Advanced glycation end products induce death of retinal neurons via activation of nitric oxide synthase.
The purpose of this study was to determine whether advanced glycation end products (AGEs) are neurotoxic for cultured retinal neurons consisting mainly of amacrine cells, and to determine whether endogenous nitric oxide (NO) is involved in the toxicity. Cultured retinal neurons obtained from fetal Wistar rats (gestational age 19 days) were maintained in culture for 10 days, and then exposed to different concentrations of AGEs (0.02, 0.1, and 0.5 mg ml(-1)) in cultured media for different lengths of time. Both trypan blue exclusion and TUNEL assay were used to determine whether AGEs were neurotoxic, and NG-nitro-L-arginine methyl ester (L-NAME, 500 microM), a nitric oxide synthase (NOS) inhibitor, was used to determine whether NO was involved. Immunohistochemical analyses were performed to determine whether specific receptors of AGEs (RAGE) are present on cultured retinal neurons; caspase-3 was activated, and 3-nitrotyrosine was expressed on neurons treated with AGEs. Nitrite levels were measured in the supernatants of the media where neurons were incubated with AGEs. AGEs induced cell death in a time- and dose-dependent manner. TUNEL-positive cells and immunoreactivity to cleaved caspase-3 were enhanced on neurons following exposure to AGEs. L-NAME significantly suppressed the AGEs-induced neurotoxicity as assessed by both trypan blue exclusion and TUNEL assays. Activation of NOS was suggested by enhanced immunoreactivity to 3-nitrotyrosine on neurons and increased nitrite levels in the media incubated with AGEs. These results indicate that AGEs are neurotoxic to retinal neurons in culture through the activation of NOS. Apoptotic pathways may be in part involved in the death of the neurons.
Department of Ophthalmology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki, Osaka 569-8686, Japan.
Takatoshi
T
Kobayashi
Hidehiro
H
Oku
Asako
A
Komori
Takashi
T
Okuno
Shota
S
Kojima
Hiroshi
H
Obayashi
Tetsuya
T
Sugiyama
Goji
G
Hasegawa
Michiaki
M
Fukui
Naoto
N
Nakamura
Tsunehiko
T
Ikeda
D025042:Amacrine Cells / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D004791:Enzyme Inhibitors; D017127:Glycation End Products, Advanced; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D019331:NG-Nitroarginine Methyl Ester