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We monitored increases in both intracellular calcium concentration ((Ca2+)i) and whole‐cell current responses induced by N‐methyl‐D‐aspartate (NMDA), applied with co‐agonist glycine, using fura‐2 digital imaging and patch‐clamp recording techniques. Extracellular application of oxidized glutathione (GSSG), but not reduced glutathione (GSH), inhibited responses mediated by activation of the NMDA subtype of glutamate receptor in cultures of rat cortical and retinal ganglion cell neurons. The NMDA responses were persistently inhibited by GSSG (500 μM to 10 mM) until introduction of a selective sulfhydryl reducing agent, dithiothreitol, which resulted in complete recovery of the responses. Exposure of the neurons to 5,5‐dithio‐bis‐2‐nitrobenzoic acid (DTNB), an efficacious oxidizing agent, also resulted in persistently smaller responses to NMDA. The addition of GSSG following exposure to DTNB, however, did not result in a further decrement in NMDA responses in our experimental paradigm. These findings suggest that a predominant action of GSSG is oxidation of vicinal thiol groups to form a peptide disulfide bond(s) comprising the redox modulatory site of the NMDA receptor–channel complex. Evidence for such regulatory sulfhydryl centers associated with the NMDA receptor has been presented previously. Moreover, the fact that DTNB produced little if any additional attenuation of the NMDA (Ca2+)i response when administered alter GSSG implies that GSSG is also an efficacious oxidant at this site. GSSG displayed little or no effect on (Ca2+)i responses elicited by high extracellular K+ or by kainate, suggesting that, at least under the conditions of the present experiments, GSSG was somewhat selective for the NMDA redox modulatory site. Based on these observations, we suggest that GSSG exerts its NMDA‐specific redox effects in a novel extracellular manner.
Journal of Neuroscience Research – Wiley
Published: Nov 1, 1991
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