Abstract
Purpose: Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Previously, the authors induced death in the RGC-5 cell line using very high concentrations (1 mM) of the excitatory amino acids glutamate (Glu) and homocysteine (Hcy) and demonstrated that the sigmaR1 ligand (+)-pentazocine ((+)-PTZ) could protect against cell death. The purpose of the present study was to establish a physiologically relevant paradigm for testing the neuroprotective effect of (+)-PTZ in retinal ganglion cells (RGCs). Methods: Primary ganglion cells (GCs) were isolated by immunopanning from retinas of 1-day-old mice, maintained in culture for 3 days, and exposed to 10, 20, 25, or 50 microM Glu or 10, 25, 50, or 100 microM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 microM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of sigmaR1 was assessed by immunocytochemistry, RT-PCR, and Western blotting. Morphologic appearance of live ganglion cells and their processes was examined over time (0, 3, 6, 18 hours) by differential interference contrast (DIC) microscopy after exposure to excitotoxins in the presence or absence of (+)-PTZ. Results: Primary GCs showed robust sigmaR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 microM Glu or 50 or 100 microM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 microM Glu and (+)-PTZ showed a marked decrease in cell death: 25 microM Glu alone, 50%; 25 microM Glu/0.5 microM (+)-PTZ, 38%; 25 microM Glu/1 microM (+)-PTZ, 20%; 25 microM Glu/3 microM (+)-PTZ, 18%. Similar results were obtained with Hcy. sigmaR1 mRNA and protein levels did not change in the presence of the excitotoxins. DIC examination of cells exposed to excitotoxins revealed substantial disruption of neuronal processes; cotreatment with (+)-PTZ revealed marked preservation of these processes. The stereoselective effect of (+)-PTZ for sigmaR1 was established in experiments in which (-)-PTZ, the levo-isomer form of pentazocine, had no neuroprotective effect on excitotoxin-induced ganglion cell death. Conclusions: Primary GCs express sigmaR1; their marked sensitivity to Glu and Hcy toxicity mimics the sensitivity observed in vivo, making them a highly relevant model for testing neuroprotection. Pretreatment of cells with 1 to 3 microM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. sigmaR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.