Sigma-1 receptors protect RGC-5 cells from apoptosis by regulating intracellular calcium, Bax levels, and caspase-3 activation

¹ Pharmacology & Neuroscience, UNT Health Science Center, Fort Worth, Texas, United States
² Pharmacology & Neuroscience, University of N. Texas Health Science Center, Fort Worth, Texas, United States

^* To whom correspondence should be addressed. E-mail: yoriot{at}hsc.unt.edu.

	Abstract

PURPOSE: Sigma-1 receptor ligands prevent neuronal death associated with glutamate excitotoxicity both in vitro and in vivo. However, the molecular mechanism of the neuroprotective effect remains to be elucidated. Presently, we determined that sigma-1 receptor agonists provide neuroprotection by decreasing glutamate-induced calcium mobilization and prevent apoptotic gene expression. METHODS: Cell death was measured using the calcein-AM/propidium iodide cell survival assay. Western blot analysis determined the expression levels of Bax and caveolin-1 in normal RGC-5 cells. Immunocytochemistry compared the subcellular expression of sigma-1 receptors and caveolin-1 in normal RGC-5 cells. Caspase-3 activation after glutamate treatment was determined using the carboxyfluorescein caspase-3 detection kit. Glutamate-induced intracellular calcium mobilization was measured using ratiometric calcium imaging. RESULTS: Sigma-1 receptor overexpressing RGC-5 (RGC-5-S1R) cells have lower glutamate-induced intracellular calcium mobilization compared to normal RGC-5 cells and the sigma-1 receptor ligand (+)-SKF10047 reduced the glutamate calcium response in normal and RGC-5-S1R cells. (+)-SKF10047 protected RGC-5 cells from glutamate-induced cell death and RGC-5-S1R cells showed a significant resistance to glutamate-induced apoptosis compared to the control RGC-5 cells. BD1047, a sigma-1 receptor antagonist, blocked the protective effect of (+)-SKF10047. Western blot showed that (+)-SKF10047 inhibited the increase of Bax and Caveolin-1 after glutamate treatments. Glutamate-mediated cell death involved activation of caspase-3, and sigma-1 receptor activation prevented an increase in caspase-3 expression. CONCLUSIONS: These results suggest that sigma-1 receptors regulate intracellular calcium levels and prevent activation of pro-apoptotic genes and promote retinal ganglion cell survival. Thus, sigma-1 ligands appear to be neuroprotective and a potential target for glaucoma therapeutics.

Key Words: ganglion cell, glaucoma pharmacology, apoptosis

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