Expression, subcellular localization and regulation of sigma receptor in retinal Muller cells. Invest Ophthamol Vis Sci.

PURPOSE. Sigma receptors (Rs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Type 1 R1 (R1) is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity, and regulation of R1 in retinal Müller cells. METHODS. Primary mouse Müller cells (MCs) were analyzed by RT-PCR, immunoblotting, and immunocytochemistry for the expression of R1, and data were compared with those of the rat Müller cell line (rMC-1) and the rat ganglion cell line (RGC-5). Confocal microscopy was used to determine the subcellular R1 location in primary mouse MCs. Membranes prepared from these cells were used for binding assays with [ 3 H]-pentazocine (PTZ). The kinetics of binding, the ability of various R1 ligands to compete with R1 binding, and the effects of donated nitric oxide (NO) and reactive oxygen species (ROS) on binding were examined. RESULTS. R1 is expressed in primary mouse MCs and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in primary mouse MCs, rMC-1, and RGC-5, the binding of PTZ was saturable. 2 Among these, type 1 R (R1) is best characterized. The cDNA encoding R1 was cloned originally from guinea pig liver 3 and subsequently from human, mouse, and rat. 4 -7 The R1 cDNA predicts a protein of 223 amino acids (M r , 25-28 kDa). 3 Initial hydropathy analysis of the deduced R1 amino acid sequence suggested a single transmembrane segment. 3, R1 distribution has been analyzed in brain, and its association with neurons is well established. 17 In retina, the role of R1 recognition sites in ischemiareperfusion injury in controlling intraocular pressure and in protection against glutamate-induced neurotoxicity has been reported. 18 -20 Binding assays making use of bovine retinal membranes suggested the presence of Rs, 21,22 but the studies did not disclose in which retinal cell types Rs were present, nor did they establish unequivocally the molecular identity of the receptor. Recently, we used molecular and biochemical methods to study R in mouse retina and reported widespread expression of R1. 23 RT-PCR analysis amplified R1 in neural retina, RPE-choroid complex, and lens. In situ hybridization studies revealed abundant expression of R1 in the ganglion cell layer, inner nuclear layer, inner segments of photoreceptor cells, and RPE cells. Immunohistochemical analysis confirmed these observations. Subsequent studies focused on ganglion cells because of their vulnerability in diabetic retinopathy and revealed that R1 continues to be expressed under hyperglycemic conditions and during diabetic retinopathy, 24 making it a promising target for neuroprotection against cell death. More recent work using the rat ganglion cell line RGC-5 showed that (ϩ)-pentazocine, a R1-specific compound, can block RGC-5 cell death induced by homocysteine and glutamate