Possible involvement of calcium channels and plasma membrane receptors on Staurosporine-induced neurite outgrowth

Staurosporine as a protein kinases inhibitor induced cell death or neurite outgrowth in PC cells. We investigated the involvement of calcium channel and plasma membrane receptors on staurosporine inducing neurite outgrowth in PC cells. PC cells were preincubated with NMDA receptor inhibitors (. mM ketamine and μM MK, treatment ) or L-Type Calcium channels ( μM nifedipine and  μM fl avoxate hydrochloride, treatment ) or calcium-calmoduline kinasses ( μM trifl uoprazine, treatment ) and nifedipine, MK, fl avoxate hydrochloride and ketamine (treatment) or without pretreatments (control). Th en, the cells were cultured in RPMI culture medium containing nM staurosporine for induction of neurite outgrowth. Th e percentage of Cell cytotoxicity and apoptotic index was assessed. Total neurite length (TNL) and fraction of cell diff erentiation were assessed. After h, the percentage of cell cytotoxicity were increased in treatments ,  and  compared with control (p<.). After h, apoptotic index was similar between all treatments. After h, apoptotic index were increased in treatment  compared with control (p<.). After h, apoptotic index were increased in treatments ,  and  compared with control (p<.). TNL were decreased in treatments ,  and  compared with control in diff erent times of assessment (,  and  h) (p<.). Th e fraction of cell diff erentiation were decreased in treatments ,  and  compared with control (p<.). 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 PC cells. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Staurosporine (STS) as a protein kinase inhibitor [, ] has dual eff ects on neuronal cells; induction of cell death and cell diff erentiation.STS induces apoptosis in high concentrations (μM) [, ] and neuronal differentiation in low concentrations (nM) by neurite extension in several types of cells [, , , ].Although the detailed mechanism of STS action as a neurogenic morphogen remains unclear, it seems that it is associated with the inhibition of some protein kinases which may contribute to neurite outgrowth [].In previous studies it has been determined that STS can inhibit PKA, Ca + / calmodulin-dependent kinase II, cyclin dependent kinases, ion channels (Kv.,L-type Ca + channel, voltage-gated K + channel) in myocyte [-].Although, 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 [,].Calcium plays an important role in the regulating a great variety of neuronal processes such as neuronal cell diff erentiation.In most neurons, 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 [-].Calcium infl ux through LTCCs is particularly eff ective in neuronal migration, activation of transcription factors (e.g CREB), changes in gene expression that underlie plasticity and adaptive neuronal responses (e.g c-fos) [, -].Although 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 diff erentiation 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.

MATERIALS AND METHODS
Cell culture PC cells were cultured in complete culture medium containing RPMI culture medium (Gibco), supplemented

Quantifi cation of cell death incidence
Hoechst / PI nuclear staining was carried out as previously described [].Briefl y, cells were plated in  well culture plates with  cells/mL density for h.Then cells were pre-treated in different treatment mediums for certain time.Th ese were grown for a range of times in diff erentiation medium (,  and h).Th en cells were incubated for  min at °C with Hoechst  dye ( mg/ml in PBS), washed twice in PBS.PI ( mg/ml in PBS) was added just before microscopy.Cells were visualized using an invertedflorescence microscope (Olympus IX-, Japan).Nuclear morphology was scored as follows: , viable cells had bluestained nuclei with smooth appearance; , viable apoptotic cells had blue-stained nuclei with multiple bright specks of condensed chromatin; , non-viable apoptotic cells had red-stained nuclei with either multiple bright specks of fragmented chromatin or one or more spheres of condensed chromatin (significantly more compact than normal nuclei); , non-viable necrotic cells had red-stained, smooth and homogeneous nuclei that were about the same size as normal (control) nuclei.The apoptotic index were calculated by the fraction of numbers of apoptotic cells on the total cell count in  ( cells), respectively.All experiments were replicated independently at least  times.Within each experiment, we replicated each condition  times.

Measurement of total neurite length
Measurement of total neurite length was conducted as reported by previous study [].The assay is based on the measurement of total neurite length.Total neurite length (length of largest neurite on  cells) was assessed.Cells were plated in  well culture plates with  cells/well density for h.Th en cells were pretreated in diff erent treatments for certain time.Th ese were then grown for a range of times at diff erentiation medium (,  and h), fi xed, and the morphology assessed by an inverted microscope (Olympus IX-, Japan).Digital photos were taken of random fi elds of neurons derived from the treatments.Total neurite length was measured (Motic software; Ver.).All experiments were replicated independently at least  times.Within each experiment, we replicated each condition  times.

Th e fraction of cell diff erentiation assessment (f ())
Fraction of cell differentiation was carried out as previous study [].PC cells were plated at a density of × cells/well on  well plates.Cells were pretreatment with different treatment mediums.These were then grown for a range of times at diff erentiation medium (,  and  h), fi xed, and the morphology microscopically assessed (Motic software; Ver.).The fraction of cell differentiation was evaluated under an inverted microscope by the fraction of neurite-bearing cells were the fraction of numbers of neurite-bearing cells with at last one neurite longer than the cell body diameter on the total cell count ( cells).All experiments were replicated independently at least  times.Within each experiment, we replicated each condition  times.

Statistical analysis
Data were expressed as Mean ± SEM.All calculations were performed by SPSS (version ; SPSS Inc.).The differences in the percentage of cytotoxicity, incidence of apoptotic index, total neurite length and fraction of cell differentiation, in PC cells between treatments were analyzed using t-test at significant level (p<.).

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.In PC cells the percentage of cytotoxicity were increased in treatments ,  and  ( ± ,  ±  and  ± , respectively) compared with control ( ± ), (p<.).The percentage of cytotoxicity in treatment  ( ± ) were decreased compared with treatments ,  and  (p<.) and was similar to control (Figure ).

Eff ects of inhibitors on apoptosis index
Th e evaluation of apoptotic index of inhibitors for PC cells cultured in culture medium containing  nM staurosporine was assessed by PI/Hoechst fl orescence staining.After h, the apoptotic index were increased in treatments  and  ( ±  and  ± ); respectively and were similar in treatment ( ± ) compared with control cells ( ± ) but these diff erences were not signifi cant.Th e apoptosis index in treatment  ( ± ) was increased compared with control and treatments - (p<.).After  h, the apoptosis index were increased in treatments  ( ± ),  ( ± ) and  ( ± ) compared with control ( ± ) (p<.).Th e apoptotic index in treatment  ( ± ) was decreased compared with treatments ,  and  (p<.) and was similar to control.After  h, the apoptosis index were increased in treatments ,  and  ( ± ,  ±  and  ± , respectively) compared with control ( ± ) (p<.).Th e apoptosis index in treatment  was increased ( ± ) compared with control but this diff erence was not signifi cant (Figures ; A and B).

Neurite outgrowth measurement
The average of total neurite length for PC cells was assessed.The total neurite length (TNL) was calculated.Af-

DISCUSSION
Th e current study investigated the involvement of calcium channel and plasma membrane receptors on staurosporine   inducing neurite outgrowth in PC cells.In this work, we used PC cells as the best cell model for study of eff ect of materials on neurite outgrowth [].PC, a neuron-like cell line, expresses voltage-dependent Ca channels appear to dihydropyridine-sensitive voltage-dependent Ca channels demonstrable by different techniques [, ].Staurosporine was employed as a strong inducer of neurite outgrowth with inhibition of protein kinases in vitro model.Th e results obtained in this study showed that nifedipine and ketamine could eff ectively inhibit neurite outgrowth induced by staurosporine and increase cell death incidence in PC cells.We observed that when cells were preincubated with nifedipine and fl avoxate hydrochloride or ketamine and MK, they dramatically suppressed the neurite outgrowth and increased cell death and cytotoxicity in PC cells.[].Previously, it has been shown that external Ca + evoke the signal transduction through the Ca + infl ux via extracellular Ca + -sensing receptor localized to neurons and their nerve terminals [].
It demonstrated that neurite outgrowth of PC is induced via the Ca + -signal transduction pathway by the Ca + infl uxes through channels [].On the other hand, recent study showed that staurosporine leads to intracellular calcium overload, which induce apoptosis in PC cells [].In the recent study, showed that staurosporine caused a large increase in [Ca + ]c even after the depletion of Ca + from the ER, the IP-sensitive Ca + store, in the absence of perfusate Ca + .Th is result indicates that IP-insensitive, non-ER compartments are responsible for the staurosporine-induced [Ca + ]c increase in rat submandibular acinar cells [].We reported previously that Staurosporine use extracellular calcium stores tend to increase intracellular calcium concentration [].
In addition, previously, it is known that cytosolic Ca + in-crease caused by staurosporine that mobilize Ca + from different sources might cause apoptosis in astrocytes [].
Ca + in DDTIMF- smooth muscle cells by influx but also by intracellular mobilization from thapsigarginsensitive and -insensitive Ca + stores.Furthermore, the high local Ca + gradient just under the plasma membrane, which can be preserved over long periods of time in Ca + -free medium despite the presence of EGTA, indicates that the efflux mechanism is also affected [].Th e stores of Ca + ion entry from extracellular into intracellular during staurosporine-induced neurite outgrowth is still not completely understood.Many studies in diff erent cells showed that staurosporine result in an increase cytosolic calcium concentration and induction of apoptosis in NGF-diff erentiated cells [, ].In another study showed that the rate of apoptotic cells is greater in diff erentiated cells than undiff erentiated cells [].Diff erent study showed that neurotrophins factors like NGF result in increase of mRNA incoding of calcium channels like voltage-dependent calcium channels and glutamate-sensitive ion channels like NMDA [-].
It has shown that compared with undifferentiated cells maybe activation of calcium channels and plasma membrane receptors by staurosporine lead to increase of staurosporine-induced apoptosis in differentiated cells.If true, these receptors and channels play important role in increasing intracellular calcium concentration during staurosporine-induced cell differentiation in PC cells.Meanwhile, We suggest it possible that staurosporine by a protein kinase-independent mechanism (PKC, PKA and CaMKs) by activation of plasma membrane Ca+ channels lead to enhance of neurite outgrowth and increases cell viability and fraction of cell diff erentiation in PC cells.

CONCLUSION
According to the results of present study, application of staurosporine with activation of calcium channels may lead to enhance of neurite outgrowth and have eff ects on neuronal cell diff erentiation in PC cells.However, more key receptors and enzymes need to be investigated in these effects.

FIGURE 1 .
FIGURE 1.The eff ects of calcium channels and receptors on cell death in PC12.(A) Morphology of cells as examined by fl uorescence microscopy a: cells without pretreated with, b: Cells pretreated with treat-ment1, c: Cells pretreated with treatment2, d: cells pretreated with treatment3, e: Cells pretreated with treatment4 for 24 hours.(B) Quantitative analysis of necrotic and apoptotic cells by fl uorescence microscopy in various treatments.Viable cell: white and short arrow, apoptotic cell: long arrow, necrotic cell: double short arrow, Control: cells without pretreatment; treatment1: pretreatment with ketamine; treatment2: pretreatment with nifedipine; treatment3: pretreatment with trifl uoperazine; treatment4: pretreatment with ketamine and nifedipine.All data represented by mean ± S.E.M (p<0.05).Magnitude is 400x.

FIGURE 3 .
FIGURE 3. The eff ects of calcium channels and receptors on PC12 cells morphology. A. without pretreatment; B. pretreatment with trifl uoperazine; C. pretreatment with ketamine; D. pretreatment with nifedipine; E. pretreatment with ketamine and nifedipine.All data represented by mean ± S.E.M (p<0.05).Magnitude is 200x.
HOSSEIN ZHALEH ET AL.: POSSIBLE INVOLVEMENT OF CALCIUM CHANNELS AND PLASMA MEMBRANE RECEPTORS ON STAUROSPORINEINDUCED NEURITE OUTGROWTHwith .  bovine serum albumin (BSA, Gibco),  NEAA (Sigma),  mM L-glutamine (Sigma),  IU/ml penicillin and  μg/ml streptomycin (Sigma) in -cm tissue culture dishes.The cultures were incubated at °C in a humidifi ed incubator containing  air and  CO.Culture medium was replaced every  days.When cell cultures reached to  confluency, they were trypsinated using trypsin-EDTA . (Sigma) and the cells were subcultured at a density of  cells/well in -well culture plates.
Bosn J Basic Med Sci2012; 12 (1): 21-25 Meanwhile, preincubation with ketamine and MK together with nifedipine and flavoxate hydrochloride result in powerful inhibition of neurite outgrowth and induce cell death in PC cells.It could be suggested that the possible involvement of voltage dependent calcium channels and NMDA receptors on staurosporine-calcium dependent signal transduction.Meanwhile, PC application of trifl uoperazine does not the same eff ects on either of cytotoxicity or neurite outgrowth.It was shown this possible that staurosporine leads to inhibition of calmodulin in  nM concentrations.It is unclear that how extracellular Ca + causes the intracellular events that leads to the differentiation in PC by staurosporine.