Abstract
Staurosporine as a protein kinases inhibitor induced cell death or neurite outgrowth in PC12 cells. We investigated the involvement of calcium channel and plasma membrane receptors on staurosporine inducing neurite outgrowth in PC12 cells. PC12 cells were preincubated with NMDA receptor inhibitors (1.8 mM ketamine and 1µM MK801, treatment 1) or L-Type Calcium channels (100 μM nifedipine and 100 µM flavoxate hydrochloride, treatment 2) or calcium-calmoduline kinasses (10 μM trifluoprazine, treatment 3) and nifedipine, MK801, flavoxate hydrochloride and ketamine (treatment4) or without pretreatments (control). Then, the cells were cultured in RPMI culture medium containing 214nM staurosporine for induction of neurite outgrowth. The percentage of Cell cytotoxicity and apoptotic index was assessed. Total neurite length (TNL) and fraction of cell differentiation were assessed. After 24h, the percentage of cell cytotoxicity were increased in treatments 1, 2 and 4 compared with control (p<0.05). After 6h, apoptotic index was similar between all treatments. After 12h, apoptotic index were increased in treatment 4 compared with control (p<0.05). After 24h, apoptotic index were increased in treatments 1, 2 and 4 compared with control (p<0.05). TNL were decreased in treatments 1, 2 and 4 compared with control in different times of assessment (6, 12 and 24 h) (p<0.05). The fraction of cell differentiation were decreased in treatments 1, 2 and 4 compared with control (p<0.05). It can be concluded that the possible involvement of L-type calcium channel and the N-methyl D-aspartate receptor on staurosporine-induced neurite outgrowth process in PC12 cells.
Highlights
Staurosporine (STS) as a protein kinase inhibitor [, ] has dual effects on neuronal cells; induction of cell death and cell differentiation
STS induced increasing of intracellular calcium in treated cells, its effect on plasma membrane and calcium channels and receptors located in the plasma membrane during neuronal differentiation and neurite outgrowth are not well known
Cell cytotoxicity The percentage of cytotoxicity of inhibitors in PC cells cultured in culture medium containing nM staurosporine was assessed by evaluation of the lactate dehydrogenase activity
Summary
Staurosporine (STS) as a protein kinase inhibitor [ , ] has dual effects on neuronal cells; induction of cell death and cell differentiation. The role of STS in the inhibition of protein kinases during neurite outgrowth was clear but its function on plasma membrane calcium channels and receptors remains to be fully known [ , ]. Multiple mechanisms exist whereby increases in intracellular calcium concentration may occur including for example in calcium entry through N-methyl-D-Aspartate (NMDA) glutamate receptors and various voltage-gated calcium channels such as L-type calcium channels (LTCC), as well as in the release of calcium from intracellular stores [ - ]. STS induced increasing of intracellular calcium in treated cells, its effect on plasma membrane and calcium channels and receptors located in the plasma membrane during neuronal differentiation and neurite outgrowth are not well known. In this study we aimed to determine whether plasma membrane calcium channels and receptors involves in staurosporine-induced neurite outgrowth
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