Sigma receptor agonists provide neuroprotection in vitro by preserving bcl-2.
BACKGROUND
Sigma (sigma)-receptor agonists attenuate brain injury after experimental focal cerebral ischemia in several species. We tested the hypothesis that the potent, prototypical sigma(1)-receptor agonist, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), protects neurons by a mechanism involving the antiapoptotic protein bcl-2.


METHODS
Primary cortical neuronal cultures were exposed to either 2 h of oxygen-glucose deprivation (OGD) or glutamate (100 microM). PPBP treatment was initiated either 15 min prior to the insult or at 15 min postinsult then continued for 24 h. In another set of experiments, cultured neurons were preincubated for 2 h prior to PPBP treatment with sigma1-receptor antagonist, rimcazole, in a dose-dependent manner. Alive and dead cells were detected with calcein-AM and propidium iodide respectively. Bcl-2 and bax expression were determined by quantitative real time reverse transcription polymerase chain reaction and western blotting, and DNA damage was detected by TUNEL staining.


RESULTS
PPBP pretreatment attenuated neuronal injury induced by OGD or glutamate (50 or 100 microM). This protection was reversed with rimcazole (cell death: OGD 48 +/- 2%, OGD plus PPBP 31 +/- 3%, OGD plus PPBP with rimcazole 46 +/- 2%). PPBP treatment increased bcl-2 but not bax mRNA levels. PPBP's ability to preserve bcl-2 protein after OGD by PPBP was fully abolished by rimcazole. Lastly, PPBP reduced the number of TUNEL-positive cells after OGD, suggesting fewer cells with overt DNA damage.


CONCLUSIONS
These data demonstrate that PPBP reduces cell death in vitro by a mechanism involving receptor-dependent preservation of protective genes such as bcl-2.
Yang
Sufang
S
Department of Anesthesiology and Peri-operative Medicine, Oregon Health Sciences University, Portland, Oregon 97239-3098, USA.
Bhardwaj
Anish
A
Cheng
Jian
J
Alkayed
Nabil J
NJ
Hurn
Patricia D
PD
Kirsch
Jeffrey R
JR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017480:Receptors, sigma / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000502:physiology*
D018696:Neuroprotective Agents; D010880:Piperidines; D019253:Proto-Oncogene Proteins c-bcl-2; D017480:Receptors, sigma; C032727:piperidine