Prevention of excitotoxicity in primary retinal ganglion cells by (+)-pentazocine, a sigma receptor-1-specific ligand

Ying Dun, Muthusamy Thangaraju, Puttur Prasad, Vadivel Ganapathy, Sylvia B. Smith

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Abstract

PURPOSE. Sigma receptors (σRs) 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 σR1 Iigand (+)-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 μM Glu or 10, 25, 50, or 100 μM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 μM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of σR1 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 σR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 μM Glu or 50 or 100 μM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 μM Glu and (+)-PTZ showed a marked decrease in cell death: 25 μM Glu alone, 50%; 25 μM Glu/0.5 μM (+)-PTZ, 38%; 25 μM Glu/1 μM (+)-PTZ, 20%; 25 μM Glu/3 μM (+)-PTZ, 18%. Similar results were obtained with Hcy. σR1 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 σR1 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 σR1; 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 μM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. σR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.

Original languageEnglish (US)
Pages (from-to)4785-4794
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume48
Issue number10
DOIs
StatePublished - Oct 1 2007

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Pentazocine
Retinal Ganglion Cells
Glutamic Acid
Homocysteine
Ligands
Ganglia
Neurotoxins
Cell Death
Neuroprotective Agents
sigma-1 receptor
Interference Microscopy
sigma Receptors
Retinal Diseases
Excitatory Amino Acids
Fluorescein
Retina
Cell Survival
Western Blotting
Immunohistochemistry
Binding Sites

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

@article{4dad860b12014ebca625801b7a66fa4f,
title = "Prevention of excitotoxicity in primary retinal ganglion cells by (+)-pentazocine, a sigma receptor-1-specific ligand",
abstract = "PURPOSE. Sigma receptors (σRs) 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 σR1 Iigand (+)-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 μM Glu or 10, 25, 50, or 100 μM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 μM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of σR1 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 σR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 μM Glu or 50 or 100 μM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 μM Glu and (+)-PTZ showed a marked decrease in cell death: 25 μM Glu alone, 50{\%}; 25 μM Glu/0.5 μM (+)-PTZ, 38{\%}; 25 μM Glu/1 μM (+)-PTZ, 20{\%}; 25 μM Glu/3 μM (+)-PTZ, 18{\%}. Similar results were obtained with Hcy. σR1 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 σR1 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 σR1; 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 μM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. σR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.",
author = "Ying Dun and Muthusamy Thangaraju and Puttur Prasad and Vadivel Ganapathy and Smith, {Sylvia B.}",
year = "2007",
month = "10",
day = "1",
doi = "10.1167/iovs.07-0343",
language = "English (US)",
volume = "48",
pages = "4785--4794",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
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}

TY - JOUR

T1 - Prevention of excitotoxicity in primary retinal ganglion cells by (+)-pentazocine, a sigma receptor-1-specific ligand

AU - Dun, Ying

AU - Thangaraju, Muthusamy

AU - Prasad, Puttur

AU - Ganapathy, Vadivel

AU - Smith, Sylvia B.

PY - 2007/10/1

Y1 - 2007/10/1

N2 - PURPOSE. Sigma receptors (σRs) 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 σR1 Iigand (+)-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 μM Glu or 10, 25, 50, or 100 μM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 μM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of σR1 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 σR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 μM Glu or 50 or 100 μM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 μM Glu and (+)-PTZ showed a marked decrease in cell death: 25 μM Glu alone, 50%; 25 μM Glu/0.5 μM (+)-PTZ, 38%; 25 μM Glu/1 μM (+)-PTZ, 20%; 25 μM Glu/3 μM (+)-PTZ, 18%. Similar results were obtained with Hcy. σR1 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 σR1 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 σR1; 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 μM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. σR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.

AB - PURPOSE. Sigma receptors (σRs) 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 σR1 Iigand (+)-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 μM Glu or 10, 25, 50, or 100 μM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 μM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of σR1 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 σR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 μM Glu or 50 or 100 μM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 μM Glu and (+)-PTZ showed a marked decrease in cell death: 25 μM Glu alone, 50%; 25 μM Glu/0.5 μM (+)-PTZ, 38%; 25 μM Glu/1 μM (+)-PTZ, 20%; 25 μM Glu/3 μM (+)-PTZ, 18%. Similar results were obtained with Hcy. σR1 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 σR1 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 σR1; 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 μM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. σR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.

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