Possible involvement of calcium channels and plasma membrane receptors on Staurosporine-induced neurite outgrowth
Hossein Zhaleh1,2, Mehri Azadbakht1, Ali Bidmeshki Pour1*
1 Department of Biology, Faculty of Science, Razi University, Taqe Bostan, Baghe Abrisham, 6714967346, Kermanshah, Iran. 2 Department of Chemical Biotechnology Engneering, Science and Research Branch, Islamic Azad University, Kermanshah, Iran Abstract
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 contain- ing nM staurosporine for induction of neurite outgrowth. Th e percentage of Cell cytotoxicity and apoptotic index was assessed. Total neu- rite 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 pos- sible 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
KEY WORDS: staurosporine, neurite outgrowth, calcium channels, plasma membrane calcium receptors, PC cell
neuronal processes such as neuronal cell diff erentiation. In INTRODUCTION most neurons, multiple mechanisms exist whereby increases in intracellular calcium concentration may occur including Staurosporine (STS) as a protein kinase inhibitor [, ] has for example in calcium entry through N-methyl-D-Aspartate dual eff ects on neuronal cells; induction of cell death and cell (NMDA) glutamate receptors and various voltage-gated diff erentiation. STS induces apoptosis in high concentrations calcium channels such as L-type calcium channels (LTCC), (μM) [, ] and neuronal differentiation in low concentra- as well as in the release of calcium from intracellular stores tions (nM) by neurite extension in several types of cells [, [-]. Calcium infl ux through LTCCs is particularly eff ec- , , ]. Although the detailed mechanism of STS action as tive in neuronal migration, activation of transcription factors a neurogenic morphogen remains unclear, it seems that it is (e.g CREB), changes in gene expression that underlie plas- associated with the inhibition of some protein kinases which ticity and adaptive neuronal responses (e.g c-fos) [, -]. may contribute to neurite outgrowth []. In previous stud- Although STS induced increasing of intracellu- ies it has been determined that STS can inhibit PKA, Ca+/ lar calcium in treated cells, its effect on plasma calmodulin-dependent kinase II, cyclin dependent kinases, membrane and calcium channels and receptors lo- ion channels (Kv., L-type Ca+ channel, voltage-gated K+ cated in the plasma membrane during neuronal channel) in myocyte [-]. Although, the role of STS in the diff erentiation and neurite outgrowth are not well known. inhibition of protein kinases during neurite outgrowth was In this study we aimed to determine whether plas- clear but its function on plasma membrane calcium channels ma membrane calcium channels and receptors in- and receptors remains to be fully known [,]. Calcium volves in staurosporine-induced neurite outgrowth. plays an important role in the regulating a great variety of MATERIALS AND METHODS * Corresponding author: Ali Bidmeshki Pour, Department of Biology, Faculty of Science, Razi University, Taqe Bostan, Baghe Abrisham, 6714967346, Kermanshah, Iran. Tel: +988314274545; Fax: +988314274545 Cell culture e-mail: [email protected] PC cells were cultured in complete culture medium con- Submitted: 29 August 2011 / Accepted: 31 October 2011 taining RPMI culture medium (Gibco), supplemented
Bosn J Basic Med Sci 2012; 12 (1): 20-25 HOSSEIN ZHALEH ET AL.: POSSIBLE INVOLVEMENT OF CALCIUM CHANNELS AND PLASMA MEMBRANE RECEPTORS ON STAUROSPORINEINDUCED NEURITE OUTGROWTH with . bovine serum albumin (BSA, Gibco), NEAA time. Th ese were grown for a range of times in diff erentia- (Sigma), mM L-glutamine (Sigma), IU/ml penicil- tion medium (, and h). Th en cells were incubated for lin and μg/ml streptomycin (Sigma) in -cm tissue min at °C with Hoechst dye ( mg/ml in PBS), culture dishes. The cultures were incubated at °C in a washed twice in PBS. PI ( mg/ml in PBS) was added just humidifi ed incubator containing air and CO. Cul- before microscopy. Cells were visualized using an inverted- ture medium was replaced every days. When cell cultures florescence microscope (Olympus IX-, Japan). Nuclear reached to confluency, they were trypsinated using morphology was scored as follows: , viable cells had blue- trypsin-EDTA . (Sigma) and the cells were subcul- stained nuclei with smooth appearance; , viable apoptotic tured at a density of cells/well in -well culture plates. cells had blue-stained nuclei with multiple bright specks of condensed chromatin; , non-viable apoptotic cells had Cell treatment red-stained nuclei with either multiple bright specks of frag- One day after plating PC cells, cells were washed with mented chromatin or one or more spheres of condensed phosphate buffer saline (PBS), pH .. For inhibition of L- chromatin (significantly more compact than normal nu- Type Calcium channel, NMDA receptor and CaM kinase, clei); , non-viable necrotic cells had red-stained, smooth cells were preincubated with, adding . mM ketamine and and homogeneous nuclei that were about the same size as μM MK, min (treatment ), μM nifedipine and normal (control) nuclei. The apoptotic index were calcu- μM fl avoxate hydrochloride, min (treatment ) and lated by the fraction of numbers of apoptotic cells on the μM trifl uoperazine, min (treatment ). In our experi- total cell count in ( cells), respectively. All experi- ment, we combined ketamine, MK, fl avoxate hydrochlo- ments were replicated independently at least times. With- ride and nifedipine for inhibition of L-Type Calcium chan- in each experiment, we replicated each condition times. nels and NMDA receptors (treatment ). Th en, cells were cultured in differentiation medium containing complete Measurement of total neurite length culture medium supplemented with nM staurosporine Measurement of total neurite length was conducted as re- for h. PC cells were cultured in diff erentiation medium ported by previous study []. The assay is based on the without inhibitor preincubation seems as control group. measurement of total neurite length. Total neurite length Th e cells were placed in the incubator at °C with CO. (length of largest neurite on cells) was assessed. Cells were plated in well culture plates with cells/well Cell cytotoxicity measurement density for h. Th en cells were pretreated in diff erent treat- Cell cytotoxicity was quantifi ed by measuring the release of ments for certain time. Th ese were then grown for a range lactate dehydrogenase (LDH) from damaged or destroyed of times at diff erentiation medium (, and h), fi xed, and cells into the medium. Cytotoxicity was measured with LDH the morphology assessed by an inverted microscope (Olym- Cytoxicity Detection Kit (Roche, Germany). Th is kit detects pus IX-, Japan). Digital photos were taken of random LDH release from dead cells. Th erefore, increase of LDH ac- fi elds of neurons derived from the treatments. Total neurite tivity in each treatment show that the treatment solution has length was measured (Motic software; Ver.). All experi- further dead cells or cytotoxicity eff ects on PC cells. Cells ments were replicated independently at least times. With- were plated in well culture plates with cells/mL densi- in each experiment, we replicated each condition times. ty for h. Th en cells were pretreated in diff erent treatments for certain time. Th en, cells were cultured by diff erentiation Th e fraction of cell diff erentiation assessment (f ()) medium for h. The percentage of cytotoxicity was mea- Fraction of cell differentiation was carried out as previ- sured by protocol of company; colorimetery of LDH activ- ous study []. PC cells were plated at a density of × ity measured by calculated the absorbance of samples at cells/well on well plates. Cells were pretreatment with or nm using an ELISA Reader (EL; USA). Th e refer- different treatment mediums. These were then grown for ences wavelength should be more than nm. All experi- a range of times at diff erentiation medium (, and h), ments were replicated independently at least times. Within fi xed, and the morphology microscopically assessed (Motic each experiment, we replicated each condition times. software; Ver. ). The fraction of cell differentiation was evaluated under an inverted microscope by the fraction of Quantifi cation of cell death incidence neurite-bearing cells were the fraction of numbers of neu- Hoechst / PI nuclear staining was carried out as previously rite-bearing cells with at last one neurite longer than the cell described []. Briefl y, cells were plated in well culture body diameter on the total cell count ( cells). All experi- plates with cells/mL density for h. Then cells were ments were replicated independently at least times. With- pre-treated in different treatment mediums for certain in each experiment, we replicated each condition times.
Bosn J Basic Med Sci 2012; 12 (1): 21-25 HOSSEIN ZHALEH ET AL.: POSSIBLE INVOLVEMENT OF CALCIUM CHANNELS AND PLASMA MEMBRANE RECEPTORS ON STAUROSPORINEINDUCED NEURITE OUTGROWTH
Statistical analysis with treatments , and (p<.) and was similar to control. Data were expressed as Mean ± SEM. All calculations After h, the apoptosis index were increased in treatments were performed by SPSS (version ; SPSS Inc.). The dif- , and ( ± , ± and ± , respectively) com- ferences in the percentage of cytotoxicity, incidence pared with control ( ± ) (p<.). Th e apoptosis index of apoptotic index, total neurite length and fraction of in treatment was increased ( ± ) compared with con- cell differentiation, in PC cells between treatments trol but this diff erence was not signifi cant (Figures ; A and B). were analyzed using t-test at significant level (p<.). Neurite outgrowth measurement RESULTS The average of total neurite length for PC cells was as- sessed. The total neurite length (TNL) was calculated. Af- Cell cytotoxicity The percentage of cytotoxicity of inhibitors in PC cells A cultured in culture medium containing nM staurospo- rine was assessed by evaluation of the lactate dehydroge- nase 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 treat- ment ( ± ) was increased compared with control and treatments - (p<.). After h, the apoptosis index were increased in treatments ( ± ), ( ± ) and ( ± B ) compared with control ( ± ) (p<.). Th e apoptotic index in treatment ( ± ) was decreased compared
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 uores- FIGURE 1. The eff ects of calcium channel and receptor on cell cence microscopy in various treatments. viability in PC12. Viable cell: white and short arrow, apoptotic cell: long arrow, treatment1: pretreatment with ketamine; treatment2: pretreat- necrotic cell: double short arrow, Control: cells without pretreat- ment with nifedipine; treatment3: pretreatment with trifl uopera- ment; treatment1: pretreatment with ketamine; treatment2: pre- zine; treatment4: pretreatment with ketamine and nifedipine. treatment with nifedipine; treatment3: pretreatment with trifl uo- Control: without pretreatment. All data represented by mean ± perazine; treatment4: pretreatment with ketamine and nifedipine. S.E.M (p<0.05). All data represented by mean ± S.E.M (p<0.05). Magnitude is 400x.
Bosn J Basic Med Sci 2012; 12 (1): 22-25 HOSSEIN ZHALEH ET AL.: POSSIBLE INVOLVEMENT OF CALCIUM CHANNELS AND PLASMA MEMBRANE RECEPTORS ON STAUROSPORINEINDUCED NEURITE OUTGROWTH
FIGURE 3. The eff ects of calcium channels and receptors on PC12 cells morphology. A. without pretreatment; B. pre- treatment 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.
FIGURE 4. The eff ects of calcium channels and receptors on total FIGURE 5. The eff ects of calcium channels and receptors on frac- neurite length in PC12 cells. tion of cell diff erentiation in PC12 cells. treatment1: pretreatment with ketamine; treatment2: pretreat- treatment1: pretreatment with ketamine; treatment2: pretreat- ment with nifedipine; treatment3: pretreatment with trifl uopera- ment with nifedipine; treatment3: pretreatment with trifl uopera- zine; treatment4: pretreatment with ketamine and nifedipine. zine; treatment4: pretreatment with ketamine and nifedipine. Control: without pretreatment; All data represented by mean ± Control: without pretreatment; All data represented by mean ± S.E.M (p<0.05). S.E.M (p<0.05). ter inhibitors preincubation, TNL significantly were de- nM staurosporine was assessed. After h, f () were not creased compared with control cells. After h, TNL were signifi cantly decreased in treatments , and ( ± , decreased in treatments , and ( ± ., ± . ± . and ± , respectively) compared with control and ± ., respectively) compared with control cells (). f () in treatment () similar to control. After ( ± .) (p<.). TNL in treatment ( ± .) h, Th e fraction of cell diff erentiation f () was decreased in was similar to control. After h, TNL were decreased treatment ( ± .) (p<.). f () were not signifi cant- in treatments , and ( ± ., ±. and ± ., ly decreased in treatments and ( ± and ± ) respectively) compared with control cells ( ± .) compared with control () (p<.). f () in treatment (p<.). TNL in treatment ( ± .) was similar to (p<.) was similar to control cells. After h, f () were de- control. After h, TNL were decreased in treatments , creased in treatments , and ( ± , ± and and ( ± ., ±. and ± ., respectively) com- ± , respectively) compared with control cells ( ± ), pared with control cells ( ± .) (p<.). TNL in treat- (p<.). f () in treatment was similar to control (Figure ). ment ( ± .) was similar to control (Figures and ). DISCUSSION Fraction of cell diff erentiation assessment Th e evaluation of the fraction of cell diff erentiation of inhibi- Th e current study investigated the involvement of calcium tors for PC cells cultured in culture medium containing channel and plasma membrane receptors on staurosporine
Bosn J Basic Med Sci 2012; 12 (1): 23-25 HOSSEIN ZHALEH ET AL.: POSSIBLE INVOLVEMENT OF CALCIUM CHANNELS AND PLASMA MEMBRANE RECEPTORS ON STAUROSPORINEINDUCED NEURITE OUTGROWTH inducing neurite outgrowth in PC cells. In this work, we crease caused by staurosporine that mobilize Ca+ from used PC cells as the best cell model for study of eff ect of different sources might cause apoptosis in astrocytes []. materials on neurite outgrowth []. PC, a neuron-like Ca+ in DDTIMF- smooth muscle cells by influx but cell line, expresses voltage-dependent Ca channels appear to also by intracellular mobilization from thapsigargin- dihydropyridine-sensitive voltage-dependent Ca channels sensitive and -insensitive Ca+ stores. Furthermore, the demonstrable by different techniques [, ]. Staurospo- high local Ca+ gradient just under the plasma mem- rine was employed as a strong inducer of neurite outgrowth brane, which can be preserved over long periods of time with inhibition of protein kinases in vitro model. Th e results in Ca+- free medium despite the presence of EGTA, in- obtained in this study showed that nifedipine and ketamine dicates that the efflux mechanism is also affected []. could eff ectively inhibit neurite outgrowth induced by stau- Th e stores of Ca+ ion entry from extracellular into intracel- rosporine and increase cell death incidence in PC cells. We lular during staurosporine-induced neurite outgrowth is still observed that when cells were preincubated with nifedipine not completely understood. Many studies in diff erent cells and fl avoxate hydrochloride or ketamine and MK, they showed that staurosporine result in an increase cytosolic cal- dramatically suppressed the neurite outgrowth and increased cium concentration and induction of apoptosis in NGF-diff er- cell death and cytotoxicity in PC cells. Meanwhile, prein- entiated cells [, ]. In another study showed that the rate of cubation with ketamine and MK together with nifedipine apoptotic cells is greater in diff erentiated cells than undiff er- and flavoxate hydrochloride result in powerful inhibition entiated cells []. Diff erent study showed that neurotroph- of neurite outgrowth and induce cell death in PC cells. It ins factors like NGF result in increase of mRNA incoding of could be suggested that the possible involvement of volt- calcium channels like voltage-dependent calcium channels age dependent calcium channels and NMDA receptors on and glutamate-sensitive ion channels like NMDA [-]. staurosporine-calcium dependent signal transduction. Mean- It has shown that compared with undifferentiated cells while, PC application of trifl uoperazine does not the same maybe activation of calcium channels and plasma mem- eff ects on either of cytotoxicity or neurite outgrowth. It was brane receptors by staurosporine lead to increase of shown this possible that staurosporine leads to inhibition of staurosporine-induced apoptosis in differentiated cells. If calmodulin in nM concentrations. It is unclear that how true, these receptors and channels play important role extracellular Ca+ causes the intracellular events that leads in increasing intracellular calcium concentration during to the differentiation in PC by staurosporine. It seems staurosporine-induced cell differentiation in PC cells. staurosporine leads to regulation of neurite outgrowth pro- Meanwhile, We suggest it possible that staurosporine by a cess with activation of diff erent plasma membrane calcium protein kinase-independent mechanism (PKC, PKA and channels and increasing of intracellular calcium concentra- CaMKs) by activation of plasma membrane Ca+ chan- tion. Development, neuronal survival and diff erentiation can nels lead to enhance of neurite outgrowth and increases be infl uenced by a variety of local signals or signals derived cell viability and fraction of cell diff erentiation in PC cells. from intermediate or fi nal target tissues []. Previously, it has been shown that external Ca+ evoke the signal transduc- CONCLUSION tion through the Ca+ infl ux via extracellular Ca+- sensing receptor localized to neurons and their nerve terminals []. According to the results of present study, application of stau- It demonstrated that neurite outgrowth of PC is induced rosporine with activation of calcium channels may lead to via the Ca+-signal transduction pathway by the Ca+ infl ux- enhance of neurite outgrowth and have eff ects on neuronal es through channels []. On the other hand, recent study cell diff erentiation in PC cells. However, more key recep- showed that staurosporine leads to intracellular calcium tors and enzymes need to be investigated in these effects. overload, which induce apoptosis in PC cells []. In the recent study, showed that staurosporine caused a large in- ACKNOWLEDGEMENTS crease 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 study was supported by Faculty of Sciences from the Th is result indicates that IP-insensitive, non-ER compart- Razi University. ments are responsible for the staurosporine-induced [Ca+]c increase in rat submandibular acinar cells []. We reported DECLARATION OF INTEREST previously that Staurosporine use extracellular calcium stores tend to increase intracellular calcium concentration []. Th ere is no confl ict of interest. In addition, previously, it is known that cytosolic Ca+ in-
Bosn J Basic Med Sci 2012; 12 (1): 24-25 HOSSEIN ZHALEH ET AL.: POSSIBLE INVOLVEMENT OF CALCIUM CHANNELS AND PLASMA MEMBRANE RECEPTORS ON STAUROSPORINEINDUCED NEURITE OUTGROWTH
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