Divergent NMDA signals leading to proapoptotic and antiapoptotic pathways in the rat retina.
OBJECTIVE
To investigate the involvement of the p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI-3) kinase-Akt signaling pathways after pathologic stimulation by N-methyl-D-aspartate (NMDA) receptors of retinal neurons in vivo.


METHODS
NMDA (2-200 nmol), SB203580 (0.2-10 nmol, an inhibitor of p38 MAP kinase), LY294002 (6 nmol, an inhibitor of PI-3 kinase), or control solution was injected into the vitreous of Long-Evans rats. To assess retinal ganglion cell (RGC) death quantitatively, we labeled RGCs retrogradely by injecting aminostilbamidine (FluoroGold) into the superior colliculus and subsequently counting fluorescently labeled RGCs in retinal wholemounts. Phosphorylation of p38 and Akt was assessed by immunoblot of whole retinal lysates, and activity was measured with in vitro kinase assays. To localize phospho-p38 and phospho-Akt, immunohistochemistry was performed. TUNEL staining coupled with morphologic assessment was performed to assess apoptotic cell death.


RESULTS
Intravitreous injection of more than 10 nmol NMDA induced RGC death. Before death, NMDA-stimulated retinas manifested increased phospho-p38 and phospho-Akt in the ganglion cell and inner nuclear layers. Subsequently, pyknotic, TUNEL-positive cells were also localized to these regions. SB203580 partially rescued RGCs, whereas LY294002 enhanced death of RGCs due to 10 nmol NMDA. SB203580 and LY294002 specifically inhibited the activity of p38 MAP kinase and Akt, respectively.


CONCLUSIONS
The p38 MAP kinase and PI-3 kinase-Akt pathways are involved in signal transduction after excessive stimulation of NMDA receptors in the retina. These inhibitor studies suggest that the p38 MAP kinase pathway is proapoptotic, whereas the PI-3 kinase-Akt pathway is antiapoptotic in RGC death induced by NMDA.
Manabe
Shin-ichi
S
Center for Neuroscience and Aging, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Lipton
Stuart A
SA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D020318:Rats, Long-Evans; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases
D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D020928:Mitogen-Activated Protein Kinases; D016202:N-Methylaspartate; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016194:Receptors, N-Methyl-D-Aspartate; D048051:p38 Mitogen-Activated Protein Kinases; C494920:Akt1 protein, rat