nmda ap-1 - marianna uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5kouhei kuribayashi... ·...

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臤膁膆腩膊腹膋膒臯膙臀臆 Vol. 33, pp. 435444, 2005 NMDA 腒腑腎腌腄腁腂腆 AP-1 腅腐腍腃腇腋腊腀腈腉腅腓腏 腋腟 腖腛腏 腍腆 腘腅 1 腊腑 腈腉 腛腐 1 腋腙 腓腏 2 腕腛腏 腛腐 腗腠 1 腚腞 腛腐 1 腑腌 腗腠 腜腊 1 腆腇 腎腓 1 : 17 9 21 腦腀臘腕腛臞膪臦膾臘膗腨腆腐腍腉腖腅臰腤腧腔腂腦腅腎腚膞臑腛腂腟腑腤腄腕腛腘 腟腐Activator protein-1 AP-1腘臂膫腙腣腥膚臢膑腊腧腦臶臋膎臄腚膍腓腕腁 膻膖膔 腛臞膪臦膾 臘膗腚膄腷膇腖腌腔 腊腣 腊腧腔腂腦膅腵腸腚 N-methyl-D- aspartate NMDA腛腗臘膗膄腷膇腙腃腂腔腛腗腀腕腚 AP-1 膲腨致腞腐AP-1 DNA 膬膹膚臢腛 Electrophoretic Gel Mobility Shift Assay 腩膀腸腀腰腱膲腛 TUNEL AP-1 TUNEL 臢膾腚膤膿腛膡臔臷膯膣腙腔腎腧腏腧膕臥腌腐NMDA control 膧腙腛腗臞膪臦膾臩腚膾臠腚至腘膱臒腖腀腛臛臩腚腬腈膑腨腠腐NMDA 200 nmol臖臄臭臲臌 6 臈膛膳腣腥腛腗腀TUNEL 臢膾膲腨24 膛腕膽臯腖腘腒腐腙腃腈腦 AP-1 DNA 膬膹膚臢腛 NMDA 24 臈膛膳腕 control 腌臙臓腨腠腐膏臨臜臧臝腙腃腂腔AP-1 腚膤膿腛 NMDA 24 臈膛膳腕腛臍腙 腛腗腀臩腙腊腤腙 TUNEL 臢膾腖腚膡自腨腠腐腉腧腤腣腥 NMDA 腛腗臘膗 腙腃腂腔 AP-1 腚臙臓腛腩膀腸腀腰腱腖腚膢腂膜腅臉膼腊腧腐腅腁腉腄 腦腀腩膀腸腀腰腱臶臋致臦膎臄臞膪臘膗AP-1NMDA 腀腆 腮膇腲膂膊臁腙腣腦臞膪臼臢腛臅臞膪臘膗腦腀 臘腢臺腃腍腛腗臕腕腚膜腅臉膼腊腧腔腂腦 14腮膇腲膂膊臁臎 臭腛 N-methyl-D-aspartate NMDA腭腪腺膊臁a-amino-3- hydroxyl-5- methylisoxazole-4-propionic acid AMPA腚腯腼腲腪腽腙腑腨腊腧腦腉腚臱腕腡 NMDA 腣腦 NMDA 臭臂膫腙腣腥腛腗腚臞膪臦膾膗腚膟腉腦腉腖腅膺腊腧臖臄臭腝腚 NMDA 腅臞膪臦膾臘膗腚膄腷膇腖腌腔膮膠腙腊腣腊腧 腔腂腦 56NMDA 腙腣腦腛腗臘膗腚膪腙腛腊腟 腋腟腘膎臄腅膜腌腔腂腦腉腖腅臰腤腧腔腂腦腅腎腚臗膾腛腂腟腑腙腤腄腕腛腘腂腛腉腧腟 腕腙 NMDA 腙腣腦腛腗臘膗腙 RhoRho kinase, NF-kappa B NF-kBMAP Kinase 腘腗腅膜腔腂腦腉腖腨膺腌腔腆腐 7-9Activator protein-1 AP-1腛臟腚膃腲膉腴腬腻腪 II 腽膉膄腀腲腀腚 cis 腫膈膃膊腸腙膬膹腍腦臶臋 致臦膎臄腖腌腔臻臵腊腧腐 11AP-1 c-Jun c- fos 腣腥膷臣腊腧腔腃腥臬腇腚膎臄腚臶臋致臦腙 腍腦臶臋致臦膎臄腚腜腖腓腕腁腦膢臡膲膩 腂腐臊膰腄腤 c-Jun c-fos 腚腾腶膉腳腪膁腀 DNA 膬膹膚臢腨膢腇臇腓腉腖腅臉腊腧腔腂 12Dominant-negative 膓臚膶臭臲腘腗 1 臤膁膆腩膊腹膋膒臯膙 膝膒膙 2 腝腥435 79

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Page 1: NMDA AP-1 - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5Kouhei Kuribayashi... · Fig. 2. Representative light microscopic photographs of rat retina at 7 days after

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Page 2: NMDA AP-1 - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5Kouhei Kuribayashi... · Fig. 2. Representative light microscopic photographs of rat retina at 7 days after

��� c-Jun��������� ��������������������13�14�� � ���c-Jun ����� ���������!�"�#$��%&'���$()%&'���*����+,-./0��� AP-1 ���1������� �&'�15�� 2�� AP-1 DNA�3���������$�45����� ��JNK 67 p38 ������16�� 8�9��NMDA �:;�� <=� JNK 67 p38 �����������>� ?� JNK 67 p38$�=5�������@�A"����$��%�9��%B%? C�DE�F� AP-1� NMDA � <=�G �H�I�5�B�� �&'"'� ?��� NMDA � <=JK,�:'&��������5� AP-1 �#$� B�5��L�� MN9�� AP-1 DNA �3��$O�%? PQRFST&O�%��

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in Ophtalmic and Vision Research” �WX%&YZ�� [��UV��\]^��_`a���b�$�&'�� ]^��� 8 c� Wistar d��ef�$�'�� ����\�g]^��hij UVk!"� lm 23�1 �C� n# 55�5�� o� 6pB oq 6p[� �r�s$t� h$u%&vChi��&'������ ��NMDA �Sigma, St. Louis, MO, U.S.A� wE'

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4��&�5%� � 67��8 1 mm ������ 9� wE'"� 40 mM NMDA �in 0.01M phosphate bu#erd salin �PBS�� $ 5 ml �:; 200nmol� ��(�%� control ���<9� wE'"�=; PBS 5 ml $(�%������ Hematoxylin-eosin ��ef� �n�10� �z/�{,|-�, �35 mg�kg,i. p. �2}C� wE'"� NMDA$(�%� 24p�q� :�7 7/>���$?�%� �,���@

��& 3p��@%� At� A�$�&�e��/BCq� 4 mm ���$DZ�� E9$�F%�� ? q hematoxylin-eosin G�$Y'� ����� 1.01.5 mm �r$H7�I�C���$��%������ TUNEL �ef� �n�25� �z/�{,|-�, �35 mg�kg,

i. p.�2}C� wE'"� NMDA$(�%� 0� 3�6� 12� 24p�q���$?�%� 10��,� /�& 24p��@%� At� A�$�&�e��/BCq� 4 mm ���$DZ�� E9$�F%�� TUNEL G�� DeadEndTM Fluorometric

TUNEL System �Promega, Madison, WI, U.S.A�$¡�%&¢~�£�,J��YZ�� E9$PBS�K¤q 20 mg�ml proteinase K� 5L¥M%� terminal dUTP transferase enzyme �& 37�C� 60L�n%�q� 2saline sodium citrate �SSC�� 15 L¥M%N¦$§O%�� FITC P¨TUNELQ�©R�ª«4�I� �LSM510META;Carl Zeiss, Jena, Germany� $�'� S¬­�%&488 nm T�­$� ��,-��{/�����,-�$¡�%&����� 1.01.5 mm �r�� $®U%������ ���TUNEL G�q� E9$ PBS �K¤%� block-ing solution �3� Bovine serum albumin �BSA� inPBS� � 30L¥M%� ¯°±' AP-1 ±' �SantaCruz Biotechnology, Santa Cruz, CA, U.S.A,

1 : 100� � 24p�¥M%�� V°±'�~�²./P¨±³´µ IgG �Cappel, Research Products,NC, U.S.A, 1 : 100� ±'�lm�& 1p�¥M%�TUNEL G��+,���$�Z�� S¬­�%& 543 nm W�­$�'� ~/+����,-�$¡�%������ Electrophoretic Gel Mobility Shift Assay�EMSA�ef� �n�10� �z/�{,|-�, �35 mg�kg,

i. p.� 2}C� wE'"� NMDA $(�%� 24p�q�� $?�%� ¶XY$ef�� B ¶©R·Z�¸f� �NE-PER Nuclear and Cytoplas-mic Extraction Reagents, Pierce Chemical, IL,

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Page 3: NMDA AP-1 - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5Kouhei Kuribayashi... · Fig. 2. Representative light microscopic photographs of rat retina at 7 days after

��� binding bu#er �20 mM HEPES-NaOH pH

7.90� 1 mM dithiotreitol, 0.3 mM EDTA, 0.2 mMEGTA, 80 mM NaCl, 10� glycerol, 0.2 mM

PMSF� ����� 2 mg poly �dI-dC�� �10,000 cpm 32 P � ���� AP-1 double-

stranded oligonucleotides �AP-1 oligonucleotides��Promega, Madison, WI, U.S.A�� 32P� ������� AP-1 oligonucleotides � 100������������������ ��� bindingbu#er� 32P ���� AP-1 oligonucleotides� !"� 32P� ������� AP-1 oligonucleotides�#$� 30 %&'()�*� DNA ��+�,�6� polyacrylamide gel �0.5 mM EDTA, 25 mMTris-borate bu#er� � apply �� 100V� 4�C� 2-./012��� 3��45()� X 678�9�:;<=()�� 78�9�>?*� Densito-graph �ATTO Corporation, Tokyo, Japan� ��@������� ���ABCBDEFG�HIJK�L��� MND

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-.*�D control c� NMDA c�!��defghijkijl�`dmkno�pqDrstu�vP�� �v�� Fig. 2�L\"]�� wxy�z{�|}� NMDA ^_,`ab* 7~� NMDA c�D control c���defghijkijl������`dmkno��q�rs� �Fig. 1, 2����� TUNELFig. 3�L\"]�� NMDA ^_,`ab 24

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D� 10���D 100��� 32P� ������� AP-1 oligonucleotide������(u����"��r��� � AP-1��� DNA ����DNMDA^_,`ab 24-.*� control������������� AP-1 �� �Fig. 5�L\"]�� NMDA ^_,`ab 24

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Fig. 1. Representative light microscopic photographs of rat retina at 24 hours after

intravitreal injection of phosphate bu#ered saline �PBS� as a control �A� or 200 nmolN-methyl-D-aspartate �NMDA� �B�. Scale bar�50 mm. RGCL: retinal ganglion cell

layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform

layer, ONL: outer nuclear layer, RPE: retinal pigment epithelium.

NMDA �"R� ¡B¢£ 437

81

Page 4: NMDA AP-1 - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5Kouhei Kuribayashi... · Fig. 2. Representative light microscopic photographs of rat retina at 7 days after

Fig. 2. Representative light microscopic photographs of rat retina at 7 days after intravitreal

injection of phosphate bu#ered saline �PBS� as a control �A� or 200 nmol N-methyl-D-aspartate �NMDA� �B�. Note loss of inner retinal thickness with 200 nmol NMDA injectionand decreased number of cell in retinal ganglion cell layer �RGCL�. Scale bar�50 mm. IPL:

inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer

nuclear layer, RPE: retinal pigment epithelium.

Fig. 3. In situ TUNEL assay. TUNEL-positive cells in the inner retina 24 hours after phosphate

bu#ered saline �PBS� as a control �A� or 200 nmol N-methyl-D-aspartate �NMDA� �B�injection. TUNEL-positive cells were localized in the retinal ganglion cell layer �RGCL� andthe inner nuclear layer �INL� of the NMDA-treated retina �B�. �C� The number ofTUNEL-positive cells in the inner retina at di#erent times after NMDA injection. Data are

expressed as means�SEM. �n�5�. IPL: inner plexiform layer, OPL: outer plexiform layer,ONL: outer nuclear layer.

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Page 5: NMDA AP-1 - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5Kouhei Kuribayashi... · Fig. 2. Representative light microscopic photographs of rat retina at 7 days after

trol� �A����� NMDA� �B� ������Fig. 5A�B� � ��� TUNEL �B,� ����� �A, � � � 2�� �C, � � ��� AP-1 ��������� TUNEL � ������� �Fig.6��

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��� !�"�� #��NMDA$%&'()�*�+,�����"�� -./0�12�34�$5678�9:��� 4�-./0�1

2�3���"� AP-1� DNA ;<=�>=�?@A��$�BC���� ��4� AP-1 �DEF���"�-.G��H�(H�IJK34�>�BC�L��� @B�� AP-1 �TUNEL ������MN>:OBA��PQ�RS�T���� 200 nmol� NMDA $%&'(�)�* 7U��� VWL��-.G��X�YZ�(�[H� \?>:OBA��NMDA ]�'�^_L`a���( Ca2� bc�de�fg-.���hiFjkl$14K�mnBA�3� NMDA �f3��-./0�hiFjkl$op��Lqr>��L�s$tu�3�mnBA�3� vw�xy��NMDA�%&'(z�)��-.G���"�� p53{N��hiFjkl.�$=�?K3���318��p53>hiFjkl$|GK3}�~�-.�������BA�3� ��� ���"��(/�"�3-.G�����hiFjkl���>��@A�3�O19�� 4AB�.����>����-.�������������3�mnBA3���� !�"�� hiFjkl�����3TUNEL �������� NMDA )� 6��*�,��� 24 ��*�v��L��� �C�� ��8�� NMDA )� 24��*��-.G��X�(�[H���?�:OBALC������NMDA )�* 7U��"�-.G��X�YZ�(�[H� \?>:OBA�� Pd�4�CB�����-.G��X�YZ�(�[H� \?>VJ?K3���� TUNEL ����>��K3�������>�3���� hiFjkl>����3���>mnBA��^��RS��� -.¡�"�¢£� ¤¥¦§��LlF¨l�fg AP-1 =��de>RS@A�320�21�� ��� ���©`a�f3ª���>AP-1��«$¬K34�>RS@A�3>22��-.G����� AP-1 �����K3RS�ZL� PQ#��� NMDA �f3 AP-1 �­®¯&��°��3 c-Jun �=��de$9:�� �un-published data� � ��#�� NMDA ��-./0$14�����"�T�� AP-1� DNA ;<=�>de��34�$�BC���� 4AB�4��±²���"�³´µ¶·¸>NMDA ]�'$¬�� c-Jun ¹º AP-1 $=�?@»�hiFjkl$�«K3�¼RS��½�

Fig. 4. Electrophoretic mobility shift assay of activator

protein-1 �AP-1� DNA binding activity in the ratretina at 24 hours after intravitreal injection of

phosphate bu#ered saline �PBS� as a control�cont.� or 200 nmol N-methyl-D-aspartate

�NMDA�. Ten excess, 10-fold excess of the AP-1unlabeled oligonucleotide; 100 excess, 100 -fold

excess of the AP-1 unlabeled oligonucleotide.

NMDA �f3hiFjkl 439

83

Page 6: NMDA AP-1 - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5Kouhei Kuribayashi... · Fig. 2. Representative light microscopic photographs of rat retina at 7 days after

���23�� ���� AP-1 �� ������������������ c-Jun����� !����"#���� AP-1 c-Jun$�% c-fos&���'��()*+,-���� !&!� AP-1��.���������� c-fos �/0�12 3���45�&���24�� 36� NMDA ���'�� AP-1 �����789:;��<=��>��?@A?� AP-1 9BC'D�EFGHI�!� NMDA � MAP kinase JKLMN� O�c-Jun N terminal kinase �JNK�PQ9��'D����"#�����25�� JNK c-Jun � N-terminal transactivation RS9MTU�!� AP-19��'D�� VWX� NMDA�Y-Z[\

6 6]^�_P+`abc%[deb� JNKc% p-JNK BC!fg� 24]^h�ij0���9kl!=9�� 3m NMDA \ 6� _P+`abc%[de`ab� AP-1 n�`a�ij!=�����b NMDA � TUNEL n�`a�o�pq'��b��� ��&#�r_P+`a�stuMT`a�sv@Nw?�x�;����"#���� h=]^y�� AP-1 DNA z{� NMDA \ 24]^6�BC!=�� ��]|��}~[b� TUNEL n�`a������� ��6�����0����������10�� ���.� JNK ���� AP-1 ���� c%sv@Nw?������ TUNEL n�`a��� ]^�

Fig. 5. Immunohistochemistry of activator protein-1 �AP-1� in rat retinas at 24 hours after intravitreal injection ofphosphate bu#ered saline �PBS� as a control �A� or 200 nmol N-methyl-D-aspartate �NMDA� �B�. Asubstantial increase in immunoreactivity after NMDA injection was noted in the retinal ganglion cell layer

�RGCL� and inner nuclear layer �INL�. Scale bar�50 mm. IPL: inner plexiform layer, ONL: outer nuclear

layer.

Fig. 6. Inner retina double-labeled with TUNEL staining and activator protein-1 �AP-1� immunostaining afterintravitreal injection of 200 nmol N-methyl-D-aspartate �NMDA�. �A� AP-1 �red�. �B� TUNEL-positive cells�green�. �C� Merged images. Scale bar�50 mm. RGCL: retinal ganglion cell layer, IPL: inner plexiform layer,

INL: inner nuclear layer.

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Abstract

NMDA Stimulates AP-1 Pathway in the Rat Retina

Kohei Kuribayashi1, Yasushi Kitaoka1, Toshio Kumai2, Yasuhiro Hayashi1,

Yasue Yomura1, Hiroyuki Takeda1, and Satoki Ueno1

Activator protein-1 �AP-1� is known to be involved in apoptotic pathways for the various stimuli. Weexamined expression of AP-1 in N-methyl-D-aspartate �NMDA�-induced neurotoxicity in the rat retina.Apoptotic cell death in the inner retina estimated by terminal deoxynucleotidyl transferase-mediated

dUTP-biotin nick-end labeling �TUNEL� staining started to appear at 6 hours and peaked at 24 hours afterintravitreal injection of NMDA �200 nmnol�. Electrophoretic mobility shift assay �EMSA� showed anincrease of AP-1 DNA binding activity in NMDA-treated retina. AP-1 immunoreactivity was observed in

the inner retina and co-localized with TUNEL positive cells after NMDA injection. These results suggest

that activation of AP-1 pathway may be pro-apoptotic in NMDA-induced neuronal cell death in the rat

retina.

1 Department of Ophthalmology St. Marianna University School of Medicine

2 Department of Pharmacology St. Marianna University School of Medicine

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