Characterization of Novel Sigma-1 Receptor (σ1-r) Ligands




Broyles, Heather V.
Park, Yong H.
Li, Linya
Ellis, Dorette Z.


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Purpose: Sigma-1 receptors (σ-1rs) are non-opioid ligand receptors that are associated with the endoplasmic reticulum. Upon agonist stimulation during stress, these receptors have the ability to translocate to the plasma membrane. σ-1rs are known to mediate ion channels such as L-type Voltage Gated Calcium Channels (VGCCs), thus facilitating neuroprotective effects in neurons such as retinal ganglion cells (RGCs). The purpose of this study was to determine if novel σ-1r agonist (PB190) and novel σ-1r antagonist (PB212) in purified RGCs have similar actions to that of known σ-1r agonists and antagonists, such as Pentazocine and BD1063, respectively. Methods: Purification and the culture of RGCs were performed by a double immunopanning technique using an antibody to Thy1.1 from P3-P7 Sprague-Dawley rats. RGCs were cultured for 5-7 days in vitro prior to experiments. Purified RGCs were incubated for 30 minutes with 1µM treatments of either Pentazocine, PB190, BD1063, PB212, or PB212 + PB190 combined. FURA-2 AM fluorescent dye was used to determine calcium concentration within RGCs. Immunoblot and immunocytochemistry were used to determine purity of RGCs and expression of σ-1r. Results: Immunocytochemistry determined 98% purity of isolated RGC cultures following 7 days in vitro. σ-1r expression was identified in purified RGCs through immunocytochemistry and immunoblot (MW~26 kDa). Intracellular calcium concentration [Ca2+]i was determined in RGCs where control (no treatment) group calcium levels were equal to 265 ± 104 nM. Pentazocine [Ca2+]i levels were equal to 115 ± 13 nM. PB190 [Ca2+]i levels were equal to 102 ± 9 nM. BD1063 [Ca2+]i levels were equal to 119 ± 13 nM. PB212 [Ca2+]i levels were equal to 118 ± 14 nM. PB190 and PB 212 co-treatment [Ca2+]i levels were equal 135 ± 15 nM. At the concentrations used (1µM), all of these σ-1r ligands significantly (Ca2+]i levels compared to control group. Conclusion: All σ-1r ligands utilized in this study modulated basal intracellular calcium levels. The new compounds PB190 and PB212 have similar effects in decreasing basal calcium levels as those seen in the known compounds Pentazocine and BD1063. Activation or inhibiting σ-1r could be modulating calcium channels involved in homeostasis such as the VGCCs. Future studies are needed to determine if these new ligands are more efficacious than that of known σ-1r ligands, and what other calcium channels they may modulate.