Lithium chloride protects PC12 pheochromocytoma cell line from morphine-induced apoptosis.
BACKGROUND
Opioid drugs are considered as important members of drugs of abuse. Opioid abusers are more likely to be infected which may be due to apoptotic effects of the drugs on immune cells. Furthermore, there are some reports on the apoptotic effect of morphine on neural cells. In the present study, the effect of morphine and lithium on apoptosis in PC12 cell line (as a model of neural cells) was examined.


METHODS
We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-fluorescein isothiocyanate test and quantitative real time RT-polymerase chain reaction for detection of necrosis and apoptosis (programmed cell death).


RESULTS
PC12 cells were exposed to different concentrations of morphine for six, 12, 24, 48, and 96 hours. Quantitative real-time RT-polymerase chain reaction revealed that mRNA expression of BAX (proapoptotic element) increased while a decrement in the mRNA expression of BCL-2 (protective element) was observed after six hours (but not after 12 or 24 hours) exposure to morphine. Furthermore, the results of MTT assay and annexin V-fluorescein isothiocyanate test indicated that morphine exposure causes an increase in the percentage of apoptotic and necrotic cells, respectively. Interestingly, the results of MTT assay and annexin V-fluorescein isothiocyanate test were observed 12 and 24 hours after morphine exposure. Thus, it can be concluded that alteration in mRNA expression is an early event rather than as a consequence of apoptosis or necrosis. On the other hand, lower concentrations of lithium elicit protective effect against apoptosis in some of mammalian cells while the higher concentrations are toxic. Despite large body of evidences on the protective effect of lithium, elucidation of downstream events are still unknown. In the present study, 72-hour preincubation of PC12 cells with 1.2 mM lithium chloride reversed the effects of morphine on the mRNA expression of BAX and BCL-2. Furthermore, the results of real time RT-polymerase chain reaction were supported by annexin V-fluorescein isothiocyanate test and MTT assay.


CONCLUSIONS
The protective effect of lithium on the morphine-induced cytotoxicity is mediated via down-regulation of BAX and up-regulation of BCL-2 mRNA expression.
Sahebgharani
Mousa
M
Department of Pharmacology, School of Medicine, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. sahebgha@sina.tums.ac.ir
Nejati
Majid
M
Sepehrizadeh
Zargham
Z
Khorramizadeh
Mohammad-Reza
MR
Bahrololoumi-Shapourabadi
Mina
M
Hashemi-Bozchlou
Saeed
S
Esmaeili
Jamileh
J
Ghazi-Khansari
Mahmoud
M
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D018692:Antimanic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D018021:Lithium Chloride / Q000494:pharmacology*; D008959:Models, Neurological; D009020:Morphine / Q000494:pharmacology; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000503:physiopathology*; D012333:RNA, Messenger / Q000187:drug effects; D051381:Rats; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000187:drug effects / Q000378:metabolism
D000701:Analgesics, Opioid; D018692:Antimanic Agents; C494826:BAX protein, human; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D009020:Morphine; D018021:Lithium Chloride