EJBS 5 (1) ● June 2012 www.ejarr.com/Volumes/Vol5/EJBS_5_04.pdf 15 The Effects of Acute Different Concentrations of Morphine on Neurite Elongation in PC12 Cells Hossein Zhaleh 1,2 , Ali Bidmeshki Pour 1 , Mehri Azadbakht 1* , Mohsen Zhaleh 3 1 Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran 2 Department of Chemical Biotechnology Engineering, Science and Research Branch, Islamic Azad University, Kermanshah, Iran 3 Department of Anatomy, Faculty of Medicine, Ahvaz Jundishapur University of Medical sciences, Ahvaz, Iran E-mail: hossain_jale@yahoo.com ABSTRACT Morphine as a mu opioid drug has some effective roles on neuronal cells. Some data suggest that morphine induces apoptosis in neurons, while other evidence shows that morphine could have beneficial effects on neuritogenesis. The present study was undertaken to evaluate the effects of different concentrations of morphine (10 -12 -10 -4 M) in acute manner on neurite elongation in PC12 cells. Cell viability, Total neurite length and Cytotoxicity were assay. Our results show that low concentrations of morphine (10 -12 -10 -10 M) in acute manner enhances neurite elongation and cell viability, and decreases cytotoxicity activity in PC12 cells with opioid receptor-dependent mechanism. Our results show that high concentrations of morphine (10 -6 -10 -4 M) in acute manner inhibits neurite elongation and cell viability, and increases cytotoxicity activity in PC12 cells with opioid receptor-dependent mechanism. Keywords: Morphine; Naltrexone; Neurite elongation; PC12 cell INTRODUCTION Opioids are some of the most effective pain-relieving drugs in the clinical management of pain [1]. These drugs at acute exposure affect a number of physiological and cellular functions including hormone secretion, neurotransmitter releasing, feeding, and reduction of intracellular cAMP and inhibition of neuronal firing [2]. It has been shown that opioids have defined at least three major types of opioid receptors designated mu, kappa and delta [3]. The opioid receptor antagonist naloxane and naltrexone is competitive antagonist at mu, kappa and delta receptors with a high affinity for mu receptor [4]. Some drugs including morphine and methadone can bind to mu (μ) receptor. Mu receptor agonists modulated survival [5], apoptosis [6], proliferation [7-13], cell growth [7] and viability [9] of neural and tumoral cells [14-16]. It has been shown that morphine also reverses manner-induced apoptosis in different cell lines [17]. Opioids have effective role in development of brain, neuritogenesis, and cell differentiation [18-22]. Further, With respect to neuritogenesis, chronic morphine has been found to exert two opposite effects that are concentration-dependent. Morphine at 1mM concentration leads to inhibit neurite elongation in PC12 cells, cerebellar granule neurons, cerebellar neuroblasts, and embryonic dorsal root ganglion neurons with naloxane-dependent mechanism whereas at 1pM it leads to enhance neurite elongation of PC12 cells , cultured rat spinal cord , cerebral cortical neurons with naloxane-independent mechanism [22-25]. However, the effects of morphine at acute exposure on neurite elongation in PC12 cells do not specified. This study was undertaken to determine whether acute exposure of morphine enhance neurite elongation in PC12 cells and whether that action dependent on mu opioid receptor? MATERIALS AND METHODS Cell culture PC12 cells were grown in RPMI 1640 culture medium (Gibco), supplemented with 10 % fetal bovine serum (FBS, Gibco), 1% non-essential amino acid (NEAA; Sigma), 2mM L-glutamine (Sigma), 100 IU/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) in 10-cm tissue culture dishes. The cultures were incubated at 37 ºC in 5% CO 2 . Medium was replaced every 2 days. When cell cultures reached 70 to 80% confluency, they were trypsinated using trypsin-EDTA 0.25% (Sigma) and were subcultured at a density of 1×10 4 cells/well in 24-well culture plates. STAUROSPORINE AND MORPHINE TREATMENT One day after plating PC12 cells, cells were washed with phosphate buffer saline (PBS), pH 7.4. There were two groups: group 1; no preincubation with naltrexone hydrochloride (Sigma) (without naltrexone) and group 2; preincubation with 100 nM naltrexone for 30 min. There were six treatments in each group, all of which were performed in the presence of staurosporine to maintaining the cell differentiation. PC12 cells were cultured in RPMI 1640 culture medium containing 214 nM staurosporine (Alexis, USA), 0.02% bovine serum albumine (BSA;