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Previously, we showed that dopamine protects cultured retinal neurons from N-methyl-D-aspartate (NMDA) receptor mediated-glutamate excitotoxicity via dopamine D1 receptor. This study has demonstrated for the first time that nitric oxide synthase (NOS) plays a crucial role in dopamine-induced neuroprotection. Our patch clamp study has shown that dopamine does not affect the NMDA-induced whole cell current. Dopamine or SKF38393 (D1 receptor agonist) inhibited ionomycin (calcium ionophore)-induced toxicity, while dopamine did not affect S-nitrosocysteine (NO donor)-induced toxicity. Biochemical analysis on enzymatic activities has shown that dopamine or cAMP (which is generated through D1 receptor stimulation) inhibits glutamate induced-NOS activation. These results suggest that dopamine inhibits glutamate induced-NOS activation via D1 receptor, resulting in the protection of retinal neurons.

Purpose: It has been shown that bradykinin (BK) protects retinal neurons against glutamate excitotoxicity, but it was not clear how BK inhibits glutamate excitotoxicity. The purpose of this study was to investigate the effect of opening the mitochondrial adenosine triphosphate (ATP)-sensitive potassium (Mit K (ATP)) channel on glutamate excitotoxicity and the protective effect of BK using cultured retinal neurons., Methods: Primary cultures were obtained from the retina of fetal rats (gestation days 17-19). Glutamate neurotoxicity was assessed by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, using the trypan blue exclusion method. BK, diazoxide (the opener of the Mit K (ATP) channel), 5HD, and glibenclamide (blockers of the Mit K (ATP) channel) were applied simultaneously with glutamate. Mitochondrial membrane potential was measured as the ratio of 590:527 nm fluorescence of JC-1., Results: Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, and glutamate induced mitochondrial depolarization of retinal neurons. BK and diazoxide protected retinal neurons against glutamate excitotoxicity and inhibited glutamate-induced mitochondrial depolarization. These actions of BK and diazoxide were inhibited by the coapplication of 5HD and glibenclamide. Furthermore, diazoxide inhibited the sodium nitroprusside (SNP, NO donor) toxicity, but did not inhibit the 3-morpholinosydnonimine (SIN-1, NO, and superoxide donor) toxicity., Conclusions: These results suggest that BK and diazoxide protect retinal neurons against glutamate excitotoxicity by opening the Mit K (ATP) channel. It is suggested that opening of the Mit K (ATP) channel inhibited glutamate-induced generation of superoxide.

Purpose: To examine the effects of acetylcholine (ACh) on glutamate-induced neurotoxicity in embryonic rat retinal neurons., Methods: Primary cultures were obtained from rat retinas at embryonic days 17 to 19. Cultured cells were exposed to glutamate for 10 minutes, followed by incubation in glutamate-free medium for 1 hour. Drugs were added to the incubation medium for 1 to 24 hours until immediately before glutamate exposure and were removed from culture medium during glutamate exposure and the postincubation period. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method., Results: Cell viability was markedly reduced by 10-minute exposure to 500 microM glutamate followed by a 1-hour incubation in glutamate-free medium. Incubating the cultures with 1 microM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 microM). The protective effect of ACh against glutamate neurotoxicity was inhibited by a nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine (0.5 microM), whereas a muscarinic acetylcholine receptor (mAChR) antagonist, atropine (0.5 microM) did not affect ACh-induced protection. In addition, a similar protection was induced by application of nicotine (1 microM), but not by muscarine (1 microM). Pretreatment with nicotine induced a protective effect in a time-dependent manner, ranging from 1 to 12 hours. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 microM induced dose-dependent protection against glutamate neurotoxicity. Furthermore, the protective action of nicotine was inhibited by simultaneous application of dopamine D1 receptor antagonist, SCH23390 (1 microM), with nicotine, whereas a dopamine D2 receptor antagonist, domperidone (1 microM), did not affect nicotine-induced protection., Conclusions: These results suggest that pretreatment of cultured rat retinal neurons with ACh or the nAChR agonists, nicotine and carbachol, has a protective action against glutamate neurotoxicity through nAChRs and that the dopamine release induced by nicotinic stimulation subsequently protects the retinal neurons by way of dopamine D1 receptors.

Purpose: To examine the effects of ifenprodil on glutamate-induced neurotoxicity in cultured retinal neurons., Methods: Primary cultures obtained from the fetal rat retina (gestation day 17-19) were used for the experiment. Neurotoxicity effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method. The cells were exposed briefly (10 min) to excitatory amino acids (EAA, 1 mM) and then were incubated for 1 h in an EAA-free medium. Ifenprodil (10 mM) was added for the 10-min exposure to EAA and the subsequent 60-min incubation in an EAA-free medium., Results: Ifenprodil dose-dependently prevented cell death induced by glutamate or NMDA, but did not affect that induced by kainate. The protective effects of ifenprodil against glutamate neurotoxicity were significantly reduced by spermidine, a polyamine modulatory site agonist, but not by glycine, a strychnine-insensitive glycine site agonist., Conclusion: The findings suggest that ifenprodil protected the cultured retinal cells we used in this study against glutamate neurotoxicity by its inhibitory action on the polyamine modulatory site of the NMDA receptor.

Purpose: To identify the localization and expression of bradykinin (BK)-B2 receptors in rat retina and examine the effects of BK on glutamate-induced neurotoxicity using cultured rat retinal neurons., Methods: An immunohistochemical study using a specific antibody against BK-B2 receptor was performed with rat retina. Primary cultures were obtained from the retina of fetal rats (gestation day 17-19). Expression of BK-B2 receptor mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA obtained from cultured retinal neurons. Cultured cells were exposed to glutamate (1 mM) for 10 minutes and followed by incubation in glutamate-free medium for 1 hour. The effects of BK were assessed by simultaneous application of BK with glutamate. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method., Results: Immunohistochemical study demonstrated that BK-B2 receptors were expressed in the ganglion cell, inner nuclear layers, and outer nuclear layers. Furthermore, BK-B2 receptor mRNA expression was observed in cultured retinal neurons. Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by a 1-hour incubation in glutamate-free medium. Simultaneous application of BK at concentrations of 0.001 to 1 microM with glutamate demonstrated dose-dependent protection against glutamate neurotoxicity. The protective action of BK (1 microM) was inhibited by simultaneous application of BK-B2 receptor antagonist, Hoe140 (1 microM). Furthermore, 1 microM BK had protective effects on neurotoxicity induced by 1 microM ionomycin, a calcium ionophore, and sodium nitroprusside (SNP, 500 microM), a nitric oxide (NO)-generating agent. However, BK did not inhibit neurotoxicity induced by 3-morpholinosydnonimine (SIN-1, 10 microM), an NO and oxygen radical donor., Conclusions: These results suggest that BK-B2 receptors were distributed in rat retinas and cultured retinal neurons and that BK had a protective action against glutamate neurotoxicity through BK-B2 receptors in cultured retinal neurons. It is suggested that BK-induced protection against glutamate neurotoxicity took place downstream to NO generation and upstream to oxygen radical generation.

Background: Both in vivo and in vitro studies suggest that excess stimulation of non-NMDA receptors can result in massive neuronal death in the retina. In particular, murine amacrine neurons have been known to show marked susceptibility to the toxic effects of kainate., Purpose: This study was designed to examine and characterize the role of N-methyl-D-aspartate (NMDA) receptor vs non-NMDA receptor in glutamate-induced neurotoxicity in the retina., Methods: Primary cultures obtained from fetal rat retina (gestation day 16-19) were used for the experiment. The neurotoxicity was assessed quantitatively using the trypan blue exclusion method. Electrophysiological studies using patch-clamp techniques were performed to record whole-cell currents evoked by these excitatory amino acids., Results: Removal of extracellular Ca2+ from the medium or application of MK-801 reduced the extent of cell death induced by the brief exposure to glutamate, NMDA, and kainate. By contrast, cell death induced by a 60-min exposure to kainate was not affected by MK-801. The electrophysiological study demonstrated that MK-801 abolished the whole-cell currents evoked by NMDA but had no effect on those induced by kainate or AMPA., Conclusion: These findings demonstrate that brief exposure to kainate induces cell death by way of activating NMDA receptors in cultured fetal retinal neurons and that NMDA receptors are the predominant route of fetal retinal neurotoxicity induced by brief glutamate exposure.

Purpose: To examine the effects of FK506 on glutamate neurotoxicity in cultured retinal neurons., Methods: Experiments were performed with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. To assess the effects of FK506 and other drugs on glutamate neurotoxicity, cultures were treated with a drug beginning 10 minutes before application of glutamate and continuing during the subsequent 10 minutes of glutamate exposure. The treated cells were then incubated for 1 hour in a drug-free and glutamate-free medium. After a 1-hour incubation, cell viability was quantitatively measured by the trypan blue exclusion method., Results: Brief exposure to glutamate markedly decreased cell viability. FK506 protected against glutamate neurotoxicity in a dose-dependent manner. Rapamycin is a competitive inhibitor of FK506 that binds FK506 binding protein. Simultaneous application of rapamycin and FK506 negated the protective effects of FK506. Cyclosporin A, which binds and inhibits calcineurin, mimicked the protective effects of FK506. Treatment with FK506 did not affect the intracellular maximum Ca2+ concentration induced by glutamate application. Although FK506 exhibited protective action against Ca2+ ionophore-induced neurotoxicity, it had no effect on nitric oxide-induced neurotoxicity. Treatment with FK506 reduced the activity of nitric oxide synthase (NOS)., Conclusion: FK506 protected against glutamate neurotoxicity by inhibiting NOS activity in cultured retinal neurons.