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Cell Calcium 43 (2008) 107–121

Mechanism of acetylcholine-induced calcium signaling duringneuronal differentiation of P19 embryonal carcinoma cells in vitro

Rodrigo R. Resende a, Katia N. Gomes a, Avishek Adhikari a,1,Luiz R.G. Britto b, Henning Ulrich a,∗

a Departamento de Bioquımica, Instituto de Quımica, Universidade de Sao Paulo, Av. Prof. Lineu Prestes 748, 05508-900 Sao Paulo, SP, Brazilb Departamento de Fisiologia e Biofısica, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Paulo, 05508-900 Sao Paulo, SP, Brazil

Received 26 September 2006; received in revised form 12 April 2007; accepted 13 April 2007Available online 26 July 2007

bstract

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronalevelopment. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinomambryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity of �2–�7, �2, �4 nAChR and M1–M5AChR subtypes during neuronal differentiation. Nicotinic �3 and �2 mRNA transcription was induced by addition of retinoic acid to19 cells. Gene expression of �2, �4–�7, �4 nAChR subunits decreased during initial differentiation and increased again when P19 cellsnderwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+ flux was observed in embryonic and neuronal-ifferentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. TheAChR-induced elevation of intracellular calcium ([Ca2+]i) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19eurons the nAChR-induced [Ca2+]i response was reduced following pretreatment with ryanodine, while the mAChR-induced response wasnaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses.

he presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initialeuronal differentiation.

2007 Elsevier Ltd. All rights reserved.

eywords: Neuronal differentiation; P19 embryonal carcinoma cells; Acetylcholine receptors; Ryanodine receptors; IP3 receptors; Ca2+ signaling

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. Introduction

The development of the nervous system is one of the mostmportant morphogenetic events occurring in the embryo.his process is accompanied by cell proliferation and dif-

erentiation as well as by tissue organization into a specific

rchitecture. Although the specific molecular pathways thatrive these events remain unresolved, it is widely believedhat proliferation and differentiation programs of the neural

∗ Corresponding author. Tel.: +55 11 3091 3810x223;ax: +55 11 3815 5579.

E-mail address: henning@iq.usp.br (H. Ulrich).1 Present address: Department of Biological Sciences, Columbia Univer-

ity, Amsterdam Av. 1212, New York, NY 10027, USA.

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143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2007.04.007

rogenitors require the interaction of extrinsic and intrinsicignals. While the function of growth factors in controllingeuronal differentiation is well documented, there also isncreasing persuasive evidence for a role of neurotransmit-ers and their respective receptors in this process [1,2]. Manyeurotransmitters are already present in the brain prior toxonogenesis and synaptogenesis, raising the possibility thathey may mediate non-classical signaling. One such neuro-ransmitter is acetylcholine (ACh), whose biological actionsre mediated by both nicotinic (nAChRs) and muscariniceceptors (mAChRs). Neuronal nAChRs are heterogeneous,

ith at least six � (�2–�7) and three � (�2–�4) subunits

reviewed in [3]). Nicotinic receptor subunits are among therst membrane proteins to appear during central nervous sys-

em (CNS) development, and their initial expression does not

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epend on nerve contact [4]. Prior to innervation, nAChRsre diffusely distributed throughout the nerve fibre surface,hile after innervation, nAChRs cluster at high concentra-

ions at neuronal junctions [5–8]. Gene expression patternsnd several functional properties of nAChRs change dur-ng differentiation and maturation of neurons [9,4,10,11].ranscription of mRNAs coding for nAChR subunits andxpression of �3, �4, �7, �2, and �4 nicotinic receptor sub-nits in the developing rat from early embryonic throughostnatal developmental stages [4] and during in vitro differ-ntiation of a P19 mouse embryonic carcinoma (EC) cell lines a model for early development [12] point at a pivotal roleuring the onset of neurogenesis.

In contrast to nAChRs which directly participate in neuro-ransmission in mature neurons, muscarinic receptor functions predominately linked to modulation of synaptic activity.ive mAChR subtypes are present in the CNS, with the5 subtype being exclusively expressed in the brain. These

eceptors activate multiple signaling pathways that are impor-ant for modulating neuronal excitability, synaptic plasticitynd feedback regulation of ACh release [13]. Muscariniceceptor participation in neurogenesis has been suggestedased on experimental evidence. For instance, treatment ofeural stem and progenitor cells with ACh or its stable ana-ogue carbamoylcholine led to increases in DNA synthesisnd accelerated neurogenesis [14]. In another study a neurob-astoma cell line, which by itself does not synthesize ACh,as transfected with a construct coding for expression of

holine acetyltransferase. The activation of ACh synthesisesulted in a higher expression of neuronal specific traitsompared to untransfected control cells. The presence ofAChRs in these transfected cells indicates the presence of

n autocrine loop that may be responsible for the advancedifferentiation state ([15], reviewed in [16]).

In the present work, we provide evidence for the pres-nce of functional nicotinic and muscarinic receptors duringn vitro differentiation of P19 EC cells as a model of earlymbryogenesis. Neuronal differentiation of this cell line iselieved to closely resemble the developmental stage of thembryonic ectoderm. The observation that in addition to dif-erential cholinergic receptor expression, the expression ofholine acetyltransferase and acetylcholine esterase with itsplice variants is regulated throughout the course of differen-iation [17,12,18,19], makes the P19 cell line a reliable modelo study cholinergic receptor function during early neuronalevelopment.

. Materials and methods

.1. Abbreviations used

4-DAMP, 4-diphenylacetoxy-N-methylpiperidine meth-odide; ACh, acetylcholine; CICR, Ca2+-induced Ca2+

elease; CPA, cyclopiazonic acid; DMEM, Dulbecco’sodified Eagle’s medium; EB, embryonic bodies; EM,

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um 43 (2008) 107–121

xtracellular medium; FBS, fetal bovine serum; Fmax,aximal fluorescence; Fmin, minimal fluorescence; IP3,

nositol-3-phosphate; mAChR, muscarinic acetylcholineeceptors; MT3, green mamba Dendroaspis angusticepsoxin; nAChR, nicotinic acetylcholine receptors; NF,eurofilament; PBS, phosphate-buffered saline; PLC, phos-holipase C; RA, “all-trans” retinoic acid; RT, reverseranscription; SERCA, sarcoplasmatic/endoplasmatic reticu-um Ca2+-ATPase; VOOC, voltage-operated Ca2+ channels.

.2. Reagents

Unless otherwise indicated, all reagents were purchasedrom Sigma (St. Louis, MO). Primary monoclonal antibodiesre from Covance (Covance Research Products, Denver, PA),at anti-�3 nAChR (MRT-611R), rat anti-�4 nAChR (MRT-13R), rat anti-�5 nAChR (MRT-623R), mouse anti-�7AChR (MMS-627R) and rat anti-�2 nAChR (MRT-639R)nd appropriate secondary biotin conjugate antibodies weresed at 1/200 dilutions (Santa Cruz Biotechnology, Heidel-erg, Germany). The anti-�2, -�6 and -�4 antibodies were aift from Dr. Jon Lindstrom (Department of Neuroscience,niversity of Pennsylvania and Philadelphia, PA). Primers

or RT-PCR reactions were synthesized by Integrated DNAechnologies, Coralville, IA.

.3. Culture and neuronal differentiation of P19 cells

P19 murine embryonal carcinoma cells were grown inMEM (Invitrogen, Carlsbad, CA) supplemented with 10%f final volume with fetal bovine serum (FBS) (Culti-ab, Campinas, Brazil), 100 units/ml penicillin, 100 �g/mltreptomycin, 2 mM l-glutamine and 2 mM sodium pyru-ate, in a humidified incubator at 5% CO2 and 37 ◦C.euronal differentiation was induced by addition of 1 �MA. Formation of EB was induced by culturing P19 cellss cell suspension in bacterial culture dishes coated with.2% of final volume with agarose for 48 h at a density of× 105 cells/ml in defined serum-free medium as describedreviously [20,21]. Completion of neuronal differentiationfter 8 days of addition of RA was confirmed by immunos-aining against neuron-specific proteins NF-200, �-3-tubulin,nd neuron-specific enolase (data not shown). Differenti-ted P19 cell cultures were kept free from glial cells byreatment with 50 �g/ml cytosine arabinoside on day 6 ofifferentiation.

.4. Reverse transcription (RT) and real-time PCR

Total RNA was isolated using the TRIzol reagent (Invit-ogen) from embryonic P19 (day 0) and differentiating P19ells on days 2–8 of differentiation. Contaminating DNA was

emoved by DNase I (Ambion Inc., Austin, TX) treatmentnd integrity of the isolated RNA was analyzed on a 2%thidium bromide-stained agarose gel. Three micrograms ofotal RNA from days 0 to 8 of differentiation were used in

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ach case as template for cDNA synthesis in the presence of0 ng of random primers and 200 units of RevertAidTM Hinus Moloney murine leukemia virus-reverse transcriptase

Fermentas Inc., Hanover, MD) in a total volume of 20 �lor 45 min at 42 ◦C. PCR reactions were carried out usinghese cDNAs as templates for amplification to verify genexpression of nAChR and mAChR subtypes. All PCRroducts were gel-purified, cloned into PGEM T-Easyectors (Promega, Madison, WI), sequenced and confirmedo be identical to mouse cholinergic receptor cDNA. Theyere electrophoresed on a 2% agarose gel, visualized by

thidium bromide staining under UV illumination, anderified for their expected size (see Table I of supplementalata). Real-time PCR was performed in order to quantifyelative mRNA transcription levels of �2, �3, �4, �5, �6, �7,2, �4 nAChR subunits and M1, M2, M3 mAChR subtypes

n embryonic P19 cells and P19 cells undergoing neuronalifferentiation. DNA templates were amplified by real timeCR on the 7000 Sequence Detection System (ABI Prism,pplied Biosystems, Foster City, CA, USA) using the Sybrreen method [22,23].

Thermal cycling conditions included an initial denatura-ion step of 95 ◦C for 10 min and 50 cycles at 95 ◦C for 15 snd 60 ◦C for 1 min. Experiments were performed in tripli-ate for each data point. Nicotinic and muscarinic receptorDNA abundance was quantified as a relative value comparedith an internal reference, �-actin cDNA, whose abundance

s believed not to change between the varying experimentalonditions [20,24]. Primer sequences used for real time PCRre detailed in Table I of supplemental data.

.5. Immunoperoxidase and immunofluorescence studies

For immunoperoxidase studies, 5 × 105 cells each frommbryonic or differentiating P19 cultures were grown onlass coverslips, fixed in 2% paraformaldehyde for 10 minnd dehydrated for 5 min at 37 ◦C on a preheated platform.or blocking non-specific antibody binding, coverslips were

ncubated for 2 h with 2% nonfat-milk containing 0.01%aponin and 2% goat or rabbit serum. Then, cells werencubated overnight at 4 ◦C with primary antibodies at 1/50ilutions in phosphate-buffered saline (PBS) in the presencef 0.3% Triton X-100 and 5% goat or rabbit serum.

Following washing with PBS, coverslips were incubatedor 1 h with biotinylated secondary antibodies at 1/200 dilu-ions. Unbound antibodies were removed by washing withBS. Following incubation with avidin–biotin complex forh, specific immunolabeling was revealed by detecting per-xidase activity in the presence of diaminobenzidine and2O2 (Aldrich, Milwaukee, WI). Negative controls were per-

ormed by repeating the above procedure in the absence of theespective primary antibody. Images were collected using a

ikon DXM1200F Digital Camera (Nikon, Surrey, UK) cou-led to an inverted Axiovert 200 microscope (Zeiss, Jena,ermany), stored into a PC, and analyzed with the NikonCT-1 version 2.62 software as described before [25].

pmui

um 43 (2008) 107–121 109

.6. Measurement of free cytosolic calciumoncentrations ([Ca2+]i)

Undifferentiated and P19 cells differentiated into neu-ons following induction with RA were incubated with 4 �Mluo3-AM in 0.5% Me2SO and 0.1% of the nonionic sur-actant pluronic acid F-127 for 30 min at 37 ◦C in EMontaining (in mM): 140 NaCl, 3 KCl, 1 MgCl2, 2.5 CaCl2,0 HEPES, pH 7.4, 10 glucose. The osmolarity of all theolutions ranged between 303 and 308 mosmol/l. After load-ng with Fluo3-AM, the cells were washed with extracellular

edium (EM) and incubated for a further 20 min at 37 ◦Co allow for complete deesterification of the dye. Calciummaging was performed with a LSM 510 confocal micro-cope (Zeiss, Jena, Germany), using image sizes of 256 × 256ixels and acquisition rates of one frame per second. Fluo-3uorescence was excited with a 488 nm line of an argon ion

aser, and the emitted light was detected using a bandpasslter at 515–530 nm [26]. At the end of each experiment,�M ionophore (4-Br-A23187) followed by 10 mM EGTAere used to determine Fmax and Fmin fluorescence values,

espectively. The [Ca2+]i values were calculated from theluo3-fluorescence emission using a self-ratio equation asescribed previously [20] assuming a Kd of 450 nM for Fluo-3alcium binding [27]. At least 10 cells were analyzed for eachata point. For [Ca2+]i measurement in Ca2+-free medium,ells were pretreated for 5 min with Ca2+-free EM supple-ented with 1 mM EGTA prior to cell stimulation. The meanmin/Fmax ratio was 0.20 ± 0.03 (mean ± S.E.M., n = 34) and.24 ± 0.03 (mean ± S.E.M., n = 38) for short- and long-termeasurements, respectively; these values are consistent with

hose reported for this dye in other systems [28,29]. The aver-ge baseline fluorescence normalized to Fmin was 0.21 ± 0.02n = 42). Data were calculated as a percentage of F/Fo with

and Fo being Fluo-3 fluorescence emissions of stimulatednd unstimulated cells, respectively. No significant effects onasal levels of fluorescence were observed after incubationith any of the antagonists or blockers used, with the excep-

ion of CdCl2. Incubation with CdCl2 produced a rather slowut sustained increase in basal fluorescence, which precludedts use in long-term measurements.

Atropine and mecamylamine were employed as spe-ific inhibitors of mAChR and nAChR-induced [Ca2+]iesponses, respectively [30,31]. Pirenzepine (0.1 �M), whicht this concentration is a M1 mAChR subtype-specificnhibitor, or 4-diphenylacetoxy-N-methylpiperidine methio-ide (4-DAMP), a mAChR antagonist with higher specificityor M3 than for other muscarinic subtypes [32,33], werepplied together with 3 �M muscarine to elucidate whether1 and M3 mAChR subtypes were functional in neuronal-

ifferentiated P19 cells. Participation of M2 and M4 subtypesn muscarine-evoked [Ca2+]i responses was studied in the

resence of 10 �M gallamine and 1 �M toxin from the greenamba D. angusticeps (MT3), respectively [33–37]. The

sed inhibitor concentrations elicited maximal effects asncreasing their concentrations by a factor of 10 did not result

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n any additional inhibition. For characterization of the sig-al transduction pathway for muscarinic receptor-mediatedobilization of Ca2+ from intracellular stores, 5 �M of theLC inhibitor U-73122 [38] was preincubated with differ-ntiated P19 cells for 5 min prior to [Ca2+]i measurements.etermination of changes in [Ca2+]i were performed in theresence of nicotine (100 �M) following preincubation of19 neurons during 14 min with cyclopiazonic acid (CPA,0 �M), a sarcoplasmatic/endoplasmatic reticulum Ca2+-TPase (SERCA) inhibitor [39,40].

Blockers of voltage-operated calcium channels (VOOC),uch as CdCl2 [41] nifedipine at 100- and 5-�M concen-rations, respectively [41,42], were used to evaluate thearticipation of these channels in nicotine-evoked increase inCa2+]i. These VOOC blockers were preincubated with theells for 15 min at room temperature in the dark prior to appli-ation of nicotine. Preincubation with 40 nM �-bungarotoxin�-Bgt) was carried out for 30 min before Ca2+ measurementsn order to evaluate the participation of �7 nAChR in [Ca2+]iesponses [29].

.7. Statistical analysis

Data represent the mean ± S.D. of five or more inde-endent experiments (each with two replicates) for RT-PCRnd real-time PCR studies. The numbers of experiments tohich the data of calcium measurements refer are indicated in

espective figure legends. Statistical significances of obtainedata were determined using the Student’s t-test or one-wayNOVA plus post hoc Tukey’s test, as stated in the figure

egends. Values of p < 0.05 were considered as statisticallyignificant.

. Results

.1. Nicotinic and muscarinic acetylcholine receptorxpression during neuronal differentiation of P19 cells

We have detected �2–�7 and �2 and �4 nAChR as wells M1–M3 mAChR mRNA transcription along P19 cellifferentiation by using RT-PCR procedures (Fig. I of sup-lemental data). Nicotinic �3 subunit mRNA transcriptionas below the detection limit of the method, and �3 and �2

ubunits were only transcribed following induction to neu-onal differentiation by RA addition. Gene expression of thehree studied M1 and M2 mAChR subtypes was detectedhroughout the differentiation period. M3 mAChR mRNAas at the limit of detection by RT-PCR in the undifferentiated

tage. However, M3 mAChR gene expression subsequentlyncreased following induction of P19 cells to neuronal differ-ntiation.

Real-time PCR revealed that gene expression of �2, �4, �5,6, �7 and �4 nAChR subtypes was already present in embry-nic P19 cells, remained then at a lower level during initialnd intermediate differentiation stages and increased signifi-

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um 43 (2008) 107–121

antly when differentiating P19 cells became postmitotic andnderwent final maturation on days 6–8. Transcription ratesor muscarinic receptors (M1–M3) showed an increase inheir abundance during neuronal differentiation of P19 cellsFig. 1).

The presence of nAChR proteins in embryonic P19 cells,19 progenitor cells on day 4, and P19 cells differentiated intoeurons on day 8 was revealed by immunoperoxidase stainingsing subtype-specific antibodies. Protein expression of allicotinic subunits besides the �3 one was detected (Fig. 2).

.2. Resting [Ca2+]i levels

Basal calculated [Ca2+]i levels were 25 ± 10 nM in embry-nic cells (n = 134) and 70 ± 14 nM in mature neuronal P19ells (n = 142).

.3. Acetylcholine receptor activity in undifferentiatednd neuronal-differentiated P19 cells

Peak values of transient elevations of [Ca2+]i in embryonicnd mature neuronal cell cultures obtained in the presence ofwide range of nicotine and muscarine concentrations werelotted as dose–response curves (Fig. 3A and B). Nicotine-nd muscarine-induced effects were dose-dependent and pro-oted highest elevations of [Ca2+]i in neuronal-differentiated

ells. Muscarine did not induce [Ca2+]i transient in embry-nic P19 cells. The concentration of nicotine necessary forctivation of 50% of the maximal receptor activity (EC50)as determined as 33.4 (9.7–115.2) �M for embryonic cells

nd 52.4 (5.9–465.5) �M (mean values and 95% confidencenterval) for neuronal-differentiated cells on day 8. TheC50 for muscarinic receptor activation in P19 neurons was4.3 (5.0–117.2) �M (mean value and 95% confidence inter-al).

Measurements of acetylcholine (ACh)-induced alterationsn [Ca2+]i provide an independent assay for detective func-ional cholinergic receptor subtypes in developing P19euronal cells. Therefore, we measured [Ca2+]i responsesfter application of cholinergic receptor agonists and aelective cholinergic antagonists in undifferentiated andeuronal-differentiated cells. Application of 300 �M AChvoked a rapid and transient increase in [Ca2+]i in undiffer-ntiated and neuronal-differentiated cells on day 8. The peakalues of [Ca2+]i in embryonic P19 cells increased in theresence of 300 �M ACh from 32 ± 1 to 78 ± 6 nM (n = 51).timulation of P19 cells on day 8 of differentiation with00 �M ACh resulted in increases of [Ca2+]i from 63 ± 1o 989 ± 39 nM (n = 62) (Fig. 3C).

The relative contributions of mAChRs and nAChRs tohe ACh-evoked [Ca2+]i in undifferentiated and differenti-ted cells were revealed by calcium imaging in the presence

f specific nicotinic and muscarinic receptor antagonists. Co-pplication of mecamylamine (3 �M), a nAChR antagonist,n combination with 300 �M ACh inhibited the ACh-inducedncrease in [Ca2+]i in embryonic P19 cells by 87 ± 10%

R.R. Resende et al. / Cell Calcium 43 (2008) 107–121 111

Fig. 1. Quantification of cholinergic receptor mRNA transcription by real-time PCR. Relative gene expression levels of receptors subtypes were corrected withthe internal control �-actin. The graphics shows nAChR and mAChR mRNA transcription levels in differentiating cells compared to those in undifferentiatedc pretreat he data*

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ells which were normalized to one. Cells on day 8 of differentiation werehe cell cultures with increasing concentrations of proliferating glial cells. Tp < 0.05, **p < 0.001 compared with control data.

�[Ca2+]i = 10 ± 0.3 nM) compared to the control measure-ent with ACh alone (�[Ca2+]i = 78 ± 6 nM). The presence

f the mAChR-specific antagonist atropine resulted in non-ignificant inhibition (12 ± 1%) of the ACh-induced increasen [Ca2+]i, (�[Ca2+]i = 69 ± 7 nM), indicating that ACh-nduced calcium responses in undifferentiated cells are

ainly due to nAChR activation (Fig. 3D). ACh-inducedncreases in [Ca2+]i of neuronal-differentiated P19 cellsere inhibited by 41 ± 2% (�[Ca2+]i = 586 ± 23 nM) or4 ± 2% (�[Ca2+]i = 456 ± 24 nM) in the presence of 3 �Mecamylamine or 1 �M atropine, respectively, compared to

ontrol (�[Ca2+]i = 993 ± 70 nM). Co-application of 3 �Mecamylamine and 1 �M atropine together with acetyl-

holine resulted in a complete inhibition of the [Ca2+]i (dataot shown). Our data indicate that ACh-induced [Ca2+]iesponses in P19 cells differentiated into neurons are dueo nAChR and mAChR activation.

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ted on day 6 with cytosine arabinoside, in order to avoid contamination ofshown are mean values ± S.E.M. of at least five independent experiments.

.4. Sources of Ca2+ in P19 neurons mobilized byAChR and nAChR activation

Relative contributions of extracellular Ca2+ and intracellu-ar Ca2+ stores to ACh-evoked [Ca2+]i responses were studiedn neuronal-differentiated P19 cells. Preincubation of neu-ons with Ca2+-free EM containing 1 mM EGTA for 1 minrior to ACh application resulted in inhibition of the [Ca2+]iesponse by 44 ± 4% (�[Ca2+]i = 493 ± 34 nM) compared tohe control experiment (�[Ca2+]i = 887 ± 39 nM) performedn the presence of extracellular Ca2+ (Fig. IIA of supplemen-al data), indicating involvement of both extracellular andntracellular Ca2+ pools in ACh-induced [Ca2+]i responses.

he inhibition of the ACh-induced [Ca2+]i in P19 neu-

ons in the absence of extracellular Ca2+ was abolishedfter reintroduction of Ca2+ into the EM (�[Ca2+]i = 955 ±5 nM).

112 R.R. Resende et al. / Cell Calcium 43 (2008) 107–121

Fig. 2. Detection of expression profiles of neuronal nicotinic acetylcholine receptors during neuronal differentiation of P19 cells by immunoperoxidase staining.Following fixation, cells were incubated with the subunit-specific primary antibody followed by addition of a biotinylated secondary antibody. The reactionw ressionm erentiat

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as revealed by immunoperoxidase staining. Nicotinic receptor subunit expiddle panels) and 8 (D8, right panels) following induction to neuronal diff

Muscarinic receptor-induced elevations of [Ca2+]i in thebsence of extracellular Ca2+ (�[Ca2+]i = 492 ± 34 nM),btained by stimulation of differentiated cells by ACh in

he presence of 3 �M mecamylamine and 1 mM EGTA�[Ca2+]i = 471 ± 18 nM) did not differ from those obtainedy ACh alone in Ca2+-containing EM. This result indi-ates that Ca2+ influx from the extracellular medium does

atflC

was verified in undifferentiated (Und., left panels) and cells on days 4 (D4,ion.

ot contribute to the mAChR-mediated [Ca2+]i response in19 neurons. In the absence of extracellular Ca2+, the ACh-

nduced [Ca2+]i response was strongly inhibited by 1 �M

tropine (�[Ca2+]i = 123 ± 11 nM), suggesting the participa-ion of internal Ca2+ sources in mAChR-mediated calciumuxes (Fig. IIB of supplemental data). As expected, influx ofa2+ from the extracellular medium participated to nAChR-

R.R. Resende et al. / Cell Calcium 43 (2008) 107–121 113

Fig. 3. [Ca2+]i responses in embryonal P19 cells and neurons on day 8 of differentiation induced by activation of nicotinic and muscarinic acetylcholinereceptors. Fluo-3 AM loaded cells were monitored for changes of [Ca2+]i by calcium imaging using confocal microscopy. (A) Dose–response (EC50) curveof activation of mAChR induced [Ca2+]i flux in neuronal-differentiated cells by muscarine. Peak fluorescence values of [Ca2+]i transients were calculated andplotted as percentage of the mean maximum response. (B) Dose–response (EC50) curve of activation of nAChR induced [Ca2+]i flux in undifferentiated andneuronal-differentiated P19 cells by nicotine. Following stimulation with 300 �M ACh during 30 s the cells were washed with culture medium for 5 min to allowfor receptor resensitization. Arrows indicate time points of agonist or agonist and antagonist application. (A) and (B) Data points are mean values ± S.E.M.from 6 to 14 independent experiments. (C) ACh-induced [Ca2+]i transients in undifferentiated (Und.) and neuronal-differentiated (Diff.) P19 cells. No receptordesensitization was noted under the used experimental conditions, as repeated ACh application did not result in a run down of [Ca2+]i responses. (D) Left upperpanels: embryonic cells were stimulated either by application of ACh alone or of ACh in the presence of 1 �M atropine or 3 �M mecamylamine. Right upperpanels: P19 cells differentiated to neurons were stimulated with 300 �M ACh in the absence or presence of ACh receptor inhibitors as indicated above. Lowerpanels indicate percentage values of [Ca2+]i increases obtained in the presence of atropine, mecamylamine compared to the control measurement in absenceof inhibitors. [Ca2+]i values in the presence of inhibitors are plotted as percentage of the uninhibited control. All antagonists were preincubated with the cellsfor at least 10 min and were then co-applied with ACh. The data shown are mean values ± S.E.M. of 8 independent experiments (n = 8). The total numbers ofcells analyzed in each experiment were 56–81. *p < 0.05, **p < 0.001 compared with control data.

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ediated elevations in [Ca 2+]i. Local perfusion of cellsith KCl (75 mM for 30 s) produced a rise in [Ca2+]i from9 ± 3 to 215 ± 33 nM (n = 6) in undifferentiated and from8 ± 8 to 715 ± 77 nM (n = 8) in neuronal-differentiated cellshich was inhibited in the absence of extracellular cal-

ium. Identical results were obtained upon KCl stimulationollowing washing of the cells for 5 min (Fig. IIA of sup-lemental data). The effects of mecamylamine and atropinen inhibiting cholinergic receptor activity were abolishedfter bathing them out (�[Ca2+]i = 942 ± 33 nM) (data nothown).

CdCl2 (100 �m) in external buffer containing 2.5 mMa2+ was used in calcium-imaging experiments in order tovaluate the participation of VOOC in the nicotine-inducedCa2+]i response in embryonic and neuronal-differentiated19 cells. The nicotine-evoked increase in [Ca2+]i was

nhibited by more than 87 ± 20% (�[Ca2+]i = 10 ± 3 nM)nd 54 ± 3% (�[Ca2+]i = 209 ± 14 nM) in undifferenti-ted (�[Ca2+]i = 78 ± 10 nM) and neuronal-differentiatedells (�[Ca2+]i = 456 ± 70 nM), respectively (Fig. IICnd D of supplemental data), indicating that VOCCediate most of the [Ca2+]i response in undifferen-

iated and about 50% in neuronal-differentiated cells.he residual nicotine-evoked increase in [Ca2+]i in

he presence of CdCl2 was significantly decreased,y 91 ± 6% (�[Ca2+]i = 7 ± 0.1 nM) and 77 ± 10%�[Ca2+]i = 19 ± 6 nM) following co-application of 40 nM-bungarotoxin (Bgt) and 100 �M CdCl2 together withicotine in embryonic and neuronal-differentiated cells,espectively (Fig. IIC and D of supplemental data).

The L-type VOCC blocker nifedipine (5 �m) inhib-ted 68 ± 7% (�[Ca2+]i = 26 ± 6 nM) and 38 ± 5%�[Ca2+]i = 151 ± 13 nM) of the nicotine-evoked increasen fluorescence in undifferentiated (�[Ca2+]i = 79 ± 25 nM)nd neuronal-differentiated cells (�[Ca2+]i = 456 ± 68 nM),espectively. In the presence of nifedipine, �-gt produced a statistically significant inhibitionf 77 ± 9% (�[Ca2+]i = 19 ± 6 nM) and 81 ± 3%�[Ca2+]i = 88 ± 9 nM) of the nicotine-induced [Ca2+]in undifferentiated and neuronal-differentiated cells, respec-ively (Fig. IIC and D of supplemental data). This resultuggests that �7 nAChR are the main mediators of theOCC-independent increase in intracellular Ca2+, as �-Bgt,t that concentration, inhibits only the �7 nAChR subtype29]. Furthermore, nAChR-mediated elevations of [Ca2+]in embryonic P19 cells were not significatively different inhe presence of nifedipine alone and nifedipine plus �-Bgt,ndicating that nicotine-activated calcium flux depends onOOC activation. However, in neuronal-differentiated P19ells about 40% of the nicotine-induced [Ca2+]i response isediated by VOCC and the remaining response is due to theobilization of internal Ca2+ pools and by calcium-influx

hrough nAChR channels themselves.Pirenzepine (0.1 �M), gallamine (10 �M), 4-DAMP

0.1 �M) and MT3 (1 �M), which at these concentrationsre specific antagonists of M1 [33], M2 [34,35] M3, [33]

FbU(

um 43 (2008) 107–121

nd M4 [36,37] mAChRs, respectively, were co-appliedogether with 300 �M muscarine, in order to elucidatehe contribution of each subtype to the mAChR-mediatedCa2+]i response in differentiated P19 cells. Among thesenhibitors, 4-DAMP is a less selective antagonist withn inhibition profile of M3 > M1 � M5 subtypes [33,34].reatment of neurons with pirenzepine resulted in an

nhibition of the muscarine-induced [Ca2+]i response by2 ± 1% (�[Ca2+]i = 492 ± 22 nM) compared to the con-rol experiment (�[Ca2+]i = 560 ± 23 nM) in the absencef the inhibitor. A higher reduction of mAChR-evokedCa2+]i response by 26 ± 2% was observed in the presencef 4-DAMP (�[Ca2+]i = 415 ± 12 nM), which at 0.1 mMoncentration preferentially inhibits the M3 subtype. Theresence of gallamine as a specific antagonist of the2 subtype also led to an inhibition of the muscarine-

nduced calcium flux by 30 ± 1% (�[Ca2+]i = 390 ± 17 nM).he M4 subtype-specific inhibitor MT3 caused the small-st inhibition (9 ± 1%) in neuronal-differentiated cellshich was not statistically significant than compared toAChR-mediated elevations of [Ca2+]i in control experi-ents (�[Ca2+]i = 511 ± 28 nM). Co-application of all four

nhibitors resulted in inhibition of the muscarine-inducedalcium response by 78 ± 4% (�[Ca2+]i = 122 ± 6 nM)Fig. 4A). The total observed inhibition in the presence ofll four inhibitors was the sum of the effects found in theresence of each inhibitor alone. The inhibition of the mus-arinic receptor response caused by the presence of a cocktailf pirenzepine, gallamine, 4-DAMP and MT3 was reversibles receptor responses recovered following washing out of thenhibitors (�[Ca2+]i = 571 ± 35 nM). The remaining [Ca2+]iesponse in the presence of the four inhibitors may be due to

5 mAChR activity which was not affected by either one ofhese mAChR antagonists.

Our data indicate that around 60% of muscarinic receptor-nduced elevation in [Ca2+]i in neuronal-differentiated P19ells is due to activation of M1, M3, and M5 subtypes,hich can simultaneously activate multiple signaling path-ays [43]. These Gq-coupled receptors exert their signal

ransduction by activating phospholipase C (PLC). Preincu-ation of P19 neurons with U-73343 (5 �M), as a specificnhibitor of PLC pathway, resulted in a significant inhi-ition, 60 ± 2% (�[Ca2+]i = 224 ± 5 nM), of the [Ca2+]iesponse induced by muscarine (�[Ca2+]i = 561 ± 21 nM),onfirming the participation of PLC in mAChR-inducedCa2+]i mobilization (Fig. 4B). The percentage of inhi-ition of muscarine-induced elevation in [Ca2+]i in theresence of blockage of PLC activity was similar to thatbserved when inhibitors for M1, M3 and M5 were co-pplied (60 ± 6%) (Fig. 4A). Following washing U-73122 outhe response to muscarine was similar to that observed in con-rol experiments (�[Ca2+]i = 517 ± 26 nM) (data not shown).

urther control experiments performed following preincu-ation of P19 neurons with the biologically inactive analog-73122 did not affect muscarine-induced [Ca2+]i responses

�[Ca2+]i = 515 ± 16 nM). These results indicate that, M1,

R.R. Resende et al. / Cell Calcium 43 (2008) 107–121 115

Fig. 4. Characterization of muscarinic acetylcholine receptor (mAChR) induced elevation of [Ca2+]i in P19 neurons. (A) The upper panel shows traces ofalterations of [Ca2+]i of P19 cells on day 8 of differentiation following stimulation with 300 �M of muscarine (Musc) alone or together with 0.1 �M pirenzipine(n = 8), 0.1 �M 4-DAMP (n = 8), 10 �M galamine (n = 8), or 1 �M MT3 (n = 8) which are specific inhibitors of M1, M3, M2 and M4 mAChR subtypes,respectively. None of the used inhibitors are antagonists of M5 mAChR activity. The remaining alteration of [Ca2+]i following stimulation by muscarine in thepresence of all four inhibitor is believed to be due to activation of the M5 subtype (n = 4). Lower panels: average [Ca2+]i values in the presence of the respectiveinhibitors were plotted as percentage of control measurements performed in the absence of inhibitors. (B) The upper panel shows traces of [Ca2+]i transientsobtained following stimulation with muscarine alone or with muscarine in the presence of 5 �M of PLC inhibitor U-73122 and its biologically inactive analogU muscarit ber ofv

Mrii

r5tiw

gRv(pt

-73343. In the lower panels, [Ca2+]i values obtained in the presence ofhe control measurements in the absence of inhibitors (n = 8). The total numalues ± S.E.M.; *p < 0.05, **p < 0.001 compared with control data.

3, and M5 mAChRs in P19 cells differentiated into neu-ons couple to PLC, thereby stimulating the production ofnositol triphosphate (IP3) followed by release of Ca2+ fromntracellular stores.

We have also determined the concentration of muscariniceceptor subtype-selective antagonists necessary to inhibit

0% of the muscarine-induced [Ca2+]i response (IC50) inhese cells. The effect of 300 �M muscarine in induc-ng elevations in [Ca2+]i in neuronal-differentiated cellsas partially and dose-dependently blocked by pirenzepine,

ttis

ne and U-73122 (n = 4) or U-73343 (n = 8) were plotted as percentage ofcells analyzed in each experiment was 56–76. Data are presented as mean

allamine, and 4-DAMP (Fig. III of supplemental data).espective potencies of the antagonists determined as pKialues with confidence intervals were 5.56 (7.3–3.8), 6.468.7–4.3) and 6.03 (6.66–5.41) for 4-DAMP, gallamine andirenzepine, respectively. The results of the IC50 determina-ion indicate that the effect of muscarine in inducing [Ca2+]i

ransients was predominately mediated by a M3-like recep-or subtype. Antagonist concentrations higher than those usedn these experiments unspecifically inhibit other muscarinicubtypes.

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16 R.R. Resende et al. / Ce

.5. Dependence of nAChR-induced [Ca 2+]i responsesn the presence of extracellular Ca2+ and Ca2+ releaserom internal Ca2+ stores

Caffeine in milimolar concentrations acts as inhibitorf intracellular receptors for IP3, as well as activator ofyanodine receptors [44], thereby depleting intracellularyanodine-sensitive Ca2+ stores [45]. In contrast, micromolaroncentrations of ryanodine inhibit caffeine-induced Ca2+

elease from intracellular Ca2+ stores. Caffeine (10 mM)-nduced Ca2+ mobilization (�[Ca2+]i = 1012 ± 41 nM)n P19 neurons was blocked by 10 �M ryanodine

f(i

ig. 5. Dependence of nicotinic receptor activation in P19 neurons on intracellularn P19 neurons on day 8 of differentiation following application of 10 mM caffeine (ith 10 �M ryanodine. (B) Representative traces of [Ca2+]i responses after applica

o-application of 300 �M ACh with 1 �M atropine or 3 �M mecamylamine followean [Ca2+]i values ± S.E.M. obtained in the presence of inhibitors were plotted as

ata shown were obtained from 4 (A) and 8 (B) independent experiments, respectiveeuronal cells were stimulated with caffeine (10 mM) in the presence or absence oistinct experiments. For each experiment 28–64 cells were used. The data are pres

um 43 (2008) 107–121

�[Ca 2+]i = 136 ± 38 nM), indicating the presence ofyanodine-sensitive intracellular Ca2+ stores (Fig. 5A).hese Ca2+ stores are responsible for Ca2+-induced Ca2+

elease CICR, which has already been known to participaten nAChR-induced elevation of [Ca2+]i.

Our data provide further evidence for participation ofyanodine-sensitive Ca2+ stores in the nAChR-mediatedCa2+]i response (Fig. 5B). Co-application of 300 �MCh, 1 �M atropine and 10 �M ryanodine to dif-

erentiated P19 neurons resulted in larger inhibition,72 ± 2% (�[Ca2+]i = 249 ± 25 nM), than that obtainedn the presence of ACh and atropine alone, (45 ± 5%

ryanodine-sensitive calcium stores. Upper panel: (A) Alterations of [Ca2+]i

during 30 s of application) with or without preincubation of cells for 30 mintion of 300 �M ACh in the absence or presence of 1 �M atropine, or after

ing preincubation of cells for 30 min with 10 �M ryanodine. Lower panels:percentages of the control value obtained in the absence of inhibitors. Thely. (C) Caffeine-induced [Ca2+]i responses of neuronal-differentiated cells.f extracellular Ca2+ (EGTA 1 mM). Typical [Ca2+]i traces were from fourented as mean values ± S.E.M.; **p < 0.001 compared with control data.

R.R. Resende et al. / Cell Calci

Table 1Inhibition of nicotine-induced [Ca2+]i responses following preincubationwith cyclopiazonic acid (CPA)

Time(min)

�[Ca2+]i (nM) Relative �[Ca2+]i

Control CPA (10 �M) Control CPA (10 �M)

0 467 ± 15 466 ± 16 1.00 ± 0.032 1.00 ± 0.0347.5 498 ± 15 503 ± 21 1.06 ± 0.033 1.08 ± 0.044

10.5 457 ± 14 403 ± 24 0.98 ± 0.029 0.86 ± 0.05113.8 450 ± 13 338 ± 24 0.96 ± 0.028 0.72 ± 0.05016.8 440 ± 15 311 ± 18 0.94 ± 0.033 0.66 ± 0.03819.5 410 ± 25 234 ± 11 0.88 ± 0.054 0.50 ± 0.023

Rp

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elative �[Ca2+]i = increases in [Ca2+]i compared to those obtained in theresence of 100 �M nicotine at time point 0.

�[Ca2+]i = 489 ± 35 nM), when compared to controlxperiments in the absence of atropine and ryanodine�[Ca2+]i = 887 ± 39 nM). No further increase in inhibitionas observed when 300 �M ACh, 3 �M mecamylamine

nd 10 �M ryanodine were co-applied to P19 neurons�[Ca2+]i = 440 ± 13 nM) in comparison to the ACh-evokedCa2+]i response in the presence of 3 �M mecamylaminelone (�[Ca2+]i = 474 ± 27 nM). These data indicate thatAChR-induced Ca2+ fluxes involve CICR mediated byyanodine receptors. The inhibitory effect caused by ryan-dine was reversible following excessive washing. Peakalues in [Ca2+]i response induced by application of 300 �MCh in the presence (�[Ca2+]i = 465 ± 41 nM) or absence

�[Ca2+]i = 878 ± 45 nM) of atropine and in the presence�[Ca2+]i = 467 ± 21 nM) or absence of mecamylamine�[Ca2+]i = 894 ± 39 nM) recovered to those that had beeneasured without ryanodine treatment. In contrast, CICRediated by activation of ryanodine receptors did not par-

icipate in mAChR-evoked increase of [Ca2+]i (Fig. 5B).Nicotine (100 �M)-induced Ca2+ fluxes were monitored

t different time points following preincubation with 10 �Myclopiazonic acid (CPA) as an inhibitor of Ca2+-ATPase ofhe endoplasmatic and sarcoplasmatic reticulum that depletesyanodine- and IP3-sensitive calcium stores [44,46]. Pre-reatment of neurons with CPA caused a time-dependentecrease of nicotine-induced Ca2+ fluxes (50 ± 2%), whichas not observed in untreated control cells (Fig. 6A). The

ontinuous decline in the amplitudes of nicotine-inducedCa2+]i response indicates slow depletion of intracellulara2+ stores due to continuous Ca2+ release in the presence ofPA. Table 1 shows the values of �[Ca2+]i and the relative[Ca2+]i induced by nicotine at various time points in the

bsence or presence of CPA treatment.The percentage of participation of IP3- and

yanodine-sensitive intracellular calcium stores in neuronal-ifferentiated cells was verified by calcium-imagingxperiments in the presence of CPA and caffeine. Activationf IP3 receptors in neurons leads to the mobilization of

a2+ from internal Ca2+ stores [46]. The [Ca2+]i response

nduced by 10 �M CPA during 20 s of application oneuronal-differentiated cells was determined to evaluate thenvolvement of IP3-sensitive stores in releasing Ca2+. CPA-

vtid

um 43 (2008) 107–121 117

pplication promoted a [Ca2+]i transient of 298 ± 32 nMn = 41) (Fig. 6B). No statistically relevant differencesetween CPA-induced [Ca2+]i elevations were observed,hen the cells had been washed for at least 10 min between

ndividual CPA-applications (data not shown).In summary, CICR via ryanodine-sensitive intracellular

a2+ stores as well as Ca2+ influx from the extracellularedium contribute to ACh-evoked elevations of [Ca2+]i in

euronal-differentiated P19 cells.

. Discussion

The presence of �3, �4, �2 nAChR subunits and M1,2, M3, M5 mAChR subtypes during neuronal differenti-

tion of P19 cells has already been reported by Cauley etl. [12] and Parnas et al. [18], respectively. Expression ofhe M3 subtype and muscarinic receptor activity were sig-ificantly augmented in P19 cells differentiated into neuronshen compared to undifferentiated cells [18,20]. However,AChR gene expression and functionality in embryonic P19ells and differential gene expression and activity of cholin-rgic receptor subtypes have not been yet studied along theourse of neuronal differentiation of this cell line.

In this study, we bring evidence that functional nicotiniceceptor channels are already present in the embryonic P19ells as well as in neural progenitor stages following stimu-ation to neuronal differentiation by RA. Gene and proteinxpression of nicotinic receptor subunits, such as �2–�7,2 and �4, were detected in these cells throughout neuronalifferentiation as evidenced by real-time PCR and immuno-istochemistry procedures. In accordance with our findings,AChR gene expression has already been detected in embry-nic tissues and cells [4]. Transcription of nAChR-subunitpecific mRNAs has also been reported from embryonic cor-ex of rodents at the 12th day of embryonic development4], and nAChR activity has been detected in BLC6 embry-nic cells, which have the ability to differentiate into cellsith characteristics of skeletal muscle [47]. The expressionf functional nAChR in embryonic cell lines capable to differ-ntiate into cell types of all three germinative layers raises theuestion about the function of these receptors in embryonicnd progenitor cells during initial brain development.

Gene expression of �2, �4, �5, �6, �7 and �4 subunits wasresent in undifferentiated P19 cells and then down-regulatedn progenitor stages when cells had already been commit-ed to neuronal differentiation. The initial upregulation ofxpression of these subunits may point to a participationf nAChRs in early proliferation events and the onset ofeuronal differentiation. We have shown that the main ACh-nduced response in undifferentiated cells was due to nAChRctivation. In agreement with our data obtained during in

itro differentiation, Atluri et al. [9] reported that func-ional nAChRs were present as early as embryonic day 10n mouse cerebral cortex, when the cortex yet consisted ofividing stem and progenitor cells. Choline present in plasma

118 R.R. Resende et al. / Cell Calcium 43 (2008) 107–121

Fig. 6. Nicotinic acetylcholine receptor activation in P19 neurons results in liberation of Ca2+ from intracellular calcium stores. (A) Upper panel: traces showthe time-dependent inhibition of nicotine (100 �M, Nic)-induced [Ca2+]i responses by 10 �M cyclopiazonic acid (CPA) present during 14 min. Lower panel:nicotine-induced alterations of [Ca2+]i obtained in the presence of CPA were normalized to those obtained in control experiments at time point zero. For eachdata point obtained by stimulation with nicotine in the presence of CPA (n = 6) (open boxes), control measurements (n = 6) were performed in the absence ofCPA (filled boxes). With exception of the first and second data point obtained at 8 min and 10.5 min of preincubation of cells with CPA, all following data pointsdemonstrated significant inhibition of nicotine-induced [Ca2+]i response in the presence of CPA. Data are presented as mean values ± S.E.M.; **p < 0.001c Ca2+]i rw ntativew

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ompared with control data. (B) Amplitudes of CPA- and caffeine-induced [ith CPA (10 �M) and caffeine (10 mM). [Ca2+]i traces shown are represeere used.

embranes could act as an endogenous activator of nAChRctivity, taking into account that it possesses high affinity to7 homomeric receptors, which are expressed by almost allells of an undifferentiated P19 population. Choline has beenhown to elicit [Ca2+]i responses in chromaffin cells [48].hanges in choline concentrations have also been shown toffect postnatal cerebral development in rats [49].

In agreements with already published studies [9,50],00 �M ACh evoked maximal cholinergic receptor-inducedncreases in [Ca2+]i. We have also demonstrated that cholin-rgic receptor-mediated Ca2+ fluxes in P19 neurons resultrom influx of Ca2+ from the extracellular medium as wells from mobilization of intracellular Ca2+ stores. In embry-nic cells, ACh-induced [Ca2+]i elevations resulted mainlyrom nAChR activation, as determined by [Ca2+]i mea-urements in the presence of atropine and mecamylamines specific inhibitors of mAChRs and nAChRs, respec-ively. In neuronal-differentiated P19 cells, ACh-inducedCa2+]i responses resulted from almost equal contributionsf mAChRs and nAChRs. ACh-induced elevations of [Ca2+]in P19 cells terminating their differentiation process rangedround 1 �M as compared to ACh-evoked [Ca2+]i responsesf 100 nM in embryonic P19 cells. The higher amplitudesf changes of [Ca2+]i in P19 neurons compared to embry-

nal cells following ACh application may not be only due tohigher fraction of active receptors in differentiated cells,

ut also to the increased expression of VOOC which arectivated following membrane depolarization caused by the

bcsd

esponse in neuronal-differentiated cells. Cells were stimulated sequentiallydata from five independent experiments. For each experiment 40–45 cells

nitial ACh-induced calcium flux and contributing to thecetylcholine receptor response [51]. Neuronal nAChRs, andarticularly homomeric nicotinic receptors formed by the �7ubtype, exhibit high relative permeability to Ca2+ [52,53].hus, opening of the nAChR channel can lead to a direct

ncrease in cytoplasmic Ca2+ concentration, whilst Na+ influxan cause neuronal depolarization and activation of VOCCnd subsequent amplification of Ca2+ transients [54,55].

The intracellular ryanodine-sensitive Ca2+ store, a CICRhannel, whose activity is regulated by cytosolic Ca2+ con-entrations, participates to the modulation and amplificationf Ca2+ signals in various neuronal cells (reviewed in [56]).e have shown that activation of ryanodine-sensitive Ca2+

tores are responsible for approximately 50% of the nAChR-ediated [Ca2+]i responses. More evidence for the presence

f CICR in P19 neuronal cells was brought by the observationhat caffeine-induced elevations in [Ca2+]i were blocked inhe presence of ryanodine, a specific inhibitor of CICR chan-els. Participation of CICR channels in regulation of [Ca2+]ias already been suggested in developing rat motor neurons46].

Ryanodine-sensitive Ca2+ stores (CICR-channels) arepparently not involved in the nAChR-mediated [Ca2+]iesponse in undifferentiated P19 cells. These results may

e explained by the absence of such stores in embryonicells and is in agreement with in vivo studies that havehown that ryanodine receptors are only expressed in theeveloping nervous system following the onset to neuronal

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ifferentiation [57]. Further support for this hypothesis wasbtained by the observation that in the presence of CdCl2 andifedipine as inhibitors of VOOC-activity nicotine-inducedCa2+]i responses in embryonic cells were almost com-letely inhibited. However, these inhibitors only promotedn approximate 50%-inhibition the nicotine-induced [Ca2+]iesponse in neuronal-differentiated P19 cells, suggestinghat CICR-channels participate in the remaining 50% of theicotine-evoked [Ca2+]i increase.

The inhibition of nicotine-induced [Ca2+]i responses fol-owing preincubation of P19 neurons with CPA as an inhibitorf endoplasmatic Ca2+-ATPase in order to deplete IP3- andyanodine-sensitive Ca2+ stores [44,46] again indicated intra-ellular Ca2+ mobilization, such as CICR, as a part of theAChR-mediated [Ca2+]i response. In agreement with ouresults, Dajas-Bailador et al. [58] demonstrated that activa-ion of nAChRs in SH-SY5Y neuroblastoma cells involvedOOC and release of Ca2+ from intracellular stores. Similar

esults were obtained for hippocampal astrocytes [29].The treatment of cells with CPA alone leads to a tran-

ient rise of cytosolic Ca2+ due to the leakage of Ca2+ fromPA-sensitive stores. In most cell types studied, this Ca2+

eakage can be rather variable, but it usually leads to a com-lete depletion of the endoplasmatic Ca2+ stores within aew minutes [59]. Ca2+ leakage from intracellular stores washown to be inhibited in the presence of Ni2+ and is presum-bly mediated by a special channel in the membrane of thendoplasmatic reticulum [60]. In the absence of extracellu-ar Ca2+, this transient Ca2+ rise may provide an estimativeuantification of the concentration of Ca2+ accumulated inPA-sensitive stores. In cultured rat dorsal root ganglia neu-

ons, concentrations of up to 270 �M Ca2+ were identifiedn endoplasmic reticular stores, by using fluorophores withow-affinity for Ca2+ [61]. Caffeine-sensitive calcium storesCICR) have been described in many vertebrate neurons initu and in vitro (reviewed in [62]). However, more thanne mechanism exists by which nAChR activation resultsn transient elevations of [Ca2+]i. For instance, Foucart etl. [63] demonstrated that nAChR activation in rat sympa-hetic neurons did not depend on intracellular Ca2+ stores.n this regard, our study has demonstrated that sources usedor nAChR-induced [Ca2+]i responses change during neu-onal differentiation of P19 cells. In undifferentiated P19ells extracellular Ca2+ was the principal source used by theicotinic component. In P19 cells differentiated into neurons,ntracellular Ca2+ stores were the main source for nAChR-s well as for mAChR-mediated Ca2+ fluxes.

The participation of the muscarinic component in theCh-induced calcium flux was monitored in the presencef the nAChR-specific inhibitor mecamylamine. Muscariniceceptor-induced elevations of [Ca2+]i remained in thebsence of extracellular Ca2+, suggesting the mAChR-

ediated [Ca2+]i response principally involves intracellulara2+ stores. In the presence of the PLC inhibitor, U-73122,ost of the mAChR-induced [Ca2+]i was abolished, again

ndicating that mAChR activation enhances PLC activity,

TIiS

um 43 (2008) 107–121 119

P3-formation, and Ca2+ release from IP3-sensitive stores.e have investigated the participation of different mAChR

ubtypes in the muscarine-induced Ca2+ mobilization. TheCa2+]i measurements in the presence of subtype-specificnhibitors indicated activities of M1, M2, M3, and M5-ubtypes in neuronal-differentiated P19 cells, which aren accordance with previously published data [18,20]. Thenvolvement of mAChRs in neuronal differentiation hasreviously been studied (reviewed in [16]). For instance, dif-erentiation of neural stem cells depended on the activity of

2 receptor subtypes, as this differentiation was blockedn the presence of the muscarinic antagonist atropine [64].nhibition of kinin-B2 receptors during the final neuronal dif-erentiation of P19 cells resulted in a subsequent decrease of

1, M2, and M3 mAChR gene expression and activity, point-ng again at a crucial function of muscarinic receptor functionn later neuronal differentiation [20].

Taken together, this work contributes to a better under-tanding of the mechanism of cholinergic receptor activationuring early embryogenesis in vitro. In vitro neuronal dif-erentiation resembles conditions of initial neuroectodermalifferentiation [65]. At the onset of differentiation, mostly7-type nAChRs, which are highly permeable for Ca2+,ontributed to [Ca2+]i transients, necessary for triggeringeurogenesis. The most expressed nAChR in undifferen-iated P19 cells was formed by �7 subunits, followed by2�4 nAChRs (data not shown). Some other nAChR typesuch as �3�4 and �4�2 may not be formed during differ-ntiation since expression of �3 and �2 subunits was lowFigs. 1 and 2). Following commitment to a neuronal phe-otype and undergoing final differentiation, nicotinic anduscarinic receptor types were functional. Ca2+ spikes reach-

ng micromolar concentrations were induced by combineda2+ fluxes from the extracellular medium and intracellulara2+ stores. These high concentrations in [Ca2+]i may be nec-ssary for final synaptogenesis, maintaining synaptogenesisnd induction of neurotransmitter release.

onflict of interest statement

None.

cknowledgments

H.U. and L.R.G.B. are grateful for grants from FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo) andNPq (Conselho Nacional de Desenvolvimento CientıficoTecnologico), Brazil. R.R’s and K.N.G.’s Ph.D. thesesere or are supported, respectively, by CAPES (Coordenacaoe Aperfeicoamento de Pessoal de Nıvel Superior), Brazil.

he Center for Applied Toxinology (CAT-CEPID), Butantan

nstitute, Sao Paulo, Brazil, is acknowledged for perform-ng DNA sequencing. We also wish to thank Adilson dailva Alves, Departamento de Fisiologia e Biofısica, Insti-

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ppendix A. Supplementary data

Supplementary data associated with this article cane found, in the online version, at doi:10.1016/j.ceca.007.04.007.

eferences

[1] X. Du, L. Iacovitti, Synergy between growth factors and transmittersrequired for catecholamine differentiation in brain neurons, J. Neurosci.15 (1995) 5420–5427.

[2] J. Zhou, H.F. Bradford, G.M. Stern, Induction of dopaminergic neuro-transmitter phenotype in rat embryonic cerebrocortex by the synergisticaction of neurotrophins and dopamine, Eur. J. Neurosci. 8 (1996)2328–2339.

[3] S. Wonnacott, N. Sidhpura, D.J. Balfour, Nicotine: from molecularmechanisms to behaviour, Curr. Opin. Pharmacol. 5 (2005) 53–59.

[4] M. Zoli, N. Le Novere, J.A. Hill Jr., J.P. Changeux, Developmentalregulation of nicotinic ACh receptor subunit mRNAs in the rat centraland peripheral nervous systems, J. Neurosci. 15 (1995) 1912–1939.

[5] P. Brehm, L. Henderson, Regulation of acetylcholine receptor channelfunction during development of skeletal muscle, Dev. Biol. 129 (1988)1–11.

[6] H.R. Brenner, V. Witzemann, B. Sakmann, Imprinting of acetylcholinereceptor messenger RNA accumulation in mammalian neuromuscularsynapses, Nature 344 (1990) 544–547.

[7] A.C. Missias, G.C. Chu, B.J. Klocke, J.R. Sanes, J.P. Merlie, Maturationof the acetylcholine receptor in skeletal muscle: regulation of the AChRgamma-to-epsilon switch, Dev. Biol. 179 (1996) 223–238.

[8] J.R. Sanes, J.W. Lichtman, Induction, assembly, maturation and main-tenance of a postsynaptic apparatus, Nat. Rev. Neurosci. 2 (2001)791–805.

[9] P. Atluri, M.W. Fleck, Q. Shen, et al., Functional nicotinic acetylcholinereceptor expression in stem and progenitor cells of the early embryonicmouse cerebral cortex, Dev. Biol. 240 (2001) 143–156.

10] I. Aubert, D. Cecyre, S. Gauthier, R. Quirion, Comparative ontoge-netic profile of cholinergic markers, including nicotinic and muscarinicreceptors, in the rat brain, J. Comp. Neurol. 369 (1996) 31–55.

11] J.A. Court, S. Lloyd, M. Johnson, et al., Nicotinic and muscariniccholinergic receptor binding in the human hippocampal formation dur-ing development and aging, Brain Res. Dev. Brain Res. 101 (1997)93–105.

12] K. Cauley, M. Marks, L.C. Gahring, S.W. Rogers, Nicotinic receptorsubunits alpha 3, alpha 4, and beta 2 and high affinity nicotine bindingsites are expressed by P19 embryonal cells, J. Neurobiol. 30 (1996)303–314.

13] L.A. Volpicelli, A.I. Levey, Muscarinic acetylcholine receptor subtypesin cerebral cortex and hippocampus, Prog. Brain Res. 145 (2004) 59–66.

14] W. Ma, D. Maric, B.S. Li, et al., Acetylcholine stimulates cortical pre-cursor cell proliferation in vitro via muscarinic receptor activation andMAP kinase phosphorylation, Eur. J. Neurosci. 12 (2000) 1227–1240.

15] S. Biagioni, A.M. Tata, A. De Jaco, G. Augusti-Tocco, Acetylcholinesynthesis and neuron differentiation, Int. J. Dev. Biol. 44 (2000)

689–697.

16] H. Ulrich, P. Majumder, Neurotransmitter receptor expression andactivity during neuronal differentiation of embryonal carcinoma andstem cells: from basic research towards clinical applications, Cell Prolif.39 (2006) 281–300.

[

um 43 (2008) 107–121

17] D. Parnas, M. Linial, Cholinergic properties of neurons differentiatedfrom an embryonal carcinoma cell-line (P19), Int. J. Dev. Neurosci. 13(1995) 767–781.

18] D. Parnas, E. Heldman, L. Branski, N. Feinstein, M. Linial, Expressionand localization of muscarinic receptors in P19-derived neurons, J. Mol.Neurosci. 10 (1998) 17–29.

19] S. Ben-Ari, D. Toiber, A.S. Sas, H. Soreq, Y. Ben-Shaul, Modulatedsplicing-associated gene expression in P19 cells expressing distinctacetylcholinesterase splice variants, J. Neurochem. 97 (2006) 24–34.

20] A.H. Martins, R.R. Resende, P. Majumder, et al., Neuronal differenti-ation of P19 embryonal carcinoma cells modulates kinin B2 receptorgene expression and function, J. Biol. Chem. 280 (2005) 19576–19586.

21] R.R. Resende, P. Majumder, K.N. Gomes, L.R. Britto, H. UlrichH, P19 embryonal carcinoma cells as in vitro model for studyingpurinergic receptor expression and modulation of N-methyl-d-aspartate–glutamate and acetylcholine receptors during neuronaldifferentiation, Neuroscience 146 (2007) 1169–1181.

22] S. Ueno, H. Yamada, T. Moriyama, et al., Measurement of dorsal rootganglion P2X mRNA by SYBR Green fluorescence, Brain Res. BrainRes. Protoc. 10 (2002) 95–101.

23] R.H. Lekanne Deprez, A.C. Fijnvandraat, J.M. Ruijter, A.F. Moorman,Sensitivity and accuracy of quantitative real-time polymerase chainreaction using SYBR green I depends on cDNA synthesis conditions,Anal. Biochem. 307 (2002) 63–69.

24] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T)) Method,Methods 25 (2001) 402–408.

25] A. Adhikari, C.A. Penatti, R.R. Resende, H. Ulrich, L.R. Britto,E.J. Bechara, 5-Aminolevulinate and 4,5-dioxovalerate ions decreaseGABA(A) receptor density in neuronal cells, synaptosomes and ratbrain, Brain Res. 1093 (2006) 95–104.

26] C.L. Curl, C.J. Bellair, T. Harris, B.E. Allman, P.J. Harris, A.G. Stewart,A. Roberts, K.A. Nugent, L.M.D. Delbridge, Refractive index measure-ment in viable cells using quantitative phase-amplitude microscopy andconfocal microscopy, Cytometry Part A 65a (2005) 88–92.

27] M.B. Hallett, R.L. Dormer, A.K. Campbell, in: K. Siddle, J.C. Hut-ton (Eds.), Peptide Hormone Action, A Practical Approach, OxfordUniversity Press, New York, 1990, pp. 115–150.

28] A. Minta, J.P.Y. Kao, R.Y. Tsien, Fluorescent indicators for cytosolicCa2+ based on rhodamine and fluorescein chromophores, J. Biol. Chem.264 (1989) 8171–8178.

29] G. Sharma, S. Vijayaraghavan, Nicotinic cholinergic signaling in hip-pocampal astrocytes involves calcium-induced calcium release fromintracellular stores, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4148–4153.

30] L. Walch, J.P. Gascard, E. Dulmet, C. Brink, X. Norel, Evidence fora M(1) muscarinic receptor on the endothelium of human pulmonaryveins, Br. J. Pharmacol. 130 (2000) 73–78.

31] L.A. Fieber, D.J. Adams, Acetylcholine-evoked currents in culturedneurons dissociated from rat parasympathetic cardiac ganglia, J. Phys-iol. 434 (1991) 215–237.

32] N.J. Buckley, T.I. Bonner, C.M. Buckley, M.R. Brann, Antagonist bind-ing properties of five cloned muscarinic receptors expressed in CHO-K1cell, Mol. Pharmacol. 35 (1989) 469–476.

33] M.P. Caulfield, N.J.M. Birdsall, International Union of Pharmacology.XVII. Classification of muscarinic acetylcholine receptors, Pharmacol.Rev. 50 (1998) 279–290.

34] A.D. Michel, R.E. Delmendo, M. Lopez, R.L. Whiting, On the interac-tion of gallamine with muscarinic receptor subtypes, Eur. J. Pharmacol.182 (1990) 335–345.

35] C.J. Doelman, R.C. Sprong, J.E. Nagtegaal, J.F. Rodrigues de Miranda,A. Bast, Prejunctional muscarinic receptors on cholinergic nerves inguinea pig airways are of the M2 subtype, Eur. J. Pharmacol. 193 (1991)

117–119.

36] M. Jolkkonen, P.L. van Giersbergen, U. Hellman, C. Wernstedt, E.Karlsson, A toxin from the green mamba Dendroaspis angusticeps:amino acid sequence and selectivity for muscarinic m4 receptors, FEBSLett. 352 (1994) 91–94.

ll Calci

[

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[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[[

[

[

[

[

[

[

[

R.R. Resende et al. / Ce

37] M.C. Olianas, A. Adem, E. Karlsson, P. Onali, Rat striatal muscarinicreceptors coupled to the inhibition of adenylyl cyclase activity: potentblock by the selective m4 ligand muscarinic toxin 3 (MT3), Br. J.Pharmacol. 118 (1996) 283–288.

38] D.I. Yule, J.A. Williams, U73122 inhibits Ca2+ oscillations in responseto cholecystokinin and carbachol but not to JMV-180 in rat pancreaticacinar cells, J. Biol. Chem. 267 (1992) 13830–13835.

39] M. Suzuki, K. Muraki, Y. Imaizumi, M. Watanabe, Cyclopiazonicacid, an inhibitor of the sarcoplasmic reticulum Ca(2+)-pump, reducesCa(2+)-dependent K+ currents in guinea-pig smooth muscle cells, Br.J. Pharmacol. 107 (1992) 134–140.

40] N. Demaurex, D.P. Lew, K.H. Krause, Cyclopiazonic acid depletesintracellular Ca2+ stores and activates an influx pathway for divalentcations in HL-60 cells, J. Biol. Chem. 267 (1992) 2318–2324.

41] S.C. Taylor, C. Peers, Store-operated Ca2+ influx and voltage-gatedCa2+ channels coupled to exocytosis in pheochromocytoma (PC12)cells, J. Neurochem. 73 (1999) 874–880.

42] J. Gafni, J.A. Munsch, T.H. Lam, M.C. Catlin, L.G. Costa, T.F. Molin-ski, I.N. Pessah, Xestospongins: potent membrane permeable blockersof the inositol 1,4,5-triphosphate receptor, Neuron 19 (1997) 723–733.

43] C.C. Felder, Muscarinic acetylcholine receptors: signal transductionthrough multiple effectors, FASEB J. 9 (1995) 619–625.

44] A. Beck, R.Z. Nieden, H.P. Schneider, J.W. Deitmer, Calcium releasefrom intracellular stores in rodent astrocytes and neurons in situ, CellCalcium 35 (2004) 47–58.

45] J. Meldolesi, Rapidly exchanging Ca2+ stores in neurons: molecu-lar, structural and functional properties, Prog. Neurobiol. 65 (2001)309–338.

46] G. Dayanithi, I. Mechaly, C. Viero, et al., Intracellular Ca2+ regulation inrat motoneurons during development, Cell Calcium 39 (2006) 237–246.

47] J. Rohwedel, V. Maltsev, E. Bober, H.H. Arnold, J. Hescheler, A.M.Wobus, Muscle cell differentiation of embryonic stem cells reflectsmyogenesis in vivo: developmentally regulated expression of myogenicdetermination genes and functional expression of ionic currents, Dev.Biol. 164 (1994) 87–101.

48] J. Fuentealba, R. Olivares, E. Ales, et al., A choline-evoked [Ca2+]i sig-nal causes catecholamine release and hyperpolarization of chromaffincells, FASEB J. 18 (2004) 1468–1470.

49] J.K. Blusztajn, J.M. Cermak, T. Holler, D.A. Jackson, Imprinting ofhippocampal metabolism of choline by its availability during gesta-

tion: implications for cholinergic neurotransmission, J. Physiol. Paris92 (1998) 199–203.

50] W. Marszalec, J.Z. Yeh, T. Narahashi, Desensitization of nicotineacetylcholine receptors: modulation by kinase activation and phos-phatase inhibition, Eur. J. Pharmacol. 514 (2005) 83–90.

[

um 43 (2008) 107–121 121

51] J. Trouslard, S.J. Marsh, D.A. Brown, Calcium entry through nicotinicreceptor channels and calcium channels in cultured rat superior cervicalganglion cells, J. Physiol. Lond. 468 (1993) 53–71.

52] P. Seguela, J. Wadiche, K. Dineley-Miller, J.A. Dani, J.W. Patrick,Molecular cloning, functional properties, and distribution of rat brain�7: a nicotinic cation channel highly permeable to calcium, J. Neurosci.13 (1993) 596–604.

53] D.S. McGehee, L.W. Role, Physiological diversity of nicotinic acetyl-choline receptors expressed by vertebrate neurons, Annu. Rev. Physiol.57 (1995) 521–546.

54] S. Vijayaraghavan, P.C. Pugh, Z.W. Zhang, M.M. Rathouz, D.K. Berg,Nicotinic receptors that bind �-bungarotoxin on neurons raise intracel-lular free Ca2+, Neuron 8 (1992) 353–362.

55] M.M. Rathouz, D.K. Berg, Synaptic-type acetylcholine receptors raiseintracellular calcium levels in neurons by two mechanisms, J. Neurosci.14 (1994) 6935–6945.

56] M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1998) 3–26.57] F. Mori, M. Fukaya, H. Abe, K. Wakabayashi, M. Watanabe, Develop-

mental changes in expression of the three ryanodine receptor mRNAsin the mouse brain, Neurosci. Lett. 285 (2000) 57–60.

58] F.A. Dajas-Bailador, A.J. Mogg, S. Wonnacott, Intracellular Ca2+ sig-nals evoked by stimulation of nicotinic acetylcholine receptors inSH-SY5Y cells: contribution of voltage-operated Ca2+ channels andCa2+ stores, J. Neurochem. 81 (2002) 606–614.

59] C. Camello, R. Lomax, O.H. Petersen, A.V. Tepikin, Calcium leak fromintracellular stores—the enigma of calcium signalling, Cell Calcium 32(2002) 355–361.

60] F. Wissing, E.P. Nerou, C.W. Taylor, A novel Ca2+-induced Ca2+ releasemechanism mediated by neither inositol triphosphate nor ryanodinereceptors, Biochem. J. 361 (2002) 605–611.

61] N. Solovyova, N. Veselovsky, E.C. Toescu, A. Verkhratsky, Ca2+

dynamics in the lumen of the endoplasmic reticulum in sensory neu-rons: direct visualization of Ca2+-induced Ca2+ release triggered byphysiological Ca2+ entry, EMBO J. 21 (2002) 622–630.

62] A. Verkhratsky, A. Shmigol, Calcium-induced calcium release in neu-rons, Cell Calcium 19 (1996) 1–14.

63] S. Foucart, S.J. Gibbons, J.R. Brorson, R.J. Miller, Increase in [Ca2+]i

by CCh in adult rat sympathetic neurons are not dependent on intracel-lular Ca2+ pools, Am. J. Physiol. 268 (1995) 829–837.

64] C. Zhou, Z.X. Wen, D.M. Shi, Z.P. Xie, Muscarinic acetylcholine recep-

tors involved in the regulation of neural stem cell proliferation anddifferentiation in vitro, Cell. Biol. Int. 28 (2004) 63–67.

65] W.A. Staines, J. Craig, K. Reuhl, M.W. McBurney, Retinoic acid treatedP19 embryonal carcinoma cells differentiate into oligodendrocytescapable of myelination, Neuroscience 71 (1996) 845–853.