6cyclohexyladenosine (CHA), Chelerythrine chloride (CHE), 8-Cyclopentyl-1,3dipropylxanthine (DPCPX), L-Glutamine, 5’-N-Ethylcarboxamidoadenosine (NECA),
Penicillin G and Streptomycin Sulfate were obtained from Sigma/RBI Chem.Co.
(Missouri, USA). Minimum Essential Media (MEM), heat-inactivated fetal bovine
serum (FBS) and Trypsin were obtained from GIBCO (New York, USA). Adenosine
deaminase (ADA) was obtained from Calbiochem (California, USA). PD 98059 and
PP1 were obtained from Biomol (Pennsylvania, USA). The horseradish peroxidaselinked anti-mouse and anti-rabbit secondaries antibodies, the polyvinylidene
difluoride (PVDF) membranes, the enhanced chemiluminescence (ECL), [3H] 8cyclopentyl-1, 3-dipropylxanthine ([3H] DPCPX) (130 Ci/mmol) were obtained from
Amersham
(Buckinghamshire,
United
Kingdom).
Mouse
monoclonal
anti-
phosphorylated ERK 1/2 antibody was obtained from Cell Signaling (Madison, USA)
and rabbit polyclonal anti-ERK 1/2 antibody was obtained from Promega (Madison,
USA). Rabbit polyclonal anti-A1 adenosine receptor antibody was supplied by
Chemicon International (CA, EUA). Mouse secondary coupled to Alexa568 and
mouse secondary coupled to Alexa488 antibodies were obtained from Molecular
Probes (Eugene, OR, USA). Mouse monoclonal 2M6 antibody was kindly supplied by
B. Schlosshauer (NMI Naturwissenschaftliches und Medizinisches Institut an der
Universität Tübingen, Markwiesenstr. 55, D-72770 Reutlingen, Germany).
All other reagents were of analytical grade.
33
2.2. Glial cell cultures
Glial cell cultures of chick retina were prepared as previously described
(Cossenza and Paes-de-Carvalho, 2000), with minor modifications. Briefly, retinas
from 11 -day-old chick embryos (White Leghorn) were dissected from other ocular
tissues, including the pigmented epithelium, and digested chemically with 0.1%
trypsin in calcium and magnesium-free Hank’s balanced salt solution (CMF), for 2025 min at 37 oC. The solution was then removed, the cells suspended in MEM
supplemented with 5% heat-inactivated fetal bovine serum, penicillin (100 U/ml) and
streptomycin (100 µg/ml) and seeded in 40 mm tissue culture plastic dishes in a
density of 3.3 x 106 cells/dish. Cultures were maintained at 37 oC in a humidified
incubator with 95% air and 5% CO2. The medium was changed every 3 days and
experiments were performed at day 21 in culture. At this time, the presence of
neurons is minimal and the glial cells turn confluent in almost whole dish.
2.3. Immunoblotting
For detection of ERK phosphorylation, cultures were washed, pre-incubated
for 5 min in Hank’s in presence or not of adenosine deaminase, and treated with
different agents for the indicated times. The medium was removed and the reaction
was stopped by adding sample buffer (10% glycerol, 100 mM 2-Mercaptoethanol, 2%
sodium dodecyl sulfate [SDS]). Cells were then scraped off from the dishes and the
material heated for 6 min at 950C. Protein concentration was determined by the
Bradford assay (Bradford, 1976). For detection of A1 adenosine receptor, cells were
scraped off from the dishes with sample buffer, heated at 95oC and the protein
concentration determined by the Bradford assay. Samples containing 45 µg protein
(ERK) or 60 µg protein (A1 receptor) were separated by electrophoresis on 9% SDS–
polyacrylamide gels (SDS-PAGE) and proteins transferred to PVDF membranes.
34
These membranes were blocked for 1h at room temperature in Tris-buffered saline
(200 mM NaCl, 20 mM Tris-HCl, pH 7,6) containing 0.1% Tween-20 (TBS-T) and 5 %
low fat milk. These membranes were incubated overnight with an antibody that
specifically recognizes ERK1/2 phosphorylated at Thr202/Tyr204 (pERK 1/2, 1:
2,000) or A1 adenosine receptor (1: 100). In the next day, membranes were washed
and incubated with horseradish peroxidase-linked anti-mouse secondary antibody
(1:5,000) (pERK ½) or horseradish peroxidase-linked anti-rabbit secondary antibody
(1:500) (A1 receptor) for 1h at room temperature and revealed by enhanced
chemiluminescence (ECL). To control for protein loading, the membranes were
stripped in a solution of glycine 0.2M pH 2.2 for 30 min under gentle agitation and
reprobed overnight with a rabbit polyclonal anti-ERK 1/2 antibody (1:5,000), washed,
incubated with horseradish peroxidase-linked anti-rabbit secondary antibody
(1:5,000) and revealed by enhanced chemiluminescence (ECL). In the present study,
the anti-pERK 1/2 antibody revealed only a single band for ERK and this was
described as ERK 2 because other reports using chick samples showed comigration
of the band with human ERK 2 (Desire et al., 2000; Sanghera et al., 1992).
Moreover, western blots of gels run with rat and chick retinas in parallel revealed that
while the rat sample showed two bands, the chick sample showed only one band in
the exact position of the rat ERK 2 band (not shown).
Quantitative analysis of blots was performed by scanning images and using
the computer program Scion Image (Scion Corporation, Frederick, MD, USA).
2.4. Immunocytochemistry
Glial cell cultures were pre-incubated in Hank’s containing adenosine
deaminase (ADA, 0.5 units/ml) for 5 minutes and then stimulated or not with N6cyclohexyladenosine (CHA, 1 µM) for 3 minutes. Immediately after this period,
35
cultures were fixed with 4 % paraformaldehyde in phosphate-buffered saline (PBS)
for 30 minutes at room temperature. Cultures were washed twice in PBS and stored
at 4oC. In the next day, cultures were washed and then blocked and permeabilized in
PBS containing 3% fetal bovine serum, 3% bovine serum albumin and 1% Triton X100 for 60 minutes at room temperature. Cells were incubated overnight at 4o C in
the same solution containing mouse monoclonal anti-pERK 1/2 antibody (1:500). In
the next day, cultures were washed three times in PBS and then incubated with a
mouse secondary antibody coupled to Alexa568 for 90 minutes at room temperature,
protected by light. After this incubation, cultures were washed three times in PBS and
incubated overnight at 4o C with mouse monoclonal antibody against 2M6 (1:200)
(specific marker of Muller glial cells). Cultures were then washed three times in PBS
and incubated with a mouse secondary antibody coupled to Alexa488 for 90 minutes
at room temperature, protected by light. Subsequently, cultured cells were washed
three times in PBS, incubated with DAPI for 30 seconds and then washed twice in
PBS. Coverslips were mounted in slides using a saturated solution of N-propyl-galate
in PBS. Imaging was performed with an Axioskop Zeiss microscope with Apotome
module coupled to a Micromax CCD camera to obtain fluorescence images. Image
analysis and processing were performed with the software Axiovision (Zeiss,
Germany) and Adobe Photoshop (Adobe Systems, USA).
2.5. Binding assays
Before the experiments, the medium was removed and the cultures rinsed
twice with Hank`s Balanced Salt Solution (140 mM NaCl, 5 mM KCl, 20 mM Hepes, 4
mM glucose, 1 mM MgCl2, 2 mM CaCl2). Cultures were then incubated with 5 nM [3H]
DPCPX in the presence of adenosine deaminase (1 U/mL) for 1 hour at 37oC.
Specific binding was calculated as total binding less non-specific binding measured
36
in the presence of 100 µM unlabelled CHA. After incubation, the cells were washed
twice with Hank´s solution and lysed with water. The radioactivity was determined by
scintillation spectroscopy. Protein was determined by the method of Lowry et al.,
(1951).
2.6. Statistical analysis
Data were analyzed by student t test or using one-way ANOVA followed by the
Bonferroni or Tukey posttest or nonlinear regression analysis using the software
Graphpad Prism.
37
3. Results
3.1. Characterization of Muller glial cell cultures and expression of A1
adenosine receptors
In order to evaluate glia-purified cultures, the Muller glial cells were identified
by an antibody against 2M6, a Muller glia cell specific antigen (Schlosshauer et al.,
1991). As shown in figure 1A, there is a strong labeling of 2M6 in these cultures.
Unlabeled cells were not observed, indicating that the Muller glia cell was the only
cell type present in the purified culture. The presence of A1 adenosine receptor in the
developing chick retina has been reported previously by Paes-de-Carvalho and de
Mello, (1985). Recently, the presence of lower level of A1 receptor was also
demonstrated in mixed-cultures of chicken retina cells (Pereira et al., 2010).
However, expression of the A1 receptor in purified cultures of Muller glial cells has
not been determined. Western blot analysis demonstrates the presence of A1
receptors in glia-purified cultures as well as in intact retina from E16 embryos
(Fig.1B). We have also detected expression of A1 adenosine receptors in Muller glia
cultures by binding assays with [3H]DPCPX (63.6 +/- 8.2 fmoles/mg protein).
3.2. A1 receptor-mediated stimulation of ERK phosphorylation by endogenous
adenosine
To investigate the role of endogenous adenosine in the modulation of the
basal level of ERK phosphorylation, cultures were incubated with different
concentrations of adenosine deaminase (ADA), an enzyme that catalyzes adenosine
deamination to inosine, and then measured pERK levels by western blot. Our results
demonstrate that incubation of cultures with ADA for 5 minutes reduced the basal
pERK level in a dose-dependent manner (Fig. 2A).
38
Figure 1: Cell-specific immunolabeling of Müller glial cells in culture and
western blots showing expression of A1 receptor in glia-purified cultures of
chicken retina and E16 intact retina. A: A representative image of
immunocytochemistry staining for 2M6, a Muller glia cell-specific marker. The arrows
indicate nuclei of glial cells. Scale bar: 10 µM B: Immunoblots for A1 receptor in E16
intact retina (1) and in glial cell cultures from the chick retina (2).
39
Figure 2: Modulation of ERK phosphorylation level in glial cell cultures from
chicken retina by endogenous adenosine. A: Glia-purified cultures were treated
with different concentrations of ADA (0.1; 0.2 and 0.5 U/ml) for 5 minutes and
processed for western blot by using anti-pERK primary antibody. Results are
expressed as percent of control and represent the mean ± SEM from three
experiments. B: Cultures were treated with 10 µM DPCPX for 5 minutes and ERK
phosphorylation was measured by western blot. Results are expressed as percent of
control and represent the mean ± SEM from three experiments. * p < 0,05 compared
to control, ** p < 0,01 compared to control.
40
To investigate the involvement of A1 receptors in the stimulation of ERK
phosphorylation by endogenous adenosine, cultures were incubated with 10 µM
DPCPX for 5 minutes. The A1 selective-receptor antagonist reduced the basal pERK
level by approximately 50 % (Fig. 2B).
To confirm involvement of the A1 receptor in the modulation of basal pERK
level, cultures were pre-incubated with ADA (0.5 U/ml) or DPCPX (10 µM) for 5
minutes and then incubated with the A1 receptor-selective agonist CHA (1 µM) for 3
minutes. CHA increased pERK level by approximately 2.5 fold in the presence of
ADA while the A1 receptor-selective antagonist DPCPX blocked CHA-induced effect
(Fig. 3). In all subsequent experiments cultures were incubated with ADA to prevent
endogenous adenosine interference.
3.3. A1 receptor-mediated stimulation of ERK phosphorylation involves MEK,
PKC and Src kinase
MEK1/2 are dual specificity protein kinases that mediate the phosphorylation
of tyrosine before threonine in ERK1 or ERK2, their only substrates (Schulte and
Fredholm, 2003). To evaluate involvement of this protein kinase in the signaling
pathway triggered by A1 receptor to stimulate ERK phosphorylation, cultures were
incubated with PD 98059, a specific MEK inhibitor. This compound reduced
significantly the basal and CHA-stimulated ERK phosphorylation, demonstrating that
MEK activation is essential for ERK stimulation induced by A1 receptor-selective
agonist (Fig. 4A).
It has been previously demonstrated that protein kinase C (PKC) can be a
mediator in the signaling pathways involved in ERK activation by phosphorylation in
mixed retinal cells in culture as well as in intact retinas from E7/E8 chick embryos
(Nunes et al., 2007; Sanches et al., 2002). To evaluate the possible involvement of
41
Figure 3: A1 receptor-mediated increase in ERK phosphorylation in glial cell
cultures. Glia-purified cultures were pre-incubated with ADA (0.5 U/ml) for 5 min, in
the absence or in the presence of DPCPX (10 µM), and incubated with CHA (1 µM)
for 3 minutes. Results are expressed as percent of control and represent the mean ±
SEM from at least three experiments. *** P<0,001 compared to control.
42
Figure 4: Involvement of MEK, PKC and Src kinase in A1 receptor-mediated
increase in pERK level in glia-purified cultures from the chicken retina. Gliapurified cultures were pre-incubated with ADA (0.5 U/ml) for 5 min, in the absence or
in the presence of 25 µM PD 98059 (MEK 1/2 inhibitor) and incubated with 1 µM
CHA (A1 receptor-selective agonist) for 3 minutes (fig. 4A). Alternatively Glia-purified
cultures were pre-incubated in the absence or in the presence 10 µM PP1 (Src
inhibitor) or 100 nM CHE (PKC inhibitor), and incubated with 1 µM CHA (A1 receptorselective agonist) for 3 minutes (fig. 4B). Results are expressed as percent of control
and represent the mean ± SEM from at least three experiments. *** P<0,001
compared to control.
43
PKC in the modulation of ERK phosphorylation by adenosine, cultures were
pre-incubated with chelerythrine chloride (CHE, 100 nM) before incubation with CHA.
The PKC-specific inhibitor blocked CHA-induced increase in ERK phosphorylation
(Fig. 4B).
Another protein kinase that has been related to modulation of ERK
phosphorylation is the Src cytoplasmic tyrosine kinase (Luttrell et al., 1996; Miñano et
al., 2008). PP1 (10 µM), a specific inhibitor of Src kinase, inhibited both basal and
CHA-induced increase in pERK level (fig. 4B).
Together, our data demonstrate that A1 receptor mediated increase in p-ERK
level by adenosine involves MEK, PKC and Src kinase in purified-glial cultures of
chicken retina.
3.4. Immunocytochemistry for ERK in glial cell cultures
It has been demonstrated previously that activation of ERK by phosphorylation
may precede translocation to the nucleus and phosphorylation of specific
transcription factors (Choe and Wang, 2001; Davis et al., 2000; Wu et al., 2001). To
evaluate if A1 receptor-induced phosphorylation of ERK induces nuclear migration,
control and CHA-stimulated cultures were prepared for immunocytochemistry by
incubation with an anti-pERK antibody. pERK was found throughout the cytoplasm of
retinal glial cells in non-stimulated cultures (Fig.5B) as well as in CHA-stimulated
cultures (Fig.5D), although the immunolabeling was significantly increased in CHAstimulated cultures. No significant labeling of nuclei was found after this period of
incubation both in control or CHA-stimulated cultures.
44
Figure 5: Immunolocalization of pERK in Muller glial cells in glia-purified
cultures. pERK staining (red) and DAPI staining (blue) in glial cells in culture.
Cultures were pre-incubated with ADA (0.5 U/ml) for 5 min and incubated (D-F) or not
(A-C) with CHA (1 µM) for 3 min. Thereafter, purified cultures were processed for
immunocytochemistry. Scale Bar: 10 µM.
45
4. Discussion
4.1. Expression of A1 receptors in glial cells
The activation of A1 receptor promotes inhibition of adenylyl cyclase in
embryonic as well as post-hatched chicken retinas (Paes-de-Carvalho, 1990b; Paesde-Carvalho and de Mello, 1985). This receptor can be detected in the chicken retina
since E10, reaching maximum levels at around E17, and after this period receptor
level decreases up to post-hatching period (Paes-de-Carvalho, 1990b). A1 receptor
is localized at the inner and outer retinal plexiforms layers (Paes-de-Carvalho et al.,
1992), suggesting a synaptic localization for this receptor. Although several reports
have shown the localization of A1 receptor in glial cells (Beraudi et al., 2003; Biber et
al., 1997, 2001; Hosli and Hosli, 1988; Van Calker et al., 1979), the expression of this
receptor subtype in retinal Muller cells has not been previously demonstrated. Some
evidences for the presence of A1 receptor in these cells comes out from studies
showing the effects of agonists on intracellular calcium levels or cell swelling (Liu and
Wakakura, 1998; Skatchkov et al., 2006; Uckerman et al., 2006). In this work it has
been shown that chicken retina glial cells in culture express A1 receptor, as detected
by radioligand binding and western blot. One additional finding was that this receptor
is involved in regulating ERK phosphorylation level in these cells.
4.2. The signaling pathway involved in ERK activation by A1 receptor
The data presented here show definitive evidences that activation of A1
receptor stimulates ERK phosphorylation in retinal Muller cells in culture. The A1
receptor-selective agonist CHA induced an increase of pERK levels while the
selective antagonist DPCPX blocked completely this effect. Previous studies have
shown the regulation of ERK by A1 receptor stimulation (Angulo et al., 2003; Canals
et al., 2005; Migita et al., 2008; Minelli et al., 2007; Schulte and Fredholm, 2000). Our
46
data suggest that PKC and the cytoplasmic tyrosine kinase of the Src family are
involved in the signaling pathway triggered by adenosine to stimulate ERK
phosphorylation. Src kinases have been previously related to the regulation of
MAPKs mediated by Gi – coupled receptors, as demonstrated in COS-7 cells (Luttrell
et al., 1996). On the other hand, Erk activation by A1 receptor in cardiomyocytes
involves PLC-dependent PKC activation by βγ subunits of Gi proteins (Germack and
Dickenson, 2004). In CHO cells, stimulation of ERKs 1/2 mediated by A1 receptor
involves MEK, Gi protein, tyrosine kinases and PI3-kinase, but not PKC (Dickenson et
al., 1998). Moreover, ERK phosphorylation in A3 receptor-transfected CHO cells has
been shown to be dependent on βγ subunit of Gi protein, Ras, PI3-kinase and MEK
(Schulte and Fredholm, 2002). Another study investigated the activation of MAPKs
by A2A receptors in CHO cells and showed a signaling pathway which involves at
least participation of Gαs, adenylyl cyclase, PKA, Rap 1, B-Raf and MEK (Seidel et
al., 1999). The proliferative effect induced by ATP in chicken retina cell cultures from
E7 embryos was blocked by inhibitors of PLC, PKC and MEK, suggesting that PKC is
also involved in ERK phosphorylation (Sanches et al., 2002). Most studies on ERK
activation report a MEK-dependent activation, despite the fact that some authors
have demonstrated the existence of signaling pathways independent of this enzyme
(Bapat et al., 2001; Grammer and Blenis, 1997). In most cases, however, inhibition
of MEK blocks cellular differentiation and proliferation involving ERK (Sanches et al.,
2002; Vaillant et al., 2002). Our results have shown that the MEK inhibitor PD 98059
blocks ERK phosphorylation induced by A1 adenosine receptor activation and also
reduces the basal levels of phosphorylation of this enzyme. Nevertheless, we do not
know
yet
physiological
processes
affected
by
adenosine-induced
ERK
phosphorylation in glial cells.
47
4.3. Localization of pERK
Our immunocytochemistry data showed that pERK is diffusely distributed throughout
the cytosol of glial cells after incubation with CHA. This stimulation occurred within 3
minutes of incubation and could reflect in a regulation of cytoskeletal molecules such
as vimentin or MAP2 which are fundamental in the control of cytoplasmatic
processes (Perlson et al., 2005; Vaillant et al., 2002). Some cells in our cultures have
not shown an increase in pERK staining but this fact could be explained by the lack
of A1 receptor expression in these cells. Chuderland et al., (2008) demonstrated that
in resting cells ERKs are associated to several cytoplasmic proteins in a calciumdependent manner. Upon stimulation, ERKs are phosphorylated and released from
the cytoplasmic anchors to allow free shuttle through the cytoplasm toward the
nucleus, and this effect is inhibited by calcium. Therefore, it is possible that activation
of A1 receptors in cultured glial cells within 3 minutes induces an increase of
intracellular calcium, which would explain an increase of pERK only in cytoplasm.
Further studies are necessary to clarify this issue.
4.4. Evidence for a role of endogenous adenosine
Our results demonstrated that ERK phosphorylation was reduced by
approximately 50 % in glia-purified cultures incubated with ADA or DPCPX (A1
receptor antagonist) indicating that endogenous adenosine released from cells in
culture could be involved in the modulation of basal pERK levels through activation of
A1 receptor.
Release of adenosine has been demonstrated previously in a variety of neural
and non-neural tissues, including the retina (Dunwiddie, 1980; Paes-de-Carvalho and
de Mello, 1982). Addition of exogenous (3H)-labeled adenosine revealed the
presence of high affinity nucleoside transporters in mixed retinal cultures or purified
48
cultures of retinal neurons or glial cells (Paes-de-Carvalho et al., 1990a; dos SantosRodrigues A. and Paes-de-Carvalho R., unpublished observations). Activation of
ionotropic glutamate receptor was able to induce release of purines from mixed
retinal cultures pre-incubated with (3H) adenosine and this process was completely
blocked by the nucleoside transport inhibitor NBMPR (Paes-de-Carvalho et al.,
2005). Therefore, it is reasonable to assume that adenosine release is under control
of signaling molecules such as glutamate. However, adenosine can also be released
in hypoxic conditions through a mechanism involving the nucleoside transporter
(Bjorklund et al., 2008; Rego et al., 1997). Regardless the mechanism involved in
accumulation of extracellular nucleoside, adenosine can trigger different mechanisms
of cell adaptation by activation of membrane receptors. ERK regulates enzymes as
well as transcription factor activities and therefore interfere in different cellular
processes. As a consequence, this protein kinase is likely involved in the modulation
of glial cell as well as retinal tissue physiology. Our data could also indicate the
possibility that endogenous adenosine, through the modulation of ERK activity,
regulates cellular proliferation and differentiation of glial cells in the retina. E11
chicken embryo retina cells have a low ability to proliferate in vivo (Prada et al.,
1991). However, glial cells in culture resume proliferation probably by a combined
action of different molecules, including growth factors, or maintenance of a high
plastic ability along its existence (Fischer and Reh, 2003).
In conclusion, the present study demonstrated that Muller glial cells from
chicken retina in glia-purified culture express A1 receptor. Furthermore, endogenous
adenosine modulates ERK phosphorylation level by A1 receptor-mediated activation
of PKC and Src kinase. The cytosolic localization of p-ERK in CHA-stimulated or non-
49
stimulated cells might indicate non-nuclear targets for this protein kinase involved in
the regulation of growth and differentiation of glial cells in the retina.
50
Acknowledgments
We acknowledge Dr. Marco A.M. Prado for help in experiments using the
Axioskop Zeiss microscope with Apotome module (PROCAD UFMG/ UFF) and Ms.
Luzeli R. de Assis and Sarah A. Rodrigues for the technical assistance. ASR, EV,
ILAS and MRP were recipients of fellowships from Capes and CNPq. RPC is a
research fellow from CNPq. This work was supported by grants from CNPq, CAPES,
FAPERJ and MCT / PRONEX.
51
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61
Modulation of adenosine transport by MAP kinase cascade in cultured avian retinal
cells
Alexandre dos Santos-Rodrigues, Jainne M. Ferreira, and Roberto Paes-de-Carvalho*
Department of Neurobiology and Program of Neurosciences, Institute of Biology, Fluminense
Federal University, Niterói, Brazil;
*Corresponding author. Tel: +55-21-2629-2263; Fax: +55-21-2629-2268; E-mail address:
[email protected] (Roberto Paes de Carvalho).
62
Abstract
Adenosine (Ado) is an important modulator of neuronal survival and differentiation in the
CNS. Our previous work showed that Ado transporters are present in cultures of chick retinal
cells, but little is known about the mechanisms regulating Ado transport in these cultures. We
have also demonstrated that activation of Ado A1 receptors promotes an increase in ERK
phosphorylation in glial cultures. Our aim in the present work was to study the participation
of the ERK pathway on Ado uptake and release, as well as on adenosine metabolism in
different types of retinal cultures. In order to characterize the Ado transporter systems present
in the cultures, we tested the effect of NBMPR, an inhibitor of equilibrative nucleoside
transporters, and found a strong reduction of uptake. Moreover, preincubation of (3H) Ado
with 2 Units/ml adenosine deaminase for 45 minutes before addition to cultures promoted a
strong inhibition of uptake in mixed or glial cultures, indicating that the uptake is
predominantly for adenosine and not inosine. Adenosine kinase inhibition with
iodotubercidin also dramatically reduced the uptake in mixed or glial cultures. In glial
cultures, ERK inhibition with PD98059 (25 µM) reduced the uptake in 37.1 ± 4.3% (n=4). In
mixed cultures, PD98059 (25 µM) or UO126 (10 µM), two MEK inhibitors, reduced the
uptake in 39.1 ± 4.5% (n=5) and 48.6 ± 2.9% (n=3), respectively. U0124 (10 µM), an
inactive UO126 analog, did not have any effect in inhibiting Ado uptake. Glutamate (Glu)
induced the release of purines from mixed cultures (136.9 ± 31.0 % increase vs control, n=3)
and this effect was not significantly inhibited by PD98059 (79.6 ± 18.8% increase vs Glu,
n=3, p <0.05). This study showed that the equilibrative nucleoside transporter (ENT1) is the
main transporter present in mixed neuronal/glial or purified cultures of retinal glial cells.
Since ERK activity is regulated by stimulation of A1 receptors in the retina, our results
suggest that adenosine regulates its own transporter activity via activation of the ERK
pathway.
63
1. Introduction
Adenosine mediates its effects via specific G-protein-coupled receptors named A1, A2A,
A2B and A3 (Schulte & Fredholm, 2003; Fredholm et al., 2005). Activation of A1 receptors
has been shown to be neuroprotective during ischemia and epilepsy (Ribeiro et al., 2002;
Boison, 2008), and among synaptic modifications induced by stress in hippocampus, it was
shown a down regulation of these receptors (Cunha et al., 2006). Nevertheless, it is also
known that A1 receptors could exacerbate neurotoxic effects induced by kainate in cultured
cortical neurons (Rebola et al., 2005). Adenosine intra and extracellular levels are regulated
by bidirectional nucleoside transporters and enzymes related to purine metabolism.
Nucleoside transporters are divided in concentrative and equilibrative. There are 3 subtypes
of concentrative transporters (CNT1, CNT2 and CNT3) which promote influx of nucleosides
against its concentration gradient through the use of energy derived from sodium
concentration gradient present in cellular membranes (Podgorska et al., 2005). On the other
hand, there are four described subtypes of equilibrative transporters, called ENT1, ENT2,
ENT3 and ENT4 (Podgorska et al., 2005), which promote sodium-independent nucleoside
transport according to its concentration gradient. However, only ENT1 and ENT2 are
expressed in the CNS (Anderson et al., 1999a; Anderson et al., 1999b). Concerning the
enzymes, adenosine kinase and adenosine deaminase are the main players in adenosine
inactivation. At physiological conditions, adenosine kinase presents a smaller Km value than
adenosine deaminase (2µM and 17-45µM, respectively) in rat brain. Adenosine kinase has
two binding sites for adenosine, one of high affinity that corresponds to a catalytic site, and
another with a lower affinity that works as a regulatory site (Arrigoni & Rosenberg, 2006).
Inhibition of ADK activity induced a larger increase of adenosine levels in hippocampal and
cortical slices than adenosine deaminase inhibition (Pak et al., 1994). Few studies have
64
shown that ADK activity can be regulated by different kinases. For example, (McNally et al.,
1997) cloned ADK and identified several potential consensus sites for PKC phosphorylation.
In DDT1 MF-2 cell line, activation of A1 adenosine receptors inhibits ADK activity through a
mechanism involving PKC (Sinclair et al., 2000). Moreover, some reports show the
modulation of ADK expression or activity in different cellular models by different kinases
and signaling molecules, such as calcineurin (Hwang et al., 2001), ERK 1/2 activated by
insulin (Pawelczyk et al., 2003) and nitric oxide (Rosenberg et al., 2000). To our knowledge,
there are no reports on the expression of ADK in the retina and few works show
measurements of ADK activity in retina (Perez et al., 1986). However, several papers
reported the presence of adenosine, its receptors and transporters in retinas from different
species, such as goldfish (Studholme & Yazulla, 1997), rabbit (Perez et al., 1986), rat
(Schaeffer & Anderson, 1981) and chicken (Perez & Bruun, 1987; Paes de Carvalho et al.,
1990). In chicken retina, the presence of ENT1 nucleoside transporters was observed using
[3H] NBTI binding and shown to be present since embryonic day 8 up to post-hatching
animals (de Carvalho et al., 1992). This work also showed the presence of [3H] NBTI binding
sites mostly in plexiform layers, in localization similar to that of A1 receptors (de Carvalho et
al., 1992). Co-localization of ENT1 and A1 receptor was also observed in other CNS
structures (Jennings et al., 2001). The presence of ENT1 was previously detected in chick
retinal mixed (neurons + Muller glia) cultures (Paes-De-Carvalho, 2002) as well as in chick
purified retinal neuronal cultures (Paes de Carvalho et al., 1990). Some additional work has
already demonstrated that nucleoside transporters are modulated by protein kinases such as
PKC (Coe et al., 2002) and PKA (Sen et al., 1999). Our previous study showed that
adenosine uptake is modulated by CAMK II (Paes-de-Carvalho et al., 2005). Here, we have
investigated the mechanisms involved in the regulation of adenosine uptake in different types
of chick retinal cultures by different protein kinases, including ERK and PKC.
65
2. Materials and Methods
2.1 Materials
Fertilized White Leghorn eggs were obtained from a local hatchery and incubated at 38 °C in
a humidified atmosphere. L-Glutamine, N6-cyclohexyladenosine (CHA), Chelerythrine
chloride (CHE), adenosine deaminase (ADA), S-(p-nitrobenzyl)-6-thioinosine (NBTI),
adenosine, 5-Iodotubericidin, U0126, PD 98059, U-73122, Penicillin G and Streptomycin
Sulfate were obtained from Sigma/RBI Chem.Co. (Missouri, USA). U0124 was obtained
from Tocris Bioscience (Ellisville, MO, USA). Minimum Essential Media (MEM), heatinactivated fetal bovine serum (FBS) and Trypsin were obtained from GIBCO (New York,
USA). [2–3H] adenosine (22–28 Ci/mmol) was obtained from GE Healthcare Life Sciences
(Buckinghamshire, United Kingdom). All other reagents were of analytical grade.
2.2 Preparation of Mixed cultures
Mixed cultures of chick retina cells were prepared as previously described (De Mello, 1978).
Briefly, retinas from 8-day-old chick embryos (White Leghorn) were dissected from other
ocular tissues, including the pigmented epithelium and digested with 0.2% trypsin, in calcium
and magnesium-free Hank’s balanced salt solution (CMF), for 15-20 min at 37o C. The cells
were suspended in minimum essential medium supplemented (MEM) supplemented with 3%
heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml) and
glutamine (2 mM) and seeded in 24 wells tissue culture plastic dishes or 40 mm tissue culture
plastic dishes (for TLC assays) in a density of 2 x 104 cells/mm2. The cells were maintained
at 37o C in a humidified incubator with 95% air and 5% CO2. The medium was changed after
1 day in culture (C1) and experiments were performed at C3–C4.
66
2.3 Preparation of Purified Muller glia cultures
Purified Muller glia cultures of chick retina were prepared as previously described (Cossenza
& Paes de Carvalho, 2000), with minor modifications. Briefly, retinas from 11 -day-old chick
embryos (White Leghorn) were dissected from other ocular tissues, including the pigmented
epithelium, and digested chemically with 0.1% trypsin in calcium and magnesium-free
Hank’s balanced salt solution (CMF), for 20-25 min at 37 oC. The solution was then
removed, the cells suspended in MEM supplemented with 5% heat-inactivated fetal bovine
serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) and seeded in 24 wells tissue
culture plastic dishes or 40 mm tissue culture plastic dishes (for TLC assays) in a density of
1.8 x 106 cells/ml. Cultures were maintained at 37 oC in a humidified incubator with 95% air
and 5% CO2. The medium was changed every 3 days and experiments were performed at day
21 in culture. At this time, the presence of neurons is minimal and the glial cells turn
confluent in almost whole dish.
2.4 [3H] Adenosine uptake assays
All adenosine uptake assays were conducted at 37oC in a total volume of 250 µL/well of
HEPES-buffered salt solution (HBSS), containing NaCl 140 mM; KCl 5 mM; HEPES 20
mM; glucose 4 mM; MgCl2 1 mM and CaCl2 2 mM, pH 7.4. In sodium-free experiments,
NaCl was isosmotically substituted by lithium chloride. Before the experiments, the medium
was removed and the wells rinsed twice with 250 µL of HBSS solution. The cultured cells
were then preincubated for 10 min in the absence or presence of different drugs, and then
[3H]adenosine (0.2 µCi/ml) was added and the cultures further incubated for 15 min. The
wells were then rinsed twice with 250 µL of HBSS solution and lysed with water for
determination of intracellular radioactivity. The radioactivity was analysed by liquid
scintillation counting, in a TRI CARB® 2100TR liquid scintillation analyzer, for
67
determination of tritium retained by cells after addition of 2 mL of scintillation cocktail
(Optiphase Hi-Safe 2, Perkin-Elmer, Foster City, CA, USA) in a scintillation vial.
2.5 [3H] Adenosine release assays
For these assays, after incubation with [3H] Adenosine (2.5 µCi/ml), cultures were rinsed
twice with HBSS and further incubated with HBSS during four periods of 3 min to
completely remove the extracellular radioactivity. The cells were then incubated for 10 min
with HBSS in order to estimate the basal release. After this time, cultures were incubated for
another period of 10 min with HBSS containing PD 98059, a MEK inhibitor. A third period
of 10 min of incubation was performed with HBSS containing only glutamate or glutamate in
the presence of PD 98059. After this time, cells were lysed with water. The radioactivity of
all these samples (three different times of 10 min) was measured by liquid scintillation
spectroscopy. The results were normalized to percent of control after calculation of the
percent fractional release, that is, the percent of radioactivity released compared to
intracellular radioactivity at each period of time.
2.6 Identification of intracellular radioactivity by TLC
In order to analyse the intracellular radioactivity after [3H]adenosine uptake, cells were lysed
with 5% TCA, removed from the dishes, the material centrifuged at 20,627 g for 15 min and
the supernatant mixed with standards, applied to TLC plates and run in a mixture of butanol,
ethyl acetate, methanol and amonium hydroxide (7:4:3:4) (Schrader & Gerlach, 1976),
1976).The spots detected under UV light were scrapped off from the plates and the
radioactivity determined by scintillation counting. The released material was mixed with 5%
TCA immediately after collected from the dishes and processed as above.
68
2.7 Statistical analysis
Data are presented as the mean ± SEM from n experiments. Statistical significance was
assessed by one-way ANOVA followed by the Bonferroni test, using GraphPad Software
(Prism, version 4.02 for Windows). Values of p < 0.05 were considered statistically
significant.
69
3. Results
First of all, we aimed to characterize the kinetic parameters involved in adenosine uptake in
mixed and purified glial cultures in order to define the time of incubation with [3H]adenosine,
as well as the adenosine concentration to be used in subsequent experiments. Adenosine
transport in mixed cultures was concentration-dependent and exhibited two components, one
with a Km1 of 0.19 µM and a Vmax1 of 61.4 ± 2.1 pmol/mg protein.min and another with a Km2
of 136.1 µM and a Vmax2 of 594.9 ± 36.04 pmol/mg protein.min (Fig. 1A). In contrast,
different Km values were obtained in purified glial cultures (Km1 of 0.037 µM and Km2 of 1.2
µM, respectively, Fig. 1B). The time courses of adenosine uptake for incubation times
ranging from 0.5 to 15 min were linear and after this time we observed a tendency to
equilibrium in both types of cultures (Figs. 1C). All subsequent experiments were performed
using the incubation time of 15 minutes for both types of cultures. We have analyzed the
relative contribution of ENTs and CNTs for [3H] Adenosine uptake by the cultures.
Adenosine transport was almost completely NBTI-sensitive and not affected by sodium
removal, suggesting that ENT1 and 2 are the primary transporters present in both cultures
(Fig. 2A). Preincubation of [3H] Adenosine with adenosine deaminase (2U/ml) before
addition to cultures strongly reduced the uptake in both mixed and glial cultures (Fig. 2B),
demonstrating a low efficiency of cells to take up inosine, the product of adenosine
deamination. We then tested the effect of different concentrations of NBTI, a selective
inhibitor of ENT1 and/or ENT2, on [3H] Adenosine uptake by mixed and purified glial
cultures (Fig. 3). IC50 values (concentration of NBTI promoting 50% inhibition of [3H]
Adenosine uptake) were 11.7 nM and 1.86 nM in mixed and purified glial cultures,
respectively. In order to verify if adenosine kinase was an important driving force for
adenosine uptake in retinal cultures, we used 5’-Iodotubercidin, a known inhibitor of
70
Figure 1: Kinetic characterization of Adenosine uptake in mixed cultures (A,C) and
purified glial cultures (B,C). (A,B) Cultures were washed and incubated with
[3H]Adenosine (0.2 µCi/mL) plus different adenosine non-labeled concentrations for 15 min
at 370 C or incubated with [3H]Adenosine (0.2 µCi/mL) for different periods of time (C),
rinsed in buffer, and lysed in water to determine intracellular radioactivity as described in
Materials and Methods. Values represent means ± SEM and the results shown are from
representative experiments performed in triplicate. The points without bars represent the
results in which the deviation from the mean was smaller than the symbol size. Inset of
(A,B): Eadie-Hofstee plot of the data. The Km1 of 0.19 µM and a Vmax1 of 61.4 ± 2.1 pmol/mg
protein.min and another with a Km2 of 136.1 µM and a Vmax2 of 594.9 ± 36.04 pmol/mg
protein.min values to mixed cultures. For purified glial cultures, the values were Km1 of 0.037
µM and Km2 of 1.2 µM.
71
A
Total
W/O Sodium
Total
W/O Sodium
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80
60
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20
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60
**
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nM
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B
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nM
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nM
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l
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tr
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l
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C
[3H]Adenosine uptake
(% of control)
[3H]Adenosine uptake
(% of control)
100
B
[ 3H]Adenosine uptake
(% of control)
100
80
ADA 2U/ml
60
***
40
***
20
0
Control
Mixed
Glia
Figure 2: (A) Characterization of relative ratio between CNTs and ENTs in mixed (left)
and purified glial cultures (right). For sodium-free experiments, the NaCl in normal HBSS
buffer was replaced with LiCl. NBTI (100 or 500 nM) was added 10 min before the addition
of [3H]adenosine; uptake was measured for 15 min. Total is uptake carried by ENTs and
CNTs in buffer containing NaCl. The equilibrative transport (uptake carried by ENT1 and
ENT2) was measured in buffer containing lithium chloride instead of NaCl. The values are
the mean ± S.E.M. from at least three separate experiments performed in triplicate. ***p <
0.001 (compared to control “total”), **p < 0.01 (compared to control “total”), ###p < 0.001
(compared to control “w/o sodium”), ##p < 0.01 (compared to control “w/o sodium”). (B)
Effect of metabolism of [3H]adenosine into inosine on adenosine uptake in mixed and
purified glial cultures. Retinal cultures were incubated with [3H]Adenosine (0.2 µCi/mL)
for 15 min at 370 C after incubation of [3H]adenosine with adenosine deaminase (2U/ml) for
45 min at 370 C prior to the exposure to the cells. The values are the mean ± S.E.M. from at
least three separate experiments performed in triplicate. ***p < 0.001 (compared to control).
72
[3H] Adenosine uptake
(% of control)
100
Mixed
Glia
80
60
40
20
-9
-8
-7
-6
-5
log [NBTI] (M)
Figure 3: NBTI inhibition of [3H]adenosine uptake in mixed and purified glial cultures.
Cells were pre-incubated in the absence or presence of graded NBTI concentrations to 10 min
and after that they were incubated with [3H]Adenosine (0.2 µCi/mL) for 15 min at 370 C. The
values are the mean ± S.E.M. from at least three separate experiments performed in triplicate
and error bars are not shown where the S.E.M. values were smaller than the size of the
symbol. Uptake values represent the percentage of [3H]adenosine uptake in the presence of
NBTI relative to that in its absence (control).
73
adenosine kinase. A significant reduction of uptake was observed in both cultures after
treatment with this compound at different concentrations (Fig. 4).
It is already known that ERK is able to modulate the expression and/or activity of
neurotransmitter transporters, as for example glutamate transporters GLT-1 and GLAST
(Matos et al., 2008) and the dopamine transporter DAT (Moron et al., 2003). The effect of
MEK inhibitors on [3H] Adenosine uptake was studied in our cultures. MEK inhibition with
PD 98059 (25 µM) induced a strong decrease on uptake in mixed, purified glial cultures and
purified neuronal cultures (Fig. 5A and C). UO126, another MEK inhibitor, produced
concentration-dependent inhibitory effects on [3H] Adenosine uptake in both mixed and
purified glial cultures (figs. 5B and C). To verify whether this observed decrease on uptake
promoted by different inhibitors was due to inhibition of adenosine metabolism, we
performed experiments measuring adenosine metabolites using TLC. No change was detected
on components of metabolism in mixed cultures after treatment with the two abovementioned MEK inhibitors (Fig. 5D).
Some reports in the literature indicate that some kinase inhibitors could have nonspecific effects on nucleoside transporters in a way independent of kinase inhibition (Huang
et al., 2002; Huang et al., 2003). We then used U0124, an analogue of U0126 that does not
inhibit MEK activity, and found no change in uptake with this inactive analogue (Fig. 6A).
Since the nucleoside transporter is bidirectional, we also tested whether inhibition of MEK
had any effect on the release of purines induced by glutamate in mixed cultures. As can be
observed in fig. 6B, a small but not significant change on release was observed when cultures
were treated with glutamate plus PD 98059 (25 µM).
74
[3H] Adenosine uptake
(% of control)
100
ITU 100 nM
80
60
ITU 300 nM
***
***
***
***
40
20
0
Control Mixed
Glia
Mixed
Glia
Figure 4: Inhibition of adenosine kinase reduces [3H]Adenosine uptake in mixed and
purified glial cultures. Cells were pre-incubated in the absence or presence of different ITU
concentrations (100 or 300 nM) to 10 min and after that they were incubated with
[3H]Adenosine (0.2 µCi/mL) for 15 min at 370 C. The values are the mean ± S.E.M. from at
least three separate experiments performed in triplicate. ***p < 0.001 (compared to control).
75
A
B
100
80
[3H] Adenosine uptake
(% of control)
[ 3H] Adenosine uptake
(% of control)
100
PD 98059 (25 µM)
60
***
40
***
***
20
0
Control
Mixed
Glia
80
60
40
∞
Neurons
0.1
1
10
100
[UO126] (µ
µM)
C
[3H] Adenosine uptake
(% of control)
90
80
70
60
50
40
0.01
0.1
1
10
log [inhibitor]( µM)
100
% of total intracellular radioactivity
D
100
100
80
Control
PD 98059
U0126
60
40
20
0
NT Ino Hx AdoAde
NT Ino Hx AdoAde
NT Ino Hx AdoAde
Figure 5: Effect of MEK inhibitors PD98059 and U0126 on the uptake and metabolism
of [3H]adenosine by cultured retinal cells. (A) Cultures were washed and preincubated in
the absence or presence of PD 98059 (25 µM) for 10 min prior to incubation with
[3H]adenosine for 15 min. Cells were then lysed and the intracellular radioactivity
determined. The asterisks (***) denote that the difference is statistically significant (p <
0.001) compared to control. (B) Mixed cultures were washed and preincubated for 10 min
with different concentrations of U0126 before incubation with [3H]adenosine for 15 min and
cell lysis for determination of intracellular radioactivity. Values represent the mean ± S.E.M.
from at least three separate experiments performed in triplicate. (C) Purified glial cultures
were washed and preincubated for 10 min with different concentrations of U0126 (circles) or
PD 98059 (squares) before incubation with [3H]adenosine for 15 min and cell lysis for
determination of intracellular radioactivity. To (A, B and C), values represent the mean ±
S.E.M. from at least three separate experiments performed in triplicate. (D) Distribution of
intracellular radioactivity after [3H]adenosine uptake in control and PD 98059 (25 µM) or
U0126 (10 µM)-treated mixed cultures. After incubation with [3H]adenosine, intracellular
radioactivity was extracted and the samples applied with standards to TLC plates. The results
are expressed in percent of total radioactivity and represent the mean ± S.E.M. from three
separate experiments.
76
A
B
80
***
60
40
20
Fractional Release
(% of Control)
240
200
160
120
80
40
PD
+
LU
G
B
A
SA
L
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01
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U
01
2
4
10
10
µM
µM
ol
on
tr
C
LU
0
0
G
[3H] Adenosine uptake
(% of control)
***
100
Figure 6: Effect of U0124, an inactive analogue of U0126, on the uptake of
[3H]adenosine and effect of PD 98059 (25 µM) on the release of [3H]purines stimulated
by glutamate in mixed cultures. (A) Cultures were washed and preincubated in the absence
or presence of U0124 (15 µM) for 10 min prior to incubation with [3H]adenosine for 15 min.
Cells were then lysed and the intracellular radioactivity determined. The asterisks (***)
denote that the difference is statistically significant (p < 0.001) compared to control. (B)
Cultures were washed and incubated with saline in the absence or presence of PD 98059 (25
µM) and stimulated with glutamate (1 mM). A slight but not significant decrease of release
stimulated by glutamate was observed in stimulus of glutamate with PD 98059.
77
We went further to investigate if other protein kinases were able to modulate [3H] Adenosine
uptake in our cultures. A strong decrease on uptake was observed in mixed or purified glial
cultures treated with the PKC inhibitor Chelerythrine chloride (CHE) (Fig. 7A). Furthermore,
inhibition of phospholipase C with U73122 also promoted a decrease on the uptake in mixed
cultures (Fig. 7B). Finally, a significant decrease on the uptake was observed with two
different CAMK II inhibitors (KN62 and KN93, both at 1 µM) (Fig. 7C) in purified glial
cultures, a similar effect than that previously observed in mixed cultures (Paes-de-Carvalho et
al., 2005).
78
Mixed
Glia
110
95
80
65
50
[3H] Adenosine uptake
(% of control)
B
[3H] Adenosine uptake
(% of control)
A
100
80
60
40
20
35
-9
-8
-7
-6
log [CHE] (M)
-5
∞
-7
-6
-5
log [U73122] (M)
C
[3H] Adenosine uptake
(% of control)
100
80
60
*
40
20
K
N
93
62
N
K
C
on
tro
l
0
Figure 7: Effect of PKC inhibitor (CHE), PLC inhibitor (U73122) and CAMK II
inhibitors (KN62 or KN93) on the uptake of [3H]adenosine by cultured retinal cells. (A)
Cells were pre-incubated in the absence or presence of graded CHE concentrations to 10 min
and after that they were incubated with [3H]Adenosine (0.2 µCi/mL) for 15 min at 370 C. The
values are the mean ± S.E.M. from at least three separate experiments performed in triplicate
and error bars are not shown where the S.E.M. values were smaller than the size of the
symbol. Uptake values represent the percentage of [3H]adenosine uptake in the presence of
CHE relative to that in its absence (control). (B) Mixed cultures were pre-incubated in the
absence or presence of graded U73122 concentrations to 10 min and after that they were
incubated with [3H]Adenosine (0.2 µCi/mL) for 15 min at 370 C. The values are the mean ±
S.E.M. from at least three separate experiments performed in triplicate and error bars are not
shown where the S.E.M. values were smaller than the size of the symbol. Uptake values
represent the percentage of [3H]adenosine uptake in the presence of U73122 relative to that in
its absence (control). (C) Purified glial cultures were washed and preincubated with the
CAMK II inhibitors (KN62 or KN93, both at 1 µM) for 10 min prior to incubation with
[3H]adenosine for 15 min. Cells were then lysed and the intracellular radioactivity
determined. Values represent the mean ± S.E.M. from at least three separate experiments
performed in triplicate. The asterisks (*) denote that the difference is statistically significant
(p < 0.05) compared to control.
79
4. Discussion
P1 purinergic markers such as different receptors and transporter system are known to be
expressed in the retina in vivo and in cultured retinal cells (Braas et al., 1987; Perez & Bruun,
1987; Paes de Carvalho et al., 1990; de Carvalho et al., 1992; Paes-de-Carvalho et al., 2005).
However, very little is known about the characteristics of [3H] Adenosine uptake and the
mechanisms involved in the regulation of intracellular or extracellular nucleoside levels. Our
previous work has already demonstrated that uptake sites labeled with [3H] NBTI and
detected using binding and autoradiographic methods are present in developing chicken
retinas from embryonic day 8 (E8) up to post-hatching animals (de Carvalho et al., 1992). At
E8, NBTI binding sites showed a diffuse distribution, but are localized to the plexiform layers
of more developed retinas, a similar localization as that found for A1 receptors (de Carvalho
et al., 1992). This co-localization between ENT1 and A1 adenosine receptors was also
observed in different brain structures (Jennings et al., 2001). In the present study, we have
shown that ENTs are the main mediators of [3H] Adenosine uptake in mixed and glia-purified
retinal cultures, in a way similar to what was found in cultured chick retinal neurons (Paes de
Carvalho et al., 1990), where [3H] Adenosine uptake was inhibited more than 80% by NBTI.
We also observed that the main nucleoside transporter expressed in mixed cultures is ENT1,
because in absence of sodium and presence of 100 nM NBTI, a concentration selective for
inhibition of only ENT1, a large decrease in [3H] Adenosine uptake (approximately 60%) was
detected. The presence of adenosine uptake sites has already been studied by binding of [3H]
NBTI in mixed retinal cultures (Paes-De-Carvalho, 2002), but there was no data about the
type of transporters expressed in these cultures. In purified glial cultures, most [3H]
Adenosine uptake is also mediated by ENTs. The adenosine uptake systems present in mixed
and in purified glial cultures are more specific to take up adenosine than inosine, since we
80
had a large decrease of uptake in both cultures when [3H] Adenosine was preincubated with
adenosine deaminase. This result is in accordance with data showing that ENT1 presents low
affinity for inosine (Km 170 µM as compared to 40 µM for adenosine), while ENT2 has an
affinity almost 4-fold higher for inosine than ENT1 (Km 50 µM) (Ward et al., 2000; Kong et
al., 2004). Accordingly, the study by (Zamzow et al., 2008) indicates that inosine is released
through ENT2.
Besides nucleoside transporters, adenosine kinase and adenosine deaminase are also
important mediators in the control of adenosine levels. Our previous work has already
demonstrated that, in mixed and purified neuronal cultures, most of the taken up [3H]
Adenosine is converted to adenine nucleotides (Paes de Carvalho et al., 1990; Paes-deCarvalho et al., 2005). A similar result was observed in mouse primary cultured cortical
astrocytes (Matz & Hertz, 1989). In purified retinal glial cultures, similarly to what was
observed in other retinal cultures, we also have an intense conversion of taken up [3H]
Adenosine to nucleotides. These data suggest that chick retinal cultures have a strong
adenosine kinase activity. On the other hand, some groups have already shown, using
different models such as chromaffin cells (Miras-Portugal et al., 1986) and mouse primary
cultured cortical neurons (Matz & Hertz, 1989), that most taken up adenosine is converted to
inosine/hypoxanthine, indicating a larger adenosine deaminase activity in respect to
adenosine kinase. We observed a significant reduction of [3H] Adenosine uptake in mixed
and purified glial cultures treated with 5’-Iodotubercidin, an adenosine kinase inhibitor.
However, we have not detected a significant decrease in the generation of nucleotides after
treatment with 5’-Iodotubercidin (data not shown).
Nucleoside transporters may have their activity regulated by activation/inhibition of
protein kinases, despite the cellular mechanisms involved in these events still remain not
81
understood. PKA (Nagy et al., 1991; Sen et al., 1999), PKC (Delicado et al., 1991; Coe et al.,
2002; Pinto-Duarte et al., 2005), CK II (Bone et al., 2007) and CAMK II (Paes-de-Carvalho
et al., 2005) are examples of kinases previously reported to modulate adenosine uptake. In
this study, PD 98059 and U0126, two different inhibitors of MEK, caused a strong and
significant decrease on [3H] Adenosine uptake in both mixed and purified glial cultures, and a
similar effect was observed in purified retinal neuronal cultures. Mouse adenosine kinase
sequence is known to have potential phosphorylation sites for several protein kinases,
including ERK (Sahin et al., 2004). However, this same work showed that ERK was not able
to phosphorylate recombinant mouse adenosine kinase in vitro. On the other hand, insulin has
been shown to modulate adenosine kinase expression in rat lymphocytes by a mechanism
mediated by MAPK cascade (Pawelczyk et al., 2003). In order to test the possibility that the
effect of MEK inhibitors on uptake of [3H] Adenosine by retinal cultures was due to
inhibition of adenosine kinase activity, we performed experiments using TLC that showed
that these inhibitors do not change the enzyme activity, raising the possibility that ERK is
directly modulating transporter activity and not adenosine metabolism. To our knowledge,
this is the first report demonstrating a role of ERK per se in the control of ENT activity.
Regarding CNTs, one report states that all-trans-retinoic Acid (ATRA)-induced human CNT3
trafficking to the plasma membrane is mediated by a TGF-β1 and ERK ½-dependent
mechanim (Fernandez-Calotti & Pastor-Anglada, 2010). (Huang et al., 2002; Huang et al.,
2003)performed a screening work to identify possible effects of different protein kinase
inhibitors on uptake of nucleosides in human erythroleukemia K562 cell line, which is a cell
line where most nucleoside transport occurs through an equilibrative NBTI-sensitive (es)
transport. They observed, using this cell model, that inhibitors of receptor tyrosine kinases,
PKC, cyclin-dependent kinases and p38 MAPK can affect nucleoside transport in an kinaseindependent fashion. It is interesting to stand out that they did not observed any effect on
82
uptake with the same MEK inhibitors used in the present work, PD 98059 and U0126.
Additionally, another report has suggested that genistein, a tyrosine kinase inhibitor, although
used in a very high concentration (100 µM), could act directly on nucleoside transporter
(Pillai & Shivakumar, 2009). U0124, an inactive analog of U0126, had no effect activity on
[3H] Adenosine uptake in our cultures, strongly indicating that the effects of different MEK
inhibitors on [3H] Adenosine uptake is due to inhibition of kinase itself, and not to eventual
unspecific effects directly on nucleoside transporters. In mixed cultures, CAMK II inhibition
was shown to decrease [3H] Adenosine uptake and also to block an increase on the release of
purines stimulated by glutamate (Paes-de-Carvalho et al., 2005). However, no significant
effect on the release of purines induced by glutamate was observed in mixed cultures. The
significance of this finding is presently unknown but it could point to a differential effect on
nucleoside transporter, depending if it is taking up or releasing the nucleoside.
We have shown that treatment of retinal cultures with a broad spectrum PKC
inhibitor, chelerythrine chloride, led to a significant decrease in [3H] Adenosine uptake.
These results are in agreement with previous reports showing the modulation of nucleoside
transporter activity by PKC (Delicado et al., 1991; Coe et al., 2002; Chaudary et al., 2004;
Pinto-Duarte et al., 2005). We also observed a strong decrease in [3H] Adenosine uptake in
mixed cultures after acute treatment with U73122, a phospholipase C inhibitor. Another
important kinase that was shown to modulate nucleoside transport in our cultures was CAMK
II, in a way similar to that previously shown in retinal mixed cultures (Paes-de-Carvalho et
al., 2005). Indeed, KN62 and KN93, two CAMK II inhibitors, inhibited [3H] Adenosine
uptake in purified glial cultures, suggesting that kinase modulates a nucleoside transporter,
but the mechanisms involved remain to be clarified.
83
Concerning the results obtained with [3H] Adenosine uptake in our retinal mixed and
purified glial cultures, it is interesting to mention that most effects observed were identical or
very similar in both cultures. This raises some possibilities to explains those results: i) the
observed effects could be operated mainly by Muller glial cells, since the results observed in
mixed cultures are similar to that observed in purified glial cultures; ii) another alternative
could be the hypothesis proposed by (Kubrusly et al., 2005; Kubrusly et al., 2008), that, in
the absence of neurons, Muller glial cells might express proteins that normally are only
associated with neurons, such as dopaminergic markers and TUJ1 (an immature neuronal
marker) as well as GABAergic markers (De Sampaio Schitine et al., 2007). In this case, the
effects observed herein could be operated mainly by neurons or a neuronal phenotype, in case
of purified glial cultures.
5. Acknowledgements
We acknowledge Ms. Luzeli R. de Assis and Ms. Sarah A. Rodrigues for the technical
assistance. This work was supported by grants from CNPq, CAPES, FAPERJ, and
PRONEX/MCT. A.S.R. was supported by a scholarship from CAPES; J.M.F. was supported
by a scholarship from CNPq. RPC is a research fellow from CNPq.
84
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Equilibrative Nucleoside Transporter 1 (ENT1)-mediated adenosine transport in
cultures of rat cortical astrocytes: Modulation by activation of adenosine A1 receptors.
Alexandre dos Santos-Rodrigues1,2, Marco Matos1, Roberto Paes-de-Carvalho2, Paula
Agostinho1 , and Rodrigo A. Cunha1,*
1
Center for Neurosciences and Cell Biology of Coimbra, University of Coimbra, 3004-504
Coimbra, Portugal;
2
Department of Neurobiology and Program of Neurosciences, Institute of Biology,
Fluminense Federal University, Niterói, Brazil;
*Corresponding author. Tel: +351-239 820190; Fax: +351-239 822776; E-mail address:
[email protected] (Rodrigo A. Cunha).
90
Abstract
Adenosine (Ado) is an important neuromodulator in the CNS, regulating synaptic
transmission and plasticity, cell proliferation and differentiation, and also cellular repair
processes and neurodegeneration. Ado acts through A1, A2A, A2B and A3 receptors, and
controls neuronal activity through a synaptic action on inhibitory A1 receptors and facilitatory
A2A receptors, although it has also been proposed to control glial functions (Fredholm et al.,
2005, Int Rev Neurobiol 63:191-270). Glial cells play fu ndamental roles in the CNS, such as
regulation of neuronal migration, neuroprotection and control of extracellular glutamate
concentrations. The extracellular levels of adenosine are tightly controlled: adenosine buildsup extracellularly as a function of neuronal activity and nucleoside (adenosine) transporters
play a key role in the rapid removal of this extracellular adenosine; this mainly occur through
Equilibrative Nucleoside Transporters, ENT1 and ENT2, which are located both in neurons
and astrocytes. In this study, we investigated if the activation of adenosine A1 receptors
affected adenosine transport and if this was due to changes in the levels of ENT1, evaluated
by Western blot and 3H-NBTI (nitrobenzylthioinosine) binding. Astrocytic cultures were
prepared from cerebral cortices of 4-5-day postnatal Wistar rats and were maintained for 20–
21 days in vitro (DIV). We found that 3H-adenosine uptake by cultured cortical astrocytes
was linear from 15 to 300 seconds. Most of 3H-adenosine uptake in these cells (73.6±7.4% of
total, n=3) was due to ENT activity. The use of NBTI (a selective antagonist of ENT1)
revealed that ENT1 was mostly responsible for this
3
H-adenosine uptake by ENTs
(68.8±8.0%, n=3). Western blot analysis confirmed the presence of ENT1 in cultured
astrocytes, and the 3H-NBTI binding was saturable and reached equilibrium after 2 minutes.
Activation of A1 receptor with its selective agonist CPA (100 nM) decreased 3H-adenosine
uptake and increased the Kd of 3H-NBTI binding. These data identify ENT1 as the main
adenosine transporter in astrocytes and show that the activation of adenosine A1 receptors
might control the rate of adenosine clearance by astrocytes.
Keywords: Cortical astrocytes primary cultures, Adenosine, Equilibrative Nucleoside
Transporters
91
1. Introduction
Adenosine is an important neuromodulator in the CNS, regulating synaptic transmission and
plasticity, cell proliferation and differentiation, and also cellular repair processes and
neurodegeneration (Stevens et al., 2002; Rebola et al., 2008; Canas et al., 2009). Adenosine
acts through interaction with A1, A2A, A2B and A3 receptors, and controls neuronal activity
through a synaptic action on inhibitory A1 receptors and facilitatory A2A receptors (for
review, (Cunha, 2008). In ischemic events, the main modulatory effect occurs through A1
receptors activation, promoting a decrease of glutamate release which is important for
controlling the excitotoxic lesion induced by excessive glutamate release (Ribeiro et al.,
2002). Adenosine can be found in intracellular medium through uptake mediated by
nucleoside transporters or can be synthesized through conversion of AMP catalysed by the
enzyme 5´- nucleotidase. In extracellular medium, adenosine can be found after release
mediated by transporters, or after the conversion of released ATP catalysed by ectonucleotidases (Dunwiddie et al., 1997). The main enzymes responsible for adenosine
metabolization are adenosine kinase, which generates AMP, and adenosine deaminase, which
metabolizes adenosine into inosine. Adenosine levels in intra and extracellular medium are
regulated by bidirectional nucleoside transporters, which are divided in concentrative and
equilibrative nucleoside transporters. There are 3 subtypes of concentrative transporters
(CNT1, CNT2 and CNT3) and these transporters promote an influx of nucleosides against its
concentration gradient through the use of energy derived from sodium concentration gradient
present in cellular membranes (Podgorska et al., 2005). At present, there are 4 subtypes of
equilibrative transporters described, called ENT1, ENT2, ENT3 and ENT4 (Podgorska et al.,
2005). These transporters are divided in sensitive and insensitive to inhibitor S-(pnitrobenzyl)-6-thioinosine (NBTI) (Podgorska et al., 2005) and can transport nucleosides to
92
inside or outside the cells according to intra and extracellular concentrations. Although it has
not yet been demonstrated whether the modulation of ENTs occurs by direct phosphorylation
and/or via interactions with secondary proteins, there are many reports on the modulation of
these transporters by different stimuli, such as nitric oxide (Farias et al., 2010), insulin and
glucose (Sakowicz et al., 2004; Grden et al., 2008), and protein kinases like PKC (Delicado
et al., 1991; Coe et al., 2002; Pinto-Duarte et al., 2005), PKA (Sen et al., 1999), casein
kinase II (Bone et al., 2007) and CAMK II (Paes-de-Carvalho et al., 2005). Some studies
have shown a close relationship between ENT1 and A1 adenosine receptors. For example,
ENT1-null mice demonstrated a tendency for a greater consumption of alcohol when
compared with their wild-type littermates and this effect seems to be correlated with a
reduction of glutamate excitatory postsynaptic currents (EPSCs) mediated by A1 receptors in
nucleus accumbens, an important region involved in regulating drug reward and selfadministration (Choi et al., 2004). Another group also observed that nucleoside transporters
and A1 receptors are able to modulate glutamatergic synaptic transmission in slices of rat
spinal cord in vitro (Ackley et al., 2003). Moreover, (Jennings et al., 2001) demonstrated a
co-localization between human ENT1 and A1 adenosine receptors in different brain structures
suggesting that this transporter plays an important role in regulating the neuromodulatory
action of A1 receptors.
Adenosine receptors and its metabolic pathways are also present in astrocytes but
their functions are still largely unknown (Fredholm et al., 2005). In the present study we
aimed to determine the relative involvement of ENT1 and ENT2 on adenosine uptake in
primary cultures of rat cortical astrocytes and to investigate the role of adenosine A1 receptor
activation on adenosine uptake mediated by nucleoside equilibrative transporters.
93
2. Experimental Procedures
2.1 Materials
N6-cyclopentyladenosine
(CPA),
adenosine
deaminase
(ADA),
S-(p-nitrobenzyl)-6-
thioinosine (NBTI), adenosine and mouse monoclonal anti-β-actin antibody were from Sigma
(St Louis, MO, USA). Dipyridamole was from Tocris (Ballwin, MO, USA). [2–3H]
adenosine (22–28 Ci/mmol), the polyvinylidene difluoride (PVDF) membranes, enhanced
chemifluorescence (ECF) and anti-mouse IgG alkaline phosphatase-conjugated secondary
antibody were from GE Healthcare Life Sciences (Buckinghamshire, United Kingdom). Goat
polyclonal anti-ENT1 antibody and alkaline phosphatase-conjugated anti-goat secondary
antibody were from Santa Cruz (Santa Cruz Biotechnology, Frilabo, Portugal). [G-3H] S-(pNitrobenzyl)-6-thioinosine ([3H] NBTI) (16.8 Ci/mmol) was from Moravek Biochemicals
(Brea, CA, USA).
2.2 Cortical astrocyte primary cultures
Primary astrocyte cultures were prepared from cerebral cortices of 3–5-day postnatal Wistar
rats according to previous described procedures (Harris et al., 1996; Saura, 2007), but with
some modifications. In brief, mice were killed by cervical dislocation and the brain was
removed. The left and right cerebral cortices were removed and isolated in ice-cold Hanks’
balanced salt solution (HBSS) (in mM: 137 NaCl, 5.4 KCl, 0.17 Na2HPO4, 0.22 KH2PO4,
2.3 NaHCO3, 10 Hepes, 2.7 D-glucose, 0.073 Phenol Red, pH 7.4). The meninges were then
removed and the cortices chopped up and incubated with a digestive medium containing
0.25% Trypsin and 0.25 mg/ml DNAse in HBSS at 37 °C for 15–20 min. Then, the
enzymatic digestion was stopped with 10% of heat-inactivated FBS and the cell suspension
centrifuged for 5 min at 180xg. Afterward, the obtained pellet was resuspended in astrocyte
94
culture medium (Dulbecco’s modified Eagle medium (DMEM)—high glucose—
supplemented with 10% FBS, penicillin (50 U/ml), streptomycin (10 mg/ml), Hepes (6 g/l),
and sodium bicarbonate (0.84 g/l)), and the number of cells in suspension counted in a
hemocytometer. Then, the cells were plated onto poly-L-lysine-coated 75-cm2 culture flasks,
at a density at 1.14x105 cells/cm2, and maintained at 37 °C in a 5% CO2/95% room-air
humidified incubator. The cell culture medium was frequently replaced, every 2–3 days, until
the mixed-glial cultures reached confluency, which was normally achieved after 13–15 days
of culture in vitro (DIV). In order to separate microglial cells from the astrocytes monolayer,
the mixed glial-cultures were shaken at 200 rpm in an orbital shaker for 4 h. Then, the
medium with the up-layer detached microglial cells was discarded and the astrocytes that
remained in the flasks washed with HBSS buffer containing EDTA (1 mM) and further
detached by a mild trypsinization procedure using HBSS with 0.1% trypsin. Finally, the cells
were reseeded with fresh astrocyte culture medium on poly-L-lysine-coated plates, at a
density of 3x104cells/cm2, and maintained in culture for 1–2 days before the experiments
beginning.
2.3 [3H] Adenosine uptake assay
All adenosine transport assays were conducted at 37oC in a total volume of 300 µL/well of
Krebs solution with the following composition: NaCl 132 mM, KCl 4 mM, NaH2PO4 1.25
mM, MgCl2 1.4 mM, CaCl2 1 mM, HEPES 10 mM, glucose 6 mM, pH 7.4. In sodium-free
experiments, NaCl was isosmotically substituted by N-Methyl-D-glucamine, which does not
have sodium. Before the experiments, the medium was removed and the wells rinsed twice
with 300 µL of Krebs solution. The cultured cells were then preincubated for 20 min in the
absence or presence of different drugs. Uptake was started by addition of [3H]adenosine (0.2
µCi/ml) during 1 min and the wells were further rinsed twice with 300 µL of Krebs solution
95
and lysed with NaOH 0.1 N for determination of intracellular radioactivity. The radioactivity
was analysed by liquid scintillation counting, in a TRI CARB® 2900TR liquid scintillation
analyzer, for determination of tritium retained by cells after addition of 2 mL of scintillation
cocktail (Optiphase Hi-Safe 2, Perkin-Elmer, Foster City, CA, USA) in a scintillation vial.
Adenosine uptake was calculated as the difference between the total amount of adenosine
taken up by cultured cells and the nonspecific component of [3H]adenosine fixation by
cultured cells, determined in the presence of dipyridamol (20 µM), NBTI (10 µM) and
adenosine (1 mM).
2.4 [3H] NBTI binding assays
All binding assays were conducted at 37oC or 4oC in a total volume of 300 µL/well of Krebs
solution with the following composition: NaCl 132 mM, KCl 4 mM, NaH2PO4 1.25 mM,
MgCl2 1.4 mM, CaCl2 1 mM, HEPES 10 mM, glucose 6 mM, pH 7.4. Cortical astrocyte
primary cultures in 24-well plates were washed twice with 300 µL of Krebs solution. All
experiments were carried out in the presence of 2 units ml-1 of adenosine deaminase (ADA)
to inhibit the effects of released endogenous adenosine. The cultured cells were then
preincubated for 20 min in ADA solution above, in the absence or presence of different
drugs. Cells were incubated with 1 nM [3H] NBTI for 10 min and the wells were further
rinsed twice with 300 µL of Krebs solution and lysed with NaOH 0.1 N for determination of
intracellular radioactivity. Specific binding was calculated as total binding less non-specific
binding, determined in the presence of 10 µM unlabeled NBTI. The radioactivity was
analysed by liquid scintillation counting, in a TRI CARB® 2900TR liquid scintillation
analyzer, for determination of tritium retained by cells after addition of 2 mL of scintillation
cocktail (Optiphase Hi-Safe 2, Perkin-Elmer, Foster City, CA, USA) in a scintillation vial.
96
2.5 Western blot analysis
Cortical astrocyte primary cultures in 12-well plates were gently scraped in lysis buffer
(50 mM KCl, 50 mM PIPES, 10 mM EGTA, 2 mM MgCl2, 0.5% Triton X-100, 1 mM
PMSF, 1 mM dithiothreitol and 5 µg/ml of a mixture of protease inhibitors containing
chymostatin, leupeptin, pepstatin A and antipain) and subject to 3 freezing cycles at − 80 °C.
Cortical astrocytes extracts were diluted at the final concentration of 2 µg protein/µl in SDS–
PAGE buffer and proteins were separated by SDS–PAGE (7.5% with a 4% concentrating gel)
under reducing conditions and electro-transferred to polyvinylidene difluoride membranes
(PVDF). After electro-transfer, membranes were blocked for 1 h at room temperature with
3% bovine serum albumin in Tris-buffered saline, pH 7.6 containing 0.1% Tween 20 (TBST), then they were incubated overnight at 4 °C with goat polyclonal anti-ENT1 antibody
(1:500). After that, membranes were washed three times for periods of 15 min with TBS-T
and incubated with an alkaline phosphatase-conjugated anti-goat secondary antibody
(1:5000) for 1 h. Membranes were washed three times for periods of 15 min and the
membranes were then analysed with a VersaDoc 3000 (Biorad) after incubation with ECF.
The membranes were then re-probed and tested for β-actin immunoreactivity to confirm that
similar amounts of protein were applied to the gels. Briefly, the membranes were incubated
for 30 min with 40% (v/v) methanol and 1 h with 0.1 M glycine buffer pH 2.2, and then
blocked as previously described before incubation with a mouse monoclonal anti-β-actin
antibody (1:5000) overnight at 4 °C. The membranes were then washed, incubated with an
anti-mouse IgG alkaline phosphatase-conjugated secondary antibody (1:10000) and analyzed
as described above.
97
2.6 Immunocitochemistry experiments in cortical astrocyte primary cultures
The cultures were washed twice with 1 ml phosphate buffer saline (PBS; 140 mM NaCl, 3
mM KCl, 20 mM Na2HPO4, 1.5 mM KH2PO4) and fixed with 4% paraformaldehyde with 4%
sucrose for 30 min at room temperature (RT). Coverslip-mounted cells were then
permeabilized with 0.2% Triton X-100 for 2 min at room temperature and further incubated
with 3% BSA in PBS to block non-specific sites for 30 min at room temperature. Then, cells
were incubated with the following primary antibodies: (i) rabbit monoclonal anti-glial
fibrillary acidic protein (GFAP) (1:400), for detection of astrocytes; ii) mouse monoclonal
anti-CD11b/OX-42 (1:200), a specific microglial marker for complement 3 receptor, in PBS
containing 3% BSA, for 90 min at RT. After the incubation with the primary antibodies, cells
were washed three times with PBS for 5 min, and further incubated was conducted with the
secondary antibodies: Alexa Fluor 594 (red) labeled donkey anti-mouse IgG antibody or
donkey anti-rabbit IgG antibody (Molecular Probes; 1 mg/ml; 1:200 dilution) and Alexa
Fluor 488 (green) labeled donkey anti-goat IgG antibody or donkey anti-rabbit IgG antibody
(Molecular Probes; 1 mg/ml; 1:200 dilution) for 90 min at room temperature. After this time,
cells were washed three times with PBS for 5 min, the coverslips were mounted using a
Prolong Antifade kit (Amersham) and, after drying, were visualized in a Zeiss Axiovert 200
fluorescence microscope equipped with a cooled CCD camera. For quantification purposes,
ten fields of several 16mm coverslips in 3 independent experiments were analysed using a
fluorescence microscope.
2.7 Statistical analyses
Data are presented as the mean results ± SEM from n experiments. When comparing twogroups of results, statistical significance was assessed using Student’s t-test. When
performing multiple comparisons, statistical significance was assessed by one-way ANOVA
98
followed by the Bonferroni correction, using GraphPad Software (Prism, version 4.02 for
Windows). Values of p < 0.05 were considered statistically significant.
99
3. Results
3.1 Characterization of primary cultures of cortical astrocytes
In order to evaluate the content of cortical astrocytes in our primary cultures, we performed
immunocitochemistry studies using specific antibodies to label astrocytes and microglia. The
astrocytes were detected by using anti-GFAP antibody, which labels an intermediate filament
protein that is specifically expressed in mature differentiated astrocytes (Fig. 1A). The
absence of significant numbers of microglia was confirmed by immunostaining using the
microglia specific antibody CD11b/OX-42 (complement receptor 3) (Fig. 1A). Our cultures
predominantly contained astrocytes (> 93%) and a small number of microglial cells (about
6%) (Fig.1B). These results indicate that cortical astrocytes are the predominant cell type
present in these cultures.
3.2 Characterization of adenosine uptake system
To evaluate the effect of drugs on adenosine uptake by cortical astrocytes, first we performed
assays in order to know the time of incubation with [3H] Adenosine. [3H] Adenosine (0.2
µCi/ml) transport was linear for incubation times ranging from 15 to 300 s (Fig. 2A). We
then decided to use a time of 60 s for all subsequent experiments. After that, different assays
were performed to test if the transporters expressed in these cells have affinity for inosine, a
product of adenosine deamination catalyzed by adenosine deaminase, and to measure the
relative amount of ENTs and CNTs expressed in these cells. Pre-treatment of [3H] Adenosine
with adenosine deaminase induced a strong decrease in transport (Fig. 2B). Additionally, a
dose-dependent decrease of [3H] Adenosine transport was observed in cultures treated with
dipyridamol (% maximal inhibition with 20 µM, 78.1 ± 9.1%, n=3, p < 0.01), a non-specific
equilibrative nucleoside transporter inhibitor, suggesting that the majority of nucleoside
100
Figure 1: Immunostaining for GFAP (astrocytes - in green) and CD11b (microglia- in
red) in astroglial-enriched cultures. Frequent medium changes and shaking, followed by
mild trypsinization of primary mixed glial cultures resulted in the isolation of enrichedastrocyte cultures. Representative image (A) shows the characteristic ratio of
astrocytes/microglia in our cultures by merging of the fluorescent views. The image was
taken in a fluorescence microscope (Magnification: 20x). The graph in B shows the average
astrocyte cell number (≈ 152) compared with the average microglial cell number (≈ 10) after
counting 10 microscopic fields in several 16mm coverslips of astrocyte cultures in 3
independent experiments. The astrocyte purity was determined to be between 92-96%.
101
B
200
[ 3H]Adenosine uptake
(% of control)
NBTI sensitive transport
(fmol/mg)
A
150
100
50
100
80
60
40
***
20
0
0
0
100
200
Control
300
ADA 2 U/ml
Time (seconds)
D
125
[ 3H]Adenosine uptake
(% of control)
100
75
50
25
80
60
40
20
µM
I1
N
B
So
/O
W
N
+
W
/O
So
di
To
ta
l
+
-3
um
-4
BI
log DIPY [M]
-5
di
-6
um
-7
1
-8
l
-9
µM
0
-10
ta
-∞
∞
100
To
[ 3H]Adenosine uptake
(% of control)
C
Figure 2: Time dependence curve of adenosine uptake, pre-treatment of [3H]Adenosine
with adenosine deaminase inhibits uptake, inhibition of adenosine transport by
dipyridamol and identification of ENTs:CNTs ratio in cortical astrocytes. (A) Adenosine
uptake was measured at different incubation times (15s to 300s). Values are mean ± SEM of
three experiments performed in triplicate. (B) [3H]Adenosine was pre-incubated with
adenosine deaminase (ADA) 2 U/ml for 45 min at 37o C and after this it was made the normal
protocol of uptake, by 1 min. The data are expressed as percent of control and the values
represent the mean from at least three separate experiments performed in triplicate. The
asterisks (***) denote that the difference is statistically significant (p < 0.001) compared to
control condition. (C) Adenosine transport was measured at 1 min with a fixed concentration
of [3H]adenosine (0.2 µCi/ml). Increasing concentrations of dipyridamol were pre-incubated
by 20 min before the incubation period itself. Values are mean ± SEM of three experiments
performed in triplicate. (D) Uptake of [3H]Adenosine was measured in normal conditions and
sodium-free experiments, and also in presence or lack of NBTI (1 µM). This drug was added
20 min before the addition of 3H nucleoside; uptake was measured for 1 min. Nonspecific
uptake was subtracted from the total uptake. Values are mean ± SEM of three experiments
performed in triplicate.
102
transporters found in these cells are of equilibrative type. Indeed, no significant changes in
adenosine transport were observed when sodium ions were removed (Fig. 2D), indicating that
concentrative transporters are not expressed in these cells or they are expressed in small
amounts. Furthermore, treatment with NBTI (1 µM) in absence of sodium promoted a strong
decrease in adenosine transport (% inhibition 67.0 ± 5.7% when compared with sodium-free
condition, n=3, p < 0.01) (Fig. 2D).
3.3 Identification of ENT1 transporters in cortical astrocytes by western blot and
immunofluorescence
We aimed to demonstrate the direct presence of ENT1 at the protein level by immunoblot
(Fig. 3A and B). We have not observed any band in blots run in absence of ENT1 primary
antibody and found a band in a region corresponding to the molecular weight of transporter
(55 kDa), which is in accordance with the predicted mass for this transporter in rats. Using
immunofluorescence, we identified a very diffuse and ubiquitous staining for virtually all
cortical astrocyte cells (Fig. 3C).
3.4 Biochemical characterization of ENT1 in cortical astrocytes by [3H] NBTI binding assays
The kinetics of association and saturation of [3H] NBTI binding to rat cortical astrocytes is
shown in Fig. 4. The equilibrium was reached after 2 min of incubation (Fig. 4A). The
saturation analysis of [3H] NBTI binding to cortical astrocytes (Fig. 4B) gave a Kd value of
1.28 ± 0.43 nM and a Bmax value of 14.1 ± 1.5 fmol/mg protein (n=3). The Scatchard
transformation of the binding data gave a linear plot indicating the existence of a single type
of NBTI-binding sites in cortical astrocytes (Fig. 4C). Displacement and reversibility studies
were performed with non-labeled NBTI to displace [3H] NBTI, allowing us to determine IC50
values. Experiments were performed at 37oC as well as at 4oC, with the aim to knowing if the
103
Figure 3: Cellular localization of ENT1 by western blot and immunofluorescence in
cortical astrocytes. (A) Control samples with no primary antibody. (B) ENT1
immunoreactive material in Western blot of rat cortical astrocytes untreated. The Western
blots of ENT1 antibody shows the immunoreactive band at 55 kDa. (C) Immunostaining for
GFAP (astrocytes - in red) and ENT1 (in green) in cultured cortical astrocytes
(Magnification: 40x). It was made immunofluorescence with no primary antibodies and we
did not observe any staining (data not shown).
104
Bound (fmol/mg)
A
60
Total
50
Specific
40
Non-specific
30
30
20
10
0
0
10 20 30 40 50 60
Time (minutes)
B
C
15
Bound/free
specific binding
(fmol/mg protein)
15
10
5
0
0.0
10
5
0
2.5
5.0
3
7.5
[ H]-NBTI (nM)
10.0
0
5
10
15
20
Bound
Figure 4: [3H] NBTI association and saturation binding in rat cortical astrocytes. (A)
Determination of association rate for [3H]-NBTI to cortical astrocytes. The association rate
was measured with 1 nM [3H]-NBTI. (B) [3H]-NBTI equilibrium binding to cortical
astrocytes. Cells were incubated with graded concentrations of [3H]NBTI, in the presence
(closed circles) or absence (open circles) of 10 µM unlabelled NBTI. Specific [3H]NBTI
binding (squares) was calculated by subtracting the non-specific component done in the
presence of unlabelled NBTI. The saturation analysis was performed using a non-linear
regression program. The kinetic parameters were: Kd value of 1.28 ± 0.43 nM and a Bmax
value of 14.1 ± 1.5 fmol/mg protein. (C) Scatchard analysis of equilibrium of [3H]NBTI
binding to cultured cortical astrocytes. This plot shows a typical experiment: each point
represents triplicate assays. B, bound: B/F, bound/free.
105
binding was essentially in cortical astrocyte cellular membranes or if it was occurring
internalization of binding sites as well. The NBTI concentration necessary to displace half of
the bound ligand (IC50) was 1.26 nM at 37o C and 0.35 nM at 4o C (Fig. 5A). We have
demonstrated that the binding was also fully reversible, since nonspecific levels were
obtained 15 min after addition of excess unlabeled NBI (10 µM) at 37oC (Fig. 5B) or after 10
min at 4o C (Fig. 5C).
3.5 Effects of A1 receptor activation on [3H] Adenosine uptake and [3H] NBTI binding
We have tested the effects of A1 receptor activation on the transport of adenosine by rat
cortical astrocytes and observed a decrease of adenosine uptake when cultures were treated
with different concentrations of CPA, an A1 receptor agonist (Fig. 6A). We then decided to
analyze the effect of CPA (100 nM) on [3H] NBTI binding. As can be observed in figure 6B,
a significant inhibitory effect is detected in cultures pretreated with CPA. The Scatchard plots
(fig. 6C) revealed that CPA induced a significant change in Kd but not in Bmax (Kd= 0.23 ±
0.05 nM and Bmax= 49.7 ± 4.2 fmol/mg protein for control cells and Kd= 0.44 ± 0.09 nM and
Bmax= 50.4 ± 4.78 fmol/mg protein for stimulated cells).
106
S p ecific b in d in g (% o f co n tr o l)
A
100
37 deg
4 deg
80
60
50
40
20
0
-∞
∞ -10
-9
-8
-7
-6
-5
log [NBTI] (M)
C
NBTI
10
5
0
10
20
30
40
50
Time (minutes)
-5
60
70
S pecific [ 3 H ]N B T I b o u n d
(fm o l/m g p ro tein)
S pecific [ 3 H ]N B T I b o u n d
(fm o l/m g p ro tein)
B
200
NBTI
150
100
50
0
0
20
40
60
80
Time (minutes)
Figure 5: Displacement and reversibility of [3H]NBTI binding in rat cortical astrocytes.
(A) Displacement of [3H]NBI binding was made with unlabelled NBTI. IC50 was determined
using 1 nM [3H]NBTI and different concentrations of displacer at 4°C or 37°C. These results
are representative of experiments performed in duplicate and conducted at least three times
with similar results. (B) and (C) Reversibility of [3H]NBTI binding at 37°C or 4°C,
respectively. Cells were incubated with [3H]NBTI (1 nM) and unlabeled NBTI (10 µM) was
added at the point indicated by the arrow; each data point is the total binding. The
experiments were repeated twice with very similar results.
107
[ 3 H ]Adenosine uptake
(% of control)
A
100
*
50
nM
0
nM
10
50
A
CP
CP
A
20
A
CP
Co
nt
ro
nM
l
0
B
C
200
40
B ound/Free
Specific binding
(fmol/m g protein)
50
30
20
10
0
0.00
Control
CPA 100 nM
150
100
50
0
0.25
0.50
3
0.75
[ H]-NBTI (nM)
1.00
0
10
20
30
40
50
Bound
Figure 6: A1 receptor activation inhibits [3H]-adenosine uptake and influences on [3H]NBTI binding in cortical astrocytes. (A) Cells were pre-incubated at 37°C for 20 min in the
presence or absence of CPA (20, 50 or 100 nM) and the NBTI-sensitive adenosine transport
was measured as described in Methods Section. The asterisk (*) denote that the difference is
statistically significant (p < 0.05) compared to control condition. To binding, (B) cells were
pre-incubated at 37°C for 20 min in the presence or absence of CPA (100 nM) and specific
[3H]NBTI binding was measured as described in Methods Section. (C) Scatchard analysis of
[3H]NBTI equilibrium binding to non-stimulated (circles) and cortical astrocytes stimulated
with CPA (100 nM) (triangles). The kinetic parameters were: Kd= 0.23 ± 0.05 nM and Bmax=
49.7 ± 4.2 fmol/mg protein for control cells and Kd= 0.44 ± 0.09 nM and Bmax= 50.4 ± 4.78
fmol/mg protein for stimulated cells. Values are the mean ± SEM of three experiments
performed in triplicate.
108
4. Discussion
Nucleoside transporters are important regulators of adenosine levels in the CNS, and play
functions in sleep, arousal, drug and alcohol addiction (for review, (King et al., 2006;
Porkka-Heiskanen & Kalinchuk, 2010)). Different nucleoside analogues and nucleoside
transport inhibitors are used in therapeutic strategies (for review, (King et al., 2006; Young et
al., 2008)). Here we report that ENT1 is the main adenosine transporter in astrocytes and that
the activation of adenosine A1 receptors might control the rate of adenosine clearance by
astrocytes. We have observed that rat cortical astrocytes do not express significant amounts
of CNTs. On the other hand, we have shown that ENTs are very important in the control of
adenosine levels by cortical astrocytes, since treatment with dipyridamol (10 µM), in a
concentration which inhibits ENT1 and ENT2 (Archer et al., 2004), promotes blockade of
adenosine uptake above 80% of control conditions. We have also shown the presence of
ENT1 by different techniques, and all results combined indicate that this transporter type is
the most important nucleoside transporter in these cells. There are many reports in the
literature demonstrating the presence of adenosine uptake systems in cultured rat cortical
astrocytes (Hertz, 1978; Bender & Hertz, 1986; Gu et al., 1996; Peng et al., 2005). However,
these experiments were not performed using cultured Wistar rat cortical astrocytes. To our
knowledge, the only study using the same preparation was the one by (Redzic et al., 2010).
We used a similar protocol for obtaining cultured rat cortical astrocytes. As in the work by
(Redzic et al., 2010), we also have identified ENT1 as the more abundant and main
nucleoside transporter in these cells. (Redzic et al., 2010) and our work are in accordance
with a previous work demonstrating the immunohistochemical localization of equilibrative
nucleoside transporters (ENT1 and 2) in the brain. In this work they showed that both
transporters were present in practically all neurons, but however, astrocytes showed
109
equilibrative nucleoside transporter 1 staining and a weak staining for equilibrative
nucleoside transporter 2 (Alanko et al., 2006). Apart from this, to our knowledge, the present
work is the first to show a modulation of ENT1 by activation of adenosine receptors in
cultures of rat cortical astrocytes. Very little is known about the mechanisms involved in the
regulation of ENT activity. As already mentioned, although some studies showed a link
between protein kinases and nucleoside transporters, there is no evidence for an interaction
between different proteins and these transporters. This may probably be related to the labile
nature of the ENT1 protein, the high turnover rate of the nucleoside transporter as well as the
unavailability of good antibodies. Our work is one of the few to demonstrate ENT1 by
immunofluorescence. We have identified a diffuse staining present in most cells, including
cells in which it was not observed staining for GFAP. One possible explanation for this fact is
that astrocytes derived from neonate rats have not already changed the cytoskeleton
composition of vimentin to GFAP (Chiu et al., 1981; Chiu & Goldman, 1984; Alonso, 2001).
One way to modulate adenosine levels is through the regulation of enzymes of purine
metabolism, mainly adenosine deaminase and/or adenosine kinase. Adenosine deaminase is
mostly cytosolic and its expression seems to be related with the presence of adenosine
transporters (Nagy et al., 1985). However, recent reports have suggested the existence of an
ecto-adenosine deaminase which could be anchored to A1 adenosine receptors, catalyzing the
degradation of adenosine at the extracellular medium (Franco et al., 1997; Latini & Pedata,
2001). Adenosine kinase, which is present in intracellular medium, promotes adenosine
phosphorylation and inhibition of this enzyme could promote an increase of adenosine
extracellular levels in hippocampal slices and a consequent modulation of synaptic
transmission. On the other hand, adenosine deaminase inhibition did not show any effect in
this model (Pak et al., 1994). The Km value for adenosine kinase is smaller than for adenosine
deaminase (2µM and 17-45µM, respectively) in rat brain (Phillips & Newsholme, 1979). At
110
adenosine physiological concentrations, adenosine kinase activity should be maximal, but at
higher concentrations this enzyme can be inhibited by its own substrate (for review, (Park &
Gupta, 2008)). In this way, the main adenosine metabolic pathway at phisyological
conditions should be the phosphorylation promoted by adenosine kinase. In situations of
metabolic stress, when occurs an increase of adenosine levels, intra and extracellular
adenosine deaminase could be the main pathway of intracellular degradation (Phillips &
Newsholme, 1979; Latini & Pedata, 2001). In our model, we used a period of 1 minute of
incubation with [3H] Adenosine in order to discard or decrease the possibility of adenosine
metabolism and then to study adenosine transport per se. However, our previous results show
a high rate of conversion of adenosine to nucleotides in retinal cultures incubated for 1
minute with [3H] Adenosine (Paes-de-Carvalho et al., 2005), but however another study
showed a lower conversion rate in a different cell type (Delicado et al., 1994). (Sinclair et al.,
2000) showed that adenosine A1 receptor activation increased nucleoside efflux from
metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase
activity. In our work, we believe this is not the case because we used a short uptake time and
then we suggested a direct effect of A1 receptor activation on nucleoside transporters. We
also have found IC50 values similar to that found by (Geiger et al., 1985). Regulation of
nucleoside transporter activity can occur at both transcriptional and post-transcriptional levels
(Kong et al., 2004). The mechanisms triggered by A1 adenosine receptor activation to induce
a reduction in the affinity for [3H] NBTI without having any effect on the number of NBTI
binding sites are presently unknown. One possibility could be a modification on the
transporter phosphorylation level , since it is already known the presence of potential sites of
phosphorylation for different kinases in this transporter, such as PKC (Delicado et al., 1991;
Coe et al., 2002; Pinto-Duarte et al., 2005), PKA (Sen et al., 1999) and casein kinase II
(Bone et al., 2007), but in our model, this needs to be investigated with more details.
111
In conclusion, the present study showed ENT1 as the main adenosine transporter in
astrocytes, and the low ability of these nucleoside transporters to take up inosine in these
cells. The presence of low levels of CNTs and high levels of ENT1 binding sites in cell
membrane is in accordance with a previous work (Alanko et al., 2006). Importantly, we have
also shown that the activation of adenosine A1 receptors might control the rate of adenosine
clearance by astrocytes.
5. Acknowledgements
This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal. A.S.R.
was supported by a scholarship from CNPq, Brazil; M.M. is recipient of a graduate student
scholarship from FCT; and R.P.C. is a research fellow from CNPq, Brazil.
112
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Alanko L, Porkka-Heiskanen T & Soinila S. (2006). Localization of equilibrative nucleoside
transporters in the rat brain. J Chem Neuroanat 31, 162-168.
Alonso G. (2001). Proliferation of progenitor cells in the adult rat brain correlates with the presence
of vimentin-expressing astrocytes. Glia 34, 253-266.
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4. Discussão
4.1 Adenosina cinase e metabolismo da adenosina em culturas de retina de galinha
Dados prévios do nosso laboratório haviam mostrado a presença de um sistema de
captação de alta afinidade para adenosina em culturas purificadas de neurônios ou culturas
mistas contendo neurônios e células gliais da retina (Paes de Carvalho et al., 1990; Paes-DeCarvalho, 2002; Paes-de-Carvalho et al., 2005). Neste trabalho, nós caracterizamos que tanto
nas culturas mistas quanto nas culturas purificadas de glia de retina de galinha ocorre a
presença de dois componentes na captação com características cinéticas distintas. A análise
de Eadie-Hofstee resultou em uma curva, indicando a existência de 2 ou mais componentes
neste sistema de captação. Isto pode representar a presença de mais de um subtipo de
transportador de nucleosídeo. Além disso, um dos componentes também pode ser relacionado
ao metabolismo de adenosina e isto é corroborado pelos resultados que demonstraram uma
redução significativa da captação de adenosina na presença do ITU, inibidor da adenosina
cinase, e também pelos dados de TLC nas culturas mistas, que demonstraram a conversão
majoritária de adenosina (> 90%) em nucleotídeos de adenina (Paes-de-Carvalho et al.,
2005). Nas culturas purificadas de glia, também há uma forte presença do componente do
metabolismo mediado pela adenosina cinase nestas células, pois ~80% da [3H] adenosina que
entra na célula é convertida em nucleotídeos de adenina (dados não-mostrados).
Igualmente constatamos que em ambos os sistemas de cultura de retina de galinha
utilizados, a captação de [3H] adenosina é mediada principalmente pelos ENTs, com o
componente que é mediado pelo ENT1 tendo uma contribuição um pouco maior nas culturas
mistas quando comparado às culturas purificadas de glia. Estes resultados são similares
àqueles observados em cultura purificada de neurônios (Paes de Carvalho et al., 1990). Em
relação à captação de inosina, sabe-se que o ENT2 de humanos possui uma afinidade um
pouco menor que 4 vezes quando comparado ao ENT1 de humanos (Km 50 µM nos hENT2s
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e 170 µM nos hENT1s) (Ward et al., 2000; Kong et al., 2004). Também observamos que nos
3 tipos de culturas aqui investigados ocorre captação de [3H] inosina, produzida a partir da
degradação de [3H] adenosina por ação da adenosina desaminase. Este transporte residual de
inosina provavelmente é mediado pelos ENT2s, pois alguns trabalhos têm demonstrado, pelo
menos por ensaios de liberação, que a inosina é transportada por estes transportadores
(Parkinson et al., 2002; Zamzow et al., 2008).
Devido à grande formação de nucleotídeos observada nas diferentes culturas de retina
de pinto, os resultados sugerem que a adenosina cinase funcione como uma rota primária para
a contínua captação de [3H] adenosina pelos ENTs, que foram caracterizados como os
principais transportadores presentes em nossas culturas. Isto é corroborado pelos relatos de
que em preparações de homogenados de cérebro de ratos a adenosina cinase possui um Km de
aproximadamente 2 µM enquanto que a adenosina deaminase possui um Km na faixa de 1745 µM (Phillips & Newsholme, 1979). Estes achados também indicam que em situações
fisiológicas, onde acredita-se que os níveis de adenosina endógena estejam na faixa de 30300 nM (Schulte & Fredholm, 2003), o metabolismo de adenosina esteja favorecido em
direção à formação de nucleotídeos de adenina por ação da ADK, enquanto que em situações
onde há aumento da atividade metabólica, como em condições isquêmicas, o metabolismo
seja deslocado para uma maior atividade da adenosina desaminase.
(Pak et al., 1994) demonstraram que a inibição da atividade da ADK induzia um
maior aumento dos níveis extracelulares de adenosina em fatias corticais e hipocampais
quando comparado à inibição da atividade da adenosina desaminase. Estes autores também
demonstraram que o aumento dos níveis extracelulares de adenosina induzia um aumento da
inibição pré-sináptica mediada pelos receptores A1. Em nosso trabalho, nós observamos uma
redução significativa da captação de [3H] adenosina após inibição da ADK nas diferentes
culturas de retina utilizadas no entanto, ainda não temos evidências dos efeitos da inibição da
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adenosina desaminase em nosso sistema. Já foi demonstrado que o cérebro imaturo de
camundongos possui uma alta expressão neuronal de ADK, enquanto que no cérebro maduro
a expressão de ADK está localizada essencialmente nos astrócitos (Studer et al., 2006). Este
padrão diferencial da ADK tem sido correlacionado com o fato de o cérebro imaturo ser mais
propenso a eventos epilépticos do que o cérebro adulto (Moshe, 2000)(para revisão, ver
(Boison, 2008)). De fato, (Studer et al., 2006) demonstraram que o tônus inibitório
adenosinérgico mediado pelos receptores A1 nos EPSPs medidos no hipocampo é maior nos
animais adultos do que em animais jovens. No nosso trabalho, quanto à redução da captação
de [3H] adenosina observada com o inibidor farmacológico da ADK, os resultados foram
bastante similares nos dois tipos de culturas de retina utilizados. No entanto, não foi estudado
o padrão de expressão da ADK nestas células em cultura.
Sabe-se que na sequência da adenosina cinase de camundongos existem sítios
potenciais de fosforilação para várias proteínas cinases, inclusive para as ERKs e PKC (Sahin
et al., 2004). No entanto, este mesmo trabalho demonstrou que a ERK não foi capaz de
fosforilar adenosina cinase recombinante in vitro. Por outro lado, (Pawelczyk et al., 2003)
demonstraram que a insulina é capaz de modular a expressão da adenosina cinase em
linfócitos de ratos por um mecanismo dependente das ERKs. Além disso, foi observado que,
na linhagem celular DDT1 MF-2, a ativação dos receptores A1 de adenosina aumentava o
efluxo de [3H] adenosina por um mecanismo dependente da inibição da atividade da
adenosina cinase mediado pela PKC (Sinclair et al., 2000).
Nossos resultados, com o uso de inibidores da MEK, demonstraram que estes
inibidores não produziram nenhum efeito no metabolismo da [3H] adenosina, indicando que a
diminuição da captação de [3H] adenosina não é resultado da inibição da adenosina cinase e
provavelmente é resultado da modulação direta ou indireta dos ENTs.
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4.2 Regulação dos transportadores de nucleosídeos por proteínas cinases
Embora até o momento nós não conheçamos nenhum trabalho demonstrando
efetivamente que o transportador equilibrativo tenha sido fosforilado por alguma proteína
cinase, diversos trabalhos demonstraram que os transportadores de nucleosídeos podem ter a
atividade regulada pela ativação/inibição de proteínas cinases, como por exemplo PKA (Nagy
et al., 1991; Sen et al., 1999), PKC (Delicado et al., 1991; Coe et al., 2002; Chaudary et al.,
2004; Pinto-Duarte et al., 2005), CK II (Bone et al., 2007) e CAMK II (Paes-de-Carvalho et
al., 2005).
Dados prévios do nosso laboratório haviam demonstrado que a inibição da CAMK II
reduz fortemente a captação de [3H] adenosina em culturas mistas de retina (Paes-deCarvalho et al., 2005). Neste trabalho, nós identificamos que o uso dos inibidores da MEK
PD 98059 e U0126 induziu uma redução significativa da captação de [3H] adenosina nas
diferentes culturas de retina. Na literatura, que seja de nosso conhecimento, existem apenas
três relatos que demonstram modulação do transporte de [3H] adenosina pelas ERKs. Um
deles demonstrou que a inibição do transporte de adenosina por glicose ocorre de modo dose
e tempo-dependente e tem a participação da PKC, óxido nítrico e ERKs (Montecinos et al.,
2000). Outro grupo demonstrou que altos níveis de glicose induzem uma diminuição dos
níveis de mRNA de ENT1 em culturas de linfócitos B por um mecanismo dependente das
ERKs (Sakowicz et al., 2005) e um resultado similar foi observado em culturas primárias de
fibroblastos do coração de ratos (Grden et al., 2008). No entanto, nosso trabalho é o primeiro
a relatar um efeito per se de inibidores da MEK nos níveis basais de atividade dos ENTs.
Existe alguns relatos de que inibidores de proteínas cinases podem ter efeitos
inespecíficos nos transportadores de nucleosídeos. Genisteína, um inibidor de tirosinas
cinases, atua diretamente nos transportadores de nucleosídeos (Pillai & Shivakumar, 2009).
Em uma linhagem celular K562, que é uma linhagem com presença majoritária dos
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transportadores equilibrativos sensíveis à NBTI, observou-se que os inibidores de receptores
tirosina cinases, PKC, cinases dependentes de ciclina (CDKs) e p38 afetavam o transporte de
nucleosídeos em uma maneira independente das cinases (Huang et al., 2002; Huang et al.,
2003). Nesses mesmos trabalhos não foi identificado qualquer efeito na captação com os
mesmos inibidores da MEK utilizados no nosso trabalho, PD 98059 e U0126. Quanto a estes
inibidores, existe um relato de efeitos inespecíficos na liberação de glutamato em
sinaptossomas hipocampais (Pereira et al., 2002), mas não foram observados efeitos
inespecíficos nos transportadores de nucleosídeos.
A ausência de efeito inibitório na captação de adenosina com o U0124, análogo
inativo do U0126, serviu para corroborar a especificidade do efeito, evidenciando que não era
devido à atuação direta nos transportadores de nucleosídeos. Em termos de sinalização, ainda
não sabemos o que está acontecendo com os transportadores, provavelmente ENT1 ou 2, para
que ocorra a diminuição da capacidade de captação de [3H] adenosina nas diferentes culturas
de retina de galinha. Já foi observado que o all-trans-ácido retinóico (ATRA) induz um
aumento da quantidade de hCNT3 na membrana plasmática de células HeLa por um
mecanismo dependente da ativação das ERKs (Fernandez-Calotti & Pastor-Anglada, 2010),
mas em nosso modelo não sabemos se há uma diminuição dos ENTs na membrana
plasmática ou se há uma diminuição em termos de afinidade dos transportadores pelo
nucleosídeo quando as ERKs estão inibidas.
Como demonstrado nas culturas purificadas de glia, a ativação dos receptores A1
induz um aumento da fosforilação da ERK por mecanismos que envolvem a PKC e a Src.
Nestas mesmas culturas, a ativação dos receptores A1 não induziu nenhuma modificação na
captação de [3H] adenosina (dados não mostrados). Este resultado pode ser decorrente do fato
de que a incubação por 15 minutos com CHA não produza mais estímulo na fosforilação da
ERK, o que por sua vez acarretaria uma ausência de estimulação na captação. Esta hipótese é
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fortalecida pelo fato de a fosforilação da ERK induzida por NECA, agonista não-seletivo dos
receptores de adenosina, nas culturas purificadas de glia ocorrer de maneira transiente e aos
15 minutos de estímulo os níveis de p-ERK estarem na mesma intensidade dos níveis basais
(dados não-mostrados).
Dados anteriores haviam demonstrado que o glutamato estimula a liberação de
adenosina em culturas mistas, por um mecanismo que envolve a ativação da CAMK II (Paesde-Carvalho et al., 2005). Em nosso trabalho, apesar de termos visto uma inibição bem
similar da captação de [3H] adenosina com os inibidores da MEK nos diferentes tipos de
culturas de retina, não observamos um efeito similar na liberação de [3H] purinas. Embora
ocorra uma tendência, a liberação de [3H] purinas estimulada por glutamato não foi inibida
significativamente pelo bloqueio concomitante da MEK com o uso de PD 98059. Este
resultado pode sugerir uma modulação diferencial dos transportadores equilibrativos de
nucleosídeos por proteínas cinases para mecanismos de captação e liberação. De fato, já foi
observado anteriormente que uma determinada região do N terminal do SERT funciona como
uma espécie de regulador conformacional entre o estado do sítio ligante voltado para o
exterior celular e o estado do sítio ligante voltado para o interior celular (Sucic et al., 2010).
Baseado nisto, podemos sugerir que determinadas drogas poderiam favorecer um maior
número de transportadores em um determinado estado mais voltado para a captação ou para a
liberação. Entretanto, mais estudos são necessários para desvendar os mecanismos celulares e
bioquímicos envolvidos nesta regulação.
Assim como outros grupos que já haviam demonstrado a influência da PKC na
captação de [3H] adenosina em diferentes modelos celulares (Delicado et al., 1991; Coe et al.,
2002; Chaudary et al., 2004; Pinto-Duarte et al., 2005), também identificamos que a PKC
modula a captação de [3H] adenosina em culturas de retina de galinha. Diferentemente dos
inibidores da MEK, a inibição da PKC induziu um aumento na liberação basal de [3H]
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purinas nas culturas mistas (dados não-mostrados). No entanto, permanece a ser caracterizado
se a via de modulação do transporte pela PKC e pela ERK é a mesma ou se é uma via
distinta.
Neste trabalho, também demonstramos pela primeira vez que a fosfolipase C (PLC)
também pode modular a atividade dos ENTs, como demonstrado nas culturas mistas de retina
de galinha. Não conhecemos nenhum relato prévio de modulação do transporte por esta
enzima. Este achado pode indicar que a captação de adenosina está sob o controle de
receptores acoplados à via da fosfolipase C e consequentemente da PKC. Como mostramos
neste trabalho que os receptores A1 de adenosina podem regular a captação de adenosina em
astrócitos cerebrais e que estes receptores podem se acoplar à via da fosfolipase C/PKC
(Biber et al., 1997; Germack & Dickenson, 2004), uma possibilidade é que a captação de
adenosina também esteja sob o controle dos receptores A1 e ativação desta via nas células da
retina.
Nós encontramos algumas similaridades entre os modelos utilizados neste trabalho, a
saber os diferentes tipos de células gliais em cultura, a glia de Müller e os astrócitos corticais.
Uma semelhança é a baixa ou inexistente presença dos CNTs e um predomínio dos ENTs
nestas células, com uma tendência a haver uma maior quantidade de ENT1s nas culturas de
astrócitos corticais do que nas culturas purificadas de glia de Müller. Os resultados em
astrócitos corticais estão de acordo com os resultados demonstrados por (Redzic et al., 2010),
também obtidos com uma cultura similar de astrócitos corticais. Além disso, (Alanko et al.,
2006) demonstraram, através de marcação por imunohistoquímica em cortes do cérebro, que
nos astrócitos havia um predomínio da marcação do ENT1 comparado a uma marcação mais
fraca para o ENT2. Observaram também que os ENT1 parecem estar localizados
principalmente na membrana plasmática, enquanto que os ENT2 parecem estar mais
localizados em membranas intracelulares e em organelas. Com relação aos ENT1, baseado
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principalmente nos resultados dos ensaios de binding a 40C, nossos dados também indicaram
uma forte presença dos sítios destes transportadores na membrana plasmática.
A co-localização entre hENT1 e receptores A1 já foi demonstrada em diferentes
regiões cerebrais, o que sugere que podem ter papéis moduladores recíprocos (Jennings et al.,
2001). Que seja de nosso conhecimento, este trabalho é o primeiro a relatar uma modulação
da atividade do ENT1 pela ativação dos receptores A1 em culturas de astrócitos corticais, o
que sugere um possível papel na remoção e/ou liberação de adenosina por estas células.
Ainda é necessário esclarecer quais são os mecanismos disparados pela sinalização mediada
pelos receptores A1. Um possível candidato é a PKC, pois já foi demonstrado que durante a
hipóxia há um aumento da adenosina extracelular e consequente ativação de receptores A1,
indução da PKCε e aumento da atividade dos ENTs (Chaudary et al., 2004). Embora nossos
resultados sugiram fortemente que a diminuição da captação observada pela ativação dos
receptores A1 seja devido a alguma alteração no funcionamento do ENT1 em função da
mudança observada no Kd, o que poderia sugerir uma
modulação por fosforilação, não
podemos descartar que os efeitos observados sejam devidos a mudanças na atividade da
ADK, como já observado por (Sinclair et al., 2000). Estes autores relataram que a ativação de
receptores A1 de adenosina em uma linhagem de células musculares DDT1 MF-2 inibe a
atividade da adenosina cinase por um mecanismo dependente da ativação de PKC. Esta
inibição da adenosina cinase acarretaria numa diminuição da captação de [3H] adenosina. No
entanto, em nosso estudo utilizamos o tempo de 1 minuto de captação para minimizar a
influência do componente do metabolismo no nosso modelo.
4.3 Regulação da ativação da ERK pelo receptor A1
Nossos dados demonstraram pela primeira vez a expressão dos receptores A1 na glia
de Müller em cultura e também identificamos que a ativação destes receptores estimula a
fosforilação da ERK nestas células. Nossos dados também mostraram que a PKC e a Src
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estão envolvidas na via de sinalização disparada pelos receptores A1 na fosforilação da ERK.
Já foi previamente demonstrado que a família das Src cinases está envolvida na regulação das
MAPKs por receptores acoplados a proteína Gi em células COS-7 (Luttrell et al., 1996). Por
outro lado, a fosforilação da ERK pela ativação dos receptores A1 em cardiomiócitos envolve
ativação da PKC, que pode ser resultante da ativação da PLC por subunidades βγ das
proteínas Gi (Germack & Dickenson, 2004). Em células CHO transfectadas com o receptor
A1, a fosforilação das ERK 1/2 foi relacionada à ativação da proteína Gi, tirosina cinases
citoplasmáticas, PI-3 cinase e MEK mas, no entanto, era insensível à inibição de PKC
(Dickenson et al., 1998). Nossos resultados recentes demonstram que também ocorre
regulação da fosforilação da ERK por receptores de adenosina em culturas mistas (dados não
mostrados). De modo interessante, a ativação aguda dos receptores A2A induz uma
diminuição da fosforilação da ERK em culturas mistas de retina (dados não publicados).
Nossos resultados também mostraram que o aumento da fosforilação da ERK induzido pelo
agonista dos receptores A1 era localizado principalmente na região citoplasmática das células
gliais, o que pode indicar a modulação de alvos não-nucleares que eventualmente possam
estar envolvidos em processos de regulação da proliferação e diferenciação das células gliais
na retina.
125
5. Perspectivas
5.1 Perspectivas Prioritárias
•
Caracterizar se o efeito da diminuição da captação de adenosina com o bloqueio da
MEK ocorre por uma diminuição do número de transportadores na membrana celular,
através da técnica de binding de [3H] NBTI, nas culturas mistas e nas culturas de glia.
•
Investigar quais são os efeitos dos inibidores da MEK na liberação de adenosina,
através de cromatografia de camada fina (ou ensaio direto da atividade da ADK) e da
técnica de binding de [3H] NBTI.
•
Investigar o tráfego do ENT1 na via de sinalização envolvida na liberação de
adenosina estimulada pelo glutamato em culturas mistas, através do uso da técnica de
binding de [3H] NBTI.
5.2 Perspectivas a longo prazo
•
Implantar um método de quantificação direta da atividade da enzima adenosina cinase
•
Investigar o efeito do tratamento crônico com Ado (em termos de sinalização celular),
com os inibidores da MEK e/ou ativadores da ERK em culturas mistas nos níveis do
ENT1, por western blot ou binding de [3H] NBTI.
•
Caracterizar por fracionamento celular a localização dos ENT1 por western blot após
o uso de inibidores da MEK
•
Identificar por imunoprecipitação se há interação física entre a ERK e o ENT1
•
Caracterizar e comparar a captação de [3H] adenosina em sinaptossomas e
homogenados totais da retina
126
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