Abstract
Purpose: :
The purpose of these studies was to investigate the neuroprotective effects of sigma receptor agonists on cultured retinal ganglion cells, which offer a unique and convenient model to study the mechanism of retinal ganglion cell death.
Methods: :
Differentiated and undifferentiated retinal ganglion cells (RGC–5) were used to determine the expression of sigma receptors. RGC–5 cells calcium response was also measured by a ratiometric technique using Fura– 2/AM. The effect of sigma ligands on endothelin–1 receptor expression in both retinal ganglion cells and human breast cancer cells (MCF–7) was also determined. A DNA fragmentation technique was used to assess retinal ganglion cell apoptosis after treatment with 1mM glutamate for 24 hours.
Results: :
Western blot and quantitative PCR have shown that both (+)–N–allylnormetazocine hydrochloride [(+) SKF10047]) and haloperidol down regulate endothelin 1 receptors A and B after 24 hours treatments. (+)–N–Allylnormetazocine hydrochloride [(+) SKF10047]) inhibited the retinal ganglion cell glutamate or KCL induced calcium influx. Haloperidol reversed the effect the (+) SKF10047 in RGC–5 cells. Endothelin–1 also potentiated KCL induced calcium influx in RGC–5 cells. Nimodipine was able to block the KCL induced calcium influx. Reverse transcriptase polymerase chain reaction (RT–PCR) demonstrated the expression of sigma receptors in both differentiated and undifferentiated RGC–5 and MCF–7 cells. In addition, we have shown that sigma 1 receptors were upregulated in retinal ganglion cells after endothelin 1, glutamate and (+)–SKF10047 treatments. DNA fragmentation studies have shown that undifferentiated RGC–5 cells were not sensitive to glutamate–induced apoptosis compared to differentiated RGC–5 cells, which were sensitive to glutamate. Quantitative PCR results show that sigma ligands down regulate both endothelin1 receptors A and B.
Conclusions: :
Sigma ligands (+) SKF10047, protected differentiated RGC–5 cells from apoptosis compared to haloperidol, which fails to protect differentiated RGC–5 cells from apoptosis induced by glutamate excitotoxicity. In addition, the calcium imaging results show that sigma 1 receptor ligands modulate intracellular calcium homeostasis by regulating voltage gated calcium channels, NMDA receptor, and endothelin A receptors. Thus Sigma 1 ligands may be neuroprotective and a target for potential glaucoma therapeutics.
Keywords: neuroprotection • ganglion cells • calcium