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S.B. Smith, W. Li, B.A. Mysona, Y. Dun, L.P. Amarnath, V. Ganapathy; Characterization of Expression and Binding Activity of Sigma Receptor–1 in Retinal Müller Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4816.
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© ARVO (1962-2015); The Authors (2016-present)
Sigma receptors (σR) are non–opioid, non–phencyclidine binding sites whose endogenous function is unknown. Two σR subtypes have been described; ligands for σR1 are thought to have neuroprotective effects. We established the molecular identity of σR1 in intact mouse retina and in the rat ganglion cell line, RGC–5 (Ola et al, 2001) and showed that the σR1 ligand pentazocine (PTZ) can prevent apoptotic RGC death in vitro (Martin et al, 2004). The presence of σR1 has been reported in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. In this study, we asked whether σR1 was expressed in retinal Müller cells and examined the σR binding activity in these cells compared with ganglion cells.
Primary Müller cells (1°MC) were isolated from 5–7 day C57BL/6 mice. RT–PCR, western blotting and immunocytochemistry were used to analyze the expression of σR1 in 1°MC, the rMC–1 (rat Müller cell line) and RGC–5 cells. Membranes were prepared from these cells and used for binding assays using [3H]–PTZ. The ability of various σR1 ligands (3–PPP, DTG, PTZ and HPD) to compete with this binding was examined in Müller and RGC–5 cells.
Using primers for σR1, RT–PCR products of appropriate size were amplified from the primary cells and cell lines. Western blotting using our antibody against σR1 detected a single protein band consistent in size with σR1 (Mr∼27 kDa) in Müller cells (both primary cells and the cell line); immunocytochemistry confirmed the presence of σR1 in these cells. Binding assays showed that in Müller cells and RGC–5 cells, the binding of PTZ was saturable. [3H]–PTZ bound with high affinity in RGC–5 cells and the binding was similarly robust in 1°MC and slightly less in the rMC–1 cells. Competition studies showed marked inhibition of [3H]–PTZ binding in the presence of various σR1–specific ligands.
Müller cells demonstrate robust σR1 binding activity. The potential of these glial cells to bind σR1 ligands may prove beneficial in retinal degenerative diseases. Future studies will analyze factors that regulate σR1 activity and expression in Müller and ganglion cells.
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