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P19 EMBRYONAL CARCINOMA CELLS AS IN VITRO MODEL FOR
STUDYING PURINERGIC RECEPTOR EXPRESSION AND MODULATION
OF N-METHYL-D-ASPARTATE– GLUTAMATE AND ACETYLCHOLINE
RECEPTORS DURING NEURONAL DIFFERENTIATION
R. R. RESENDE,
a1
P. MAJUMDER,
a1
K. N. GOMES,
a
L. R. G. BRITTO
b
AND H. ULRICH
a
*
a
Departamento de Bioquímica, Instituto de Química, Universidade de
São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP,
Brazil
b
Departamento de Fisiologia e Biofísica, Instituto de Ciências Bi-
omédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1524,
São Paulo, SP 05508-900, Brazil
Abstract—The in vitro differentiation of P19 murine embryo-
nal carcinoma cells to neurons resembles developmental
stages which are encountered during neuronal development.
Three days following induction to neuronal differentiation by
retinoic acid, most cells of the P19 population lost expression
of the stage specific embryonic antigen (SSEA-1) and ex-
pressed the neural progenitor cell specific antigen nestin.
Beginning from day 4 of differentiation nestin expression was
down-regulated, and expression of neuron-specific enolase
as marker of differentiated neurons increased. The molecular
mechanisms underlying neuronal differentiation are poorly
understood. We have characterized the participation of puri-
nergic ionotropic (P2X) and metabotropic (P2Y) receptors at
mRNA transcription and protein levels as well as ATP-in-
duced Ca
2ⴙ
transients during neuronal differentiation of P19
cells. Gene and protein expression of P2X
2
, P2X
6
, P2Y
2
, and
P2Y
6
receptors increased during the course of differentiation,
whereas P2X
3
, P2X
4
, P2Y
1
and P2Y
4
receptor expression was
high in embryonic P19 cells and then decreased following
induction of P19 cells to differentiation. P2X
1
receptor protein
expression was only detected on days 2 and 4 of differenti-
ation. Although P2X
5
and P2X
7
mRNA transcription was
present, no protein expression for this receptor subunit
could be detected throughout the differentiation process. In
undifferentiated cells, mainly ionotropic P2X receptors con-
tributed to the ATP-induced Ca
2ⴙ
-response. In neuronal-dif-
ferentiated P19 cells, the ATP-induced Ca
2ⴙ
-response was
increased and the metabotropic component predominated.
Purinergic receptor function is implicated to participate in
neuronal maturation, as cholinergic and glutamate–N-methyl-
D-aspartate (NMDA) induced calcium responses were af-
fected when cells were differentiated in the presence of pu-
rinergic receptor antagonists pyridoxalphosphate-6-azophe-
nyl-2=,4=-disulphonic acid (PPADS), suramin or reactive
blue-2. Our data suggest that inhibition of P2Y
1
and possibly
P2X
2
receptors led to a loss of NMDA receptor activity
whereas blockade of possibly P2X
2
and P2Y
2
purinergic re-
ceptors during neuronal differentiation of P19 mouse led to
inhibition of cholinergic receptor responses. © 2007 IBRO.
Published by Elsevier Ltd. All rights reserved.
Key words: purinergic receptors, P19 embryonal carcinoma
cells, neuronal differentiation, maturation of synapses, intrin-
sic modulation of cholinergic and glutamate–NMDA receptor
activity, ATP.
Brain development is one of the most important morpho-
genetic events occurring in the embryo and is a complex
process involving cell proliferation and differentiation as
well as tissue organization into a specific architecture. The
understanding of the mechanism of neuronal differentia-
tion lies in the determination of how a small number of
tissue-restricted transcription factors can establish a com-
plex pattern of expression of developmental and tissue-
specific genes. Previous studies have identified several
growth factors and neurotransmitters that influence the
proliferation of embryonic or adult mammalian neural stem
cells (NSCs) (Gage et al., 1995; Cameron et al., 1998;
Erlandsson et al., 2001), but little is known about the
signaling molecules that determine the self-renewal capac-
ity of NSCs. Purinergic receptors, activated by ATP and
other nucleotides, have been attributed to several func-
tions including neurotransmission and neuromodulation in
synapses (Zimmermann, 1994; Neary et al., 1996; Franke
and Illes, 2006). However, purinergic receptor expression
and activity are not only found in the adult brain but also in
early stages of embryonic development (Franke and Illes,
2006) suggesting that these receptors may play a role in
embryogenesis. ATP binds to and activates ligand-gated
ionotropic purinergic ionotropic receptors (P2X) and G-
protein-coupled metabotropic purinergic metabotropic re-
ceptors (P2Y) (Abbracchio and Burnstock, 1994), thereby
inducing a variety of cellular functions, including differentia-
tion and proliferation. Nowadays seven subtypes of iono-
tropic (P2X
1
–P2X
7
) and eight subtypes (P2Y
1,2,4,6,11–14
)of
metabotropic purinoreceptors have been identified by mo-
lecular cloning. Purinergic receptors have been shown to
be essential for the proliferation and migration of oligoden-
drocytes progenitors in the CNS (Agresti et al., 2005),
1
These authors equally contributed to the work.
*Corresponding author. Tel: ⫹55-11-3091-3810; fax: ⫹55-11-38155579.
E-mail address: henning@iq.usp.br (H. Ulrich).
Abbreviations: [Ca
2⫹
]
i
, intracellular free calcium concentration; CCh,
carbamoylcholine; DMEM, Dulbecco’s modified Eagle’s medium; EB,
embryonic body; EC, embryonal carcinoma; EGTA, ethylene glycol
tetraacetic acid; FBS, fetal bovine serum; Hepes, N-(2-hydroxyethyl)-
piperazine-N=-2-ethanesulfonic acid; NEL, neuron-specific enolase;
NF-160, medium-molecular weight neurofilaments; NF-200, high-
molecular weight neurofilaments; NMDA, N-methyl-D-aspartate; NSC,
neural stem cells; PB, phosphate buffer; PC12, pheochromocy-
toma cells; PPADS, pyridoxalphosphate-6-azophenyl-2=,4=-disul-
phonic acid; P2X, purinergic ionotropic receptors; P2Y, purinergic
metabotropic receptors; RA, retinoic acid; RNAi, RNA interference;
SSEA-1, stage specific embryonic antigen.
Neuroscience 146 (2007) 1169–1181
0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2007.02.041
1169
control of hippocampus neurogenesis (Shukla et al., 2005)
and direction of migration events as demonstrated by tro-
phic roles of extracellular ATP on astrocytes and neurons
(Abbracchio et al., 1994; Neary et al., 1996).
Complex developmental processes can be studied in a
simplified environment using pluripotent cells as in vitro
models which can differentiate into a defined phenotype.
The P19 murine embryonal carcinoma (EC) cell line is
such an in vitro model that can be differentiated into vari-
ous cell types by cellular aggregation in presence of the
differentiating agent retinoic acid (RA). The differentiation
of this cell line emulates the molecular and morphological
events occurring during early embryonic development
(McBurney et al., 1982). Following treatment with RA, P19
cells differentiate into various neuroectodermal derivatives
(McBurney et al., 1982) and express neuronal markers,
including high-molecular weight neurofilaments (NF-200),

-3-tubulin, neuron-specific enolase (NEL) (Niemann and
Schaller, 1996; Martins et al., 2005), glutamate receptors
(MacPherson et al., 1997), GABA receptors (Reynolds et
al., 1996) and proteins specific for cholinergic neurons
(Jones-Villeneuve et al., 1982; Cauley et al., 1996; Parnas
et al., 1998) indicating that the establishment of a neuronal
phenotype proceeds with similar molecular patterning dur-
ing neuronal differentiation of P19 cells and in vivo devel-
opment (Bayer and Altman, 1995; Wiese et al., 2004; Wu
and Chow, 2005). The expression of neurotransmitters
and their receptors becomes upregulated during precursor
stages and synapse formation in cortical and subcortical
regions (Nguyen et al., 2001). This time-regulated expres-
sion and activity of neurotransmitter receptors can also be
observed during neuronal differentiation of P19 cells; for
instance, cholinergic, NMDA (N-methyl-D-aspartate), en-
dothelin-B, and kinin-B2 receptors are already present in
neural precursor cell stages and become fully functional in
P19 neurons (Martins et al., 2005; Monge et al., 1995;
Ulrich and Majumder, 2006).
We have used the P19 cell line as in vitro model for
studying expression and activity of purinergic P2X and
P2Y receptors during differentiation from embryonic to
neural precursor stages and finally to neurons expressing
functional NMDA-glutamate receptors and muscarinic and
nicotinic acetylcholine receptors. We have verified the par-
ticipation of purinergic receptor function by analyzing the
fate of neuronal differentiation in the presence of the puri-
nergic receptor antagonists pyridoxalphosphate-6-azophe-
nyl-2=,4=-disulphonic acid (PPADS), reactive blue-2 and
suramin. We show that loss of NMDA or cholinergic recep-
tor responses in differentiated neurons is due to inhibition
of P2Y
1
and possibly P2X
2
or P2Y
2
and possibly P2X
2
receptor activity, respectively.
EXPERIMENTAL PROCEDURES
Materials
If not otherwise noticed, all reagents were purchased from Sigma (St.
Louis, MO, USA) in highest available purity. The P19 murine EC cell
line was kindly provided by Dr. H. C. Schaller, Center for Molecular
Neurobiology, Hamburg, Germany. P2X
1
- (PC378) and P2X
3
(PC411)-specific antibodies were purchased from Oncogene Re-
search Products (Cambridge, MA, USA). P2X
4
(sc-15187), P2X
6
(sc-15197), P2Y
1
(sc-15203), P2Y
4
(sc-17634) receptor-specific an-
tibodies were from Santa Cruz Biotechnologies (Heidelberg, Ger-
many). P2X
7
receptor-specific antibodies (506107) and anti-

-tubulin
mouse mAb (DM1B) were obtained from Calbiochem (San Diego,
CA, USA). P2X
2
(ab5244), P2X
5
(ab9226), P2Y
2
(ab5931 and
ab5816) and P2Y
6
(ab5715) receptor-specific antibodies were from
Chemicon International (Temecula, CA, USA).
Mice (Mus musculus, Balbi-C strain) were obtained from the
Animal Facility, Institute of Chemistry, University of São Paulo.
The protocols for keeping and killing of these animals were ap-
proved by the Bioethics Committee of the Chemistry Institute,
Universidade de São Paulo, Brazil, and are in accordance with the
National Institutes of Health Guide for the Care and Use of Lab-
oratory Animals. All efforts were made to minimize the number of
animals used and their suffering.
In vitro differentiation of P19 cells into neurons
P19 mouse EC cells were cultured and differentiated to neurons as
described previously (Tárnok and Ulrich, 2001; Martins et al., 2005).
Briefly, P19 cell cultures were maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supple-
mented with 10% fetal bovine serum (FBS, Cultilab, Campinas,
Brazil), 100 units/ml penicillin, 100
g/ml streptomycin, 2 mM L-
glutamine and 2 mM sodium pyruvate. For induction of neuronal
differentiation, 5⫻10
5
P19 cells/ml in defined medium, containing
DMEM medium supplemented with 2 mM glutamine, 2 mM sodium
pyruvate, 2.4 mg/ml sodium bicarbonate, 5
g/ml insulin, 30
g/ml
human apo-transferrin, 20
M ethanolamine, 30 nM sodium selenite,
100 U/ml penicillin, 100
g/ml streptomycin, and 10 mM Hepes, pH
7.2, were treated with 1
M “all-trans” RA and plated into bacterial
dishes previously coated with 0.5% agarose, to avoid adhesion of the
cell culture to plastic surfaces. After 2 days of culture in suspension
in the presence of RA, P19 cells formed embryonic body stages
(EBs). EBs were collected from suspension cultures and replated in
adherent culture flasks in DMEM medium with 10% FBS for 48 h.
The serum-containing medium was replaced with defined medium
on day 4, followed by four more days of culture until neuronal mat-
uration was completed as determined by neuron-specific protein
expression (neurofilament-200 and

-3-tubulin) according to Martins
et al., 2005. Glial cells were eliminated from differentiating neuron
cultures by addition of cytosine-arabinoside (5
g/ml) 2 days before
cell removal.
Immunohistochemistry
Cells at a density of 5⫻10
5
cells/ml were grown on glass cover-
slips, fixed in 2% paraformaldehyde for 10 min and dehydrated on
a platform heated at 37 °C. Incubations were performed as de-
scribed below for the immunohistochemistry procedure. Goat or
rabbit antibodies raised against purinergic receptor subtypes (see
Materials) and mouse anti-medium-molecular weight neurofila-
ments (NF-160) and mouse anti-NF-200 antibodies were used at
1:150, 1:500 and 1:500 dilutions, respectively. Goat anti-rabbit
and goat anti-mouse IgG were used at 1:200 dilutions. Conjugated
secondary antibodies were purchased from Jackson Immunore-
search Laboratories (West Grove, PA, USA). The cells were col-
lected in phosphate buffer (PB) and incubated overnight with goat
anti-P2X or P2Y receptor antibodies (1:150) in the presence of
0.3% Triton X-100 and 5% normal goat serum. After washing with
PB, cells were incubated with biotinylated goat anti-rabbit antibod-
ies at 1:200 dilution during 1 h. Following washing with PB cells
were incubated with an avidin–biotin complex for 1 h. Peroxidase
activity was detected using diaminobenzidine as a chromogen and
H
2
O
2
(Adhikari et al., 2006). Negative controls were obtained by
repeating the procedure above in the absence of the primary
antibody. Images were collected from an optical microscope
equipped with the Nikon Digital Camera DXM1200F and analyzed
R. R. Resende et al. / Neuroscience 146 (2007) 1169–11811170
with the Nikon ACT-1 version 2.62 software (Surrey, UK) as
described elsewhere (Adhikari et al., 2006).
Reverse transcription and PCR
Total RNA was isolated using TRIzol (Invitrogen) from undifferen-
tiated P19 cells and P19 cells from days 2–8 following induction to
neuronal differentiation in the presence of RA. Integrity of the
isolated RNA was verified by separation of an aliquot of the
extracted RNA on a 2% ethidium bromide–stained agarose gel.
DNA was removed from RNA samples by incubation with DNase
I (Ambion Inc., Austin, TX, USA).
Five micrograms of total RNA were reverse transcribed to
cDNA with 200 U of RevertAid
TM
H Minus M-MuLV-reverse tran-
scriptase (Fermentas Inc., Hanover, MD, USA). Obtained cDNA
was amplified by PCR using TaqDNA polymerase (Fermentas),
and analyzed as described previously (Martins et al., 2005).
Primer sequences for reverse transcription, PCR and real-time
PCR amplification of

-actin, P2X and P2Y subtype coding
cDNAs, are listed in Table 1.
For purinergic receptor and

-actin cDNA amplification, the
PCR reaction was cycled 35 times (95 °C for 60 s, 55–64 °C for
60 s, and 72 °C for 60 s) plus a final extension of 10 min at 72 °C.
Real time PCR
Real time PCR was performed in order to quantify P2X and P2Y
receptor mRNA transcription in embryonal P19 cells and during
the course of differentiation of P19 cells to neurons. Total RNAs
(750 ng) were reverse-transcribed to cDNA, and the reaction
product was amplified by real time PCR on the 7000 Sequence
Detection System (ABI Prism, Applied Biosystems, Foster City,
CA, USA) using the Sybr-Green method The thermal cycling
conditions composed an initial denaturation step of 95 °C for 10
min and 50 cycles at 95 °C for 15 s and 60 °C for 1 min.
Experiments were performed in triplicate for each data point. P2X
and P2Y receptor subtype mRNA abundance was quantified as a
relative value compared with an internal reference,

-actin, whose
abundance remained unchanged under varying experimental con-
ditions. Primers used for real time PCR are specified in Table 1.
Analysis of relative P2X and P2Y receptor mRNA transcription
rates was done as detailed elsewhere (Livak and Schmittgen,
2001; Martins et al., 2005).
Immunofluorescence studies
For immunofluorescence detection, undifferentiated P19 cells or
EBs recovered after 48 h of induction to differentiation with RA
were seeded (2.5⫻10
5
cells/ml) in chamber slides containing six
wells (Laboratory-Tek
®
, Naperville, IL, USA). The cells were fixed
with 2% paraformaldehyde in PB for 10 min at 4 °C and then
dehydrated on a preheated platform at 37 °C. Primary antibodies
against stage-specific embryonic antigen (SSEA-1), nestin or NEL
at 1:50 dilutions in PB containing 0.3% Triton X-100 and 5%
normal goat or rabbit serum were added to the cells followed by
overnight incubation. Slides were washed three times for 2 min
each with 1 ml of PB containing 0.3% Triton X-100 and 5% normal
rabbit serum. Anti-NEL, -nestin and -SSEA-1 immunofluores-
cence was detected with anti-rabbit IgG-Alexa-Fluor 488 (Molec-
ular Probes, Eugene, OR, USA), anti-goat IgG-Cy5 (Zymed Tech-
nologies., Invitrogen), and anti-rabbit IgG-Cy3 (Abcam Inc., Cam-
bridge, MA, USA) secondary antibodies for 120 min at dilutions
recommended by the manufacturer at room temperature in PB
containing 2% rabbit serum albumin. Following washing of the
slides with PB, coverslips were mounted with Vectashield (Vector
Laboratories, Burlingame, CA, USA), and images were collected
using a Nikon DXM1200F Digital Camera coupled to an inverted
Axiovert 200 microscope (Zeiss, Jena, Germany), stored to a PC,
and analyzed with the software MetaMorph version 6.3r0.
Western blot assay
Undifferentiated P19 cells and cultures from days 4, 6 and 8 after
induction of neuronal differentiation were homogenized in ice-cold
lysis buffer containing 10 mM Tris–HCl (pH 7.5), 150 mM NaCl,
20 mM EDTA, 1% Triton X-100, 8 M urea and protease inhibitors.
Proteins were separated by SDS PAGE and transferred onto a
nitrocellulose membrane. The procedure of incubation with pri-
mary and secondary antibodies was essentially the same as
described by Martins et al. (2005). The following polyclonal anti-
bodies were used: anti-P2X
2
(1:500), anti-P2Y
1
(1:400), anti-P2Y
2
(1:200) and anti-

-tubulin (1:400), detecting proteins of 52–53,
42–45, 57– 60, or 60 kDa, respectively. The amount of loaded
proteins was in the 20–50
g/ml range.
Ca
2ⴙ
imaging by confocal microscopy
Undifferentiated and neuronal differentiated P19 cells on days 8
and 9 following induction with RA were loaded with 5
M Fluo-3
AM for 30 min at 37 °C in 140 mM NaCl, 3 mM KCl, 1 mM MgCl
2
,
2 mM CaCl
2
, 10 mM Hepes, 10 mM glucose at pH 7.4, in 0.5%
Me
2
SO and 0.1% of the nonionic surfactant pluronic acid F-127.
After loading with Fluo-3 AM, the cells were washed with incuba-
tion buffer and incubated for a further 20 min to ensure complete
deesterification of the dye. Ca
2⫹
imaging was performed with the
LSM 510 confocal microscope (Zeiss). Fluo-3-fluorescence emis-
sion was excited with a 488 nm line of an argon ion laser, and the
emitted light at 515 nm was detected using a bandpass filter.
Images were collected every 1.5 s. Following recording of 20
images of intracellular free calcium concentration [Ca
2⫹
]
i
of un-
stimulated cells, 50 or 100
M of ATP, 1.5 mM carbamoylcholine
(CCh) or 100
M of NMDA was added to the cells to stimulate
purinergic, cholinergic or glutamate–NMDA receptor responses,
respectively, and receptor-induced changes in [Ca
2⫹
]
i
were mon-
itored for further 3 min. At the end of each experiment, 5
MCa
2⫹
ionophore (4-Br-A23187) followed by 10 mM EGTA was used to
determine maximal (F
max
) and minimal (F
min
) fluorescence values,
respectively. Free cytosolic calcium concentration [Ca
2⫹
]
i
was
calculated from the Fluo-3 AM fluorescence emission using a
self-ratio equation as described previously (Martins et al., 2005),
assuming a K
d
of 450 nM for Fluo-3 AM-calcium binding (Hallett et
al., 1990). The Ca
2⫹
imaging data in the present study were
obtained at 20–22 °C. Concentrations were calculated for cell
populations containing at least each 10 cells in five to seven
independent experiments.
Inhibition of purinergic receptors during neuronal
differentiation
One micromole of each purinergic receptor antagonist PPADS,
reactive blue-2 or suramin was added on day 3 following induction
to differentiation. The respective inhibitor was again added to the
cell cultures with each medium change in intervals of 48 h until day
8 of differentiation. Control reactions were set up under identical
conditions in the absence of purinergic receptor antagonists. Two
hours prior to calcium measurements cells were washed with
fresh culture medium for removal of inhibitors.
Statistical analysis
Data are reported as mean values⫾standard errors (S.E.M.) from
data obtained in at least five independent experiments, with data
from each independent real time PCR experiment being deter-
mined at least in duplicate. For Ca
2⫹
measurements, the numbers
of independent experiments are indicated in the respective figure
legends. Statistical differences among groups having received
different treatment were evaluated by Student’s t-test or one-way
analysis of variance (ANOVA), followed by Tukey’s post hoc anal-
ysis. The level of significance was set to be at least P⬍0.05.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–1181 1171
RESULTS
P19 cells were induced to differentiate into neurons by
addition of RA and keeping them in suspension culture. As
modification of the originally published differentiation pro-
tocol (Jones-Villeneuve et al., 1982; Jonk et al., 1988; Berg
and McBurney, 1990), cells were differentiated in serum-
free defined medium in order to limit proliferation of fibro-
blasts and glial cells (Martins et al., 2005; da Silva et al.,
2007). In order to characterize the initial phases of differ-
entiation in defined medium, we have performed immuno-
Table 1. Primers for amplification of P2X and P2Y receptor subtype DNAs by RT-PCR and real-time PCR (qRT-PCR)
Gene Access number Primer Sequence (5=-3=) Length (bp)
qRT-PCR

-actina NM_007393 FWD CTG GCC TCA CTG TCC ACC TT 76
REV CGG ACT CAT CGT ACT CCT CTT
P2X
1
AF250121, X84896, NM_012997 FWD GAG AGT CGG GCC AGG ACT TC 233
REV GCG AAT CCC AAA CAC CTT CA
P2X
2
NM_153400, NM_053656 FWD TCC CTC CCC CAC CTA GTC AC 149
REV CAC CAC CTG CTC AGT CAG AGC
P2X
3
NM_145526, NM_031075 FWD CTT CCT AAC CTCACCGACAAG 150
REV AAT GCC CAG AAC TCC ACC C
P2X
4
NM_011026, NM_031594 FWD CCC ACT GCC TGC CCA GAT AT 145
REV ACA CTC ACC AAG GCA TAT GG
P2X
5
NM_033321, NM_080780 FWD GGA AGA TAA TGT TGA GGTTGA 81
REV TCC TGA TGA ACC CTC TCC AGT
P2X
6
X92070, NM_012721 FWD CCC AGA GCA TCC TTC TGT TCC 152
REV GGC ACC AGC TCC AGA TCT CA
P2X
7
NM_011027 FWD GCA CGA ATT ATG GCA CCG TC 171
REV CCC CAC CCT CTG TGA CAT TCT
P2Y
1
XM003033, NM_008772 FWD CGT GCT GGT GTG GCT CAT T 67
REV GGA CCC CGG TAC CTG AGT AGA
P2Y
2
XM002564, BC012104.1, AY136753.1 FWD TTC CTG CCA TTC CAC GTC A 73
REV TTG AGG GTG TGG CAG CTG A
P2Y
4
X91852, NM_020621 FWD TGT CCT TTT CCT CAC CTG CAT 63
REV TGC CCG AAG TGG GYG G
P2Y
6
X97058, AF298899 FWD CCT GCC CAC AGC CAT CTT 67
REV GGC TGA GGT CAT AGC AGA CAG TG
RT-PCR
P2X
1
AF250121, X84896, NM_012997 FWD AGG ATA CCA GAC CTC AAG TG 701
REV GGA GAC AGG TTC TTC TCC C
P2X
2
NM_153400, NM_053656 FWD CAA AGT GTG GGA CGT GGA G 700
REV CAT AGG CTT TGA TGA GAG TTC
P2X
3
NM_145526, NM_031075 FWD GGC CGC TGC GTG AAC TAC 665
REV CTG GCT TTG TAG TGA TCA GC
P2X
4
NM_011026, NM_031594 FWD CTG TTC GAG TAC GAC ACG C 860
REV CCA AAC ACG ATG ATG TCA AAG
P2X
5
NM_033321, NM_080780 FWD CTG ATA AAG AAG AGT TAT CAG G 936
REV GAG GTA GAT AAG TAC CAG GTC
P2X
6
X92070, NM_012721 FWD CGA TTC ACT CTC CAG TCC G 427
REV GGT CCT CCA GTA GAA ACC G
P2X
7
NM_011027 FWD GGT ATC GAG ATC TAT TGG GA 1019
REV TTG AAG CCA CTG TAC TGC CC
P2Y
1
XM003033, NM_008772 FWD CCA GAA ATG TGT GAT TTC AAC 365
REV G CAC ACA CTG GTC TTT TGG
P2Y
2
XM002564, BC0121041, AY136753.1 FWD ACC CGC ACC CTC TAC TAC T 175
REV GCT TGG CAT CTC GGG CAA A
P2Y
4
X91852, NM_020621 FWD GTC CCT GGA CTG GAC TAA G 391
REV GG ACA CTG CAG TAG AGG TTC
P2Y
6
X97058, AF298899 FWD CTT CCA TCT TGC ATG AGA C 379
REV GGC ATA GAA GAG GAA GCG

-
Actin
NM_007393 FWD AGG AAG AGG ATG CGG CAG TGG 535
REV CGA GGC CCA GAG CAA GAG AG
FWD, forward primer; REV, reverse primer.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–11811172
fluorescence studies using antibodies against proteins
specific for undifferentiated cells, progenitor cells and dif-
ferentiated neurons (Fig. 1). On day 6 following induction
of differentiation, more than 95% of the cells in the culture
did not express any more SSEA-1 as a marker of undiffer-
entiated cells (Wu and Chow, 2005). A peak of nestin
expression as a marker for neural progenitors expressed in
almost every cell (Ho and Liem, 1996) was observed on
day 4 of differentiation. Nestin expression was down-reg-
ulated when differentiating P19 cells underwent final dif-
ferentiation from days 6– 8. At this stage, most cells ex-
pressed NEL (Fig. 1), NF-160 (data not shown) and NF-
200 (Martins et al., 2005).
Expression of purinergic receptors in P19 cells
during neuronal differentiation
We have studied induction of gene and protein expression
of P2X and P2Y receptor subunits during P19 cell differ-
entiation into neuronal progenitor cells followed by matu-
ration of functional neurons. cDNAs reverse transcribed
from total RNAs of cells collected on days 0– 8 of differ-
entiation were amplified by PCR in the presence of P2X- or
P2Y-subunit specific primers. DNA sequencing confirmed
homologies of the amplified cDNAs with mouse purinergic
receptor cDNA sequences available in the GenBank
(http://www.ncbi.nlm.nih.gov/). Relative mRNA transcrip-
tion of purinergic receptor subtypes was quantified by real-
time PCR.
Gene expression of P2X receptor subunits was regu-
lated during the course of differentiation. Gene expression
of P2X
2
, P2X
7
(Figs. 2 and 3)and P2X
6
subunits (da Silva
et al., 2007) increased when cells differentiated into pro-
genitor cells and neurons.
P2X
3
and P2X
4
receptor gene expression being up-
regulated in undifferentiated cells decreased when cells
became functional neurons. Gene expression of P2X
1
and
P2X
5
receptors could not be detected in embryonic P19
cells and was only present at basal levels following induc-
tion of differentiation by RA. Transcription rates of mRNAs
coding for metabotropic purinergic P2Y
1
and P2Y
4
recep-
tors were high in the initial stage, but then decreased
during ongoing differentiation. Gene expression of P2Y
2
and P2Y
6
receptors already present in undifferentiated
cells augmented during the differentiation process.
Immunohistochemical detection of P2X and P2Y re-
ceptor proteins was performed with undifferentiated cells,
cells of the neural progenitor stage day (day 4), during final
differentiation (day 6) and differentiated neurons (day 8)
(Fig. 4A). Protein expression of P2X
2
, P2X
3
, P2X
4
and
P2X
6
receptor subunits was in agreement with mRNA
transcription levels as determined by real-time PCR. How-
ever, P2X
5
and P2X
7
receptor proteins could not be de-
tected in any stage of differentiation. These results were
confirmed by Western-blot analysis (data not shown).
P2Y
1
and P2Y
4
receptor protein expression decreased
when cells underwent differentiation, whereas expression
levels of P2Y
2
and P2Y
6
receptor proteins remained un-
changed during the period of differentiation. Expression of
P2X
2
, P2Y
1
and P2Y
2
receptors was confirmed by West-
ern blot.
P2X
1
and P2X
4
receptor immunoreactivity was only
present in a part of the cell population. Immunohistochem-
Fig. 1. Detection of cell phenotype during neuronal differentiation of P19 cells. Expression of SSEA-1, nestin and NEL in undifferentiated and
RA-treated P19 cells on days 4, 6 and 8 of differentiation was determined by immunofluorescence procedures as detailed in the Experimental
Procedures section.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–1181 1173
ical staining of the P2X
4
receptor subunit in undifferenti-
ated P19 cells was the strongest when compared with that
of other P2X and P2Y receptor subtypes. On day 4 of
differentiation, cells in the progenitor stage showed pro-
nounced immunostaining for P2X
2
and P2X
6
ionotropic as
well as for P2Y
1
, P2Y
2
, P2Y
4
and P2Y
6
metabotropic
receptors. In this stage a reduction of expression of P2X
4
receptor subunits was observed, and low immunostaining
for P2X
1
receptor subunits was detected. However, on the
sixth day of differentiation P2X
4
subunit expression in-
creased again. Increased expression was also observed
for the P2X
6
receptor subunit. At the same time, a strong
reduction of P2X
3
and P2Y
4
receptor expression was no-
ticed. Expression levels of the other purinergic receptors
maintained when embryonic cells transformed to neural
progenitors cells. The fact that some receptors appeared,
such as P2X
1
subtypes, and the expression of others
became below detection limit, such as P2X
3
, P2Y
1
and
P2Y
4
receptors, may be explained by phenotype modifica-
tions which occur during the progress of differentiation. In
differentiated neurons no immunostaining against P2X
1
,
P2X
3
, P2Y
1
and P2Y
4
receptors could be observed. P2X
4
receptor expression was reduced when compared with its
expression level on day 6.
Purinergic receptor elicited elevation in [Ca
2ⴙ
]
i
in
P19 cells
Calcium imaging experiments were carried out to verify
purinergic receptor activity in undifferentiated P19 and P19
neurons on day 8 of differentiation (Fig. 5). These experi-
ments were performed in extracellular media with 2.5 mM
calcium or without extracellular calcium in the presence of
1 mM EGTA. In the presence of extracellular calcium,
application of 100
M ATP to the cells induced a transient
elevation of [Ca
2⫹
]
i
. In calcium-free extracellular medium
containing 1 mM EGTA, ATP-induced calcium responses
were reduced when compared with those obtained in cal-
cium measurements performed in the presence of extra-
cellular calcium. The typical response observed in the
presence of P2X receptor activity including a peak of
[Ca
2⫹
]
i
elevation and a following plateau of the calcium
response (Wood et al., 1975) was abolished in the ab-
sence of extracellular calcium. The calcium responses
obtained in calcium-free extracellular medium were similar
to typical calcium responses elicited by G-protein-coupled
seven transmembrane receptors (An et al., 1999).
The application of 100
M ATP induced elevations of
[Ca
2⫹
]
i
of 367⫾14 nM (n⫽70) and 625⫾36 nM (n⫽72) in
undifferentiated P19 cells and P19 neurons on day 8 of
differentiation, respectively (P⬍0.0001). Due to inhibition
of P2X receptor participation, the ATP-induced calcium
response in undifferentiated and neuronal-differentiated
P19 cells in the absence of extracellular calcium was re-
duced to 36⫾1% (n⫽65) and 59⫾3% (n⫽68) of the re-
sponses obtained in calcium-containing medium, respec-
tively (P⬍0.0001).
Cholinergic and NMDA receptor activity in the
presence of purinergic antagonists during
maturation of neural progenitor to neurons
P19 cells on days 8 and 9 following induction to differen-
tiation responded with a transient increase in [Ca
2⫹
]
i
to
CCh, NMDA (Martins et al., 2005; Ulrich and Majumder,
2006) and ATP application (Figs. 5 and 6). In order to verify
the role of purinergic receptor signaling in directing differ-
entiation from the neural progenitor stage on day 3 to
neurons, P19 cell differentiation was performed in the
presence of 1
M of each purinergic antagonist PPADS,
reactive blue-2 or suramin. PPADS strongly inhibits iono-
tropic P2X
2
receptors, and is a less potent inhibitor of
metabotropic P2Y
1
receptors. PPADS inhibits to an even
lower degree P2Y
2
, P2Y
4
and P2Y
6
metabotropic puriner-
gic receptor subtypes. Reactive blue-2 is a partial inhibitor
Fig. 2. Detection of the gene expression profile of P2X and P2Y
receptor subtypes during neuronal differentiation of P19 cells by RT-
PCR. Gene expression of P2X and P2Y receptors was detected by
RT-PCR using total RNA extracted from various days of differentiation.
RT-PCR as detailed in the Experimental Procedures section was
performed in the presence of primers coding for P2X
1
–P2X
7
and
P2Y
1,2,4,6
subtypes. Amplified DNA fragments were confirmed by DNA
sequencing to match to purinergic receptor subtypes cDNAs. Total
RNAs isolated from mouse brain (MB) were used as positive controls
for RT-PCR procedures. RT-PCR reactions with

-actin (Act) mRNA
were employed to verify the integrity of respective isolated total RNAs.
The amplification of cDNAs of various sizes resulted from alternative
splicing of mRNAs coding for P2X
1
, P2X
4
and P2Y
6
receptor subtypes.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–11811174
of P2Y
6
receptors and to a less degree of P2Y
2
and P2Y
4
receptors. This purinergic receptor antagonist does not
inhibit P2Y
1
receptors. Suramin partially inhibits P2X
2
, P2Y
1
and P2Y
2
receptors, is less effective in blocking P2Y
4
and
P2Y
6
receptors and does not have any effect on P2X
6
recep-
tors (Kennedy, 1990; Lambrecht, 2000; von Kugelgen and
Wetter, 2000; von Kugelgen, 2006).
The activities of purinergic receptors, cholinergic and
NMDA-glutamate receptor in inducing calcium transients
were evaluated in P19 neurons on day 8 or 9 of differen-
tiation. Representative images of changes in [Ca
2⫹
]
i
fol-
lowing receptor stimulation by their respective ligands in
P19 cells which had been differentiated to neurons in the
absence or presence of purinergic receptor antagonists
are shown in Fig. 6A. No inhibition of purinergic receptor
activity was observed when cells had been differentiated in
the presence of 1
M PPADS (Fig. 6A, B).
The CCh-induced cholinergic receptor response, how-
ever, was inhibited when reactive blue-2 had been present
during neuronal differentiation. NMDA-induced calcium
transients were absent in cells differentiated in the pres-
ence of suramin (Fig. 6A, C). The presence of reactive
blue-2 or PPADS during differentiation also resulted in a
significant inhibition of NMDA-receptor activity (Fig. 6A, D).
Fig. 3. Quantification of purinergic receptor mRNA transcription during neuronal differentiation of P19 cells by real-time PCR. Relative expression
levels were corrected with the internal control

-actin. The graphics show P2X and P2Y mRNA transcription levels in differentiating P19 cells compared
with those in undifferentiated P19 cells which were normalized to 1. Cells at day 8 of differentiation were pretreated on day 6 with cytosine arabinoside,
in order to avoid contamination of the cell cultures with increasing concentrations of proliferating glial cells. Error bars indicate mean values⫾S.E.M.;
*P⬍0.05 compared with control data, ** P⬍0.0001 compared with control data.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–1181 1175
Fig. 4. Detection of expression profiles of neuronal purinergic receptors during neuronal differentiation of P19 cells by immunohistochemical staining.
(A) Following fixation, cells were incubated with the subtype-specific primary antibody followed by addition of a biotinylated secondary antibody. The
reaction was revealed by immunoperoxidase staining. Purinergic receptor subtype expression was verified in undifferentiated P19 cells (Und; left
panels) and P19 cells on days 4 and 6 (middle panels) and 8 (right panels) following induction to neuronal differentiation. (B) Detection of P2Y
1
, P2Y
2
,
P2X
2
receptor expression on days 0– 8 of differentiation was performed by Western blot analysis as described in the Experimental Procedures section
and by Martins et al. (2005).

-Actin expression was detected as internal control for integrity of the protein extract.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–11811176
Taking into account the expression of P2X
1–4
, P2X
6
,
P2Y
1
, P2Y
2
, P2Y
4
and P2Y
6
purinergic receptors in the
neural progenitor stage of P19 cells and the pharmacolog-
ical properties of the inhibitors of purinergic receptors, our
results suggest that inhibition of P2Y
1
and possibly P2X
2
receptors along with the differentiation of P19 neural pre-
cursor cells to neurons resulted in loss of NMDA receptor
activity, whereas inhibition of metabotropic P2Y
2
and pos-
sibly P2X
2
receptors led to inhibition of cholinergic receptor
responses. The possible participation of P2Y
11–14
metabo-
tropic purinergic receptor subtypes in the process of neu-
ronal differentiation has not been evaluated.
DISCUSSION
We have determined P2X and P2Y receptor gene and
protein expression throughout in vitro differentiation of P19
EC cells in order to characterize changes in purinergic
receptor expression when undifferentiated cells transform
into neural progenitor cells and finally functional neurons.
Primary cultures of astrocytes (Fumagalli et al., 2003),
oligodendrocytes (Agresti et al., 2005), as well as estab-
lished cell lines such as rat pheochromocytoma cells
(PC12) (Arslan et al., 2000) were previously used for
studying purinergic receptor expression. Neural progenitor
cells as well as astrocytes and oligodendrocytes were
shown to express enzymes involved in hydrolysis of nu-
cleoside tri- and diphosphates such as ecto-nucleoside
triphosphate diphosphohydrolase 2 (NTPDase2), and
ecto-ATPase (Shukla et al., 2005) and express high
amounts of purinergic receptors (Fumagalli et al., 2003;
Agresti et al., 2005) suggesting participation of the puri-
nergic system in differentiation.
The involvement of ATP-induced calcium fluxes in the
regulation of cell proliferation and neuronal differentiation
was observed in adult neural progenitor cells in the sub-
ventricular zone progenitor and progenitor cells of the den-
tate gyrus of the hippocampus (Mishra et al., 2006; Hogg
et al., 2004). Moreover, Mishra et al., 2006 showed that
nucleotides participate in growth-factor-mediated induction
of proliferation. The constitutive release of ATP and mainly
P2Y
1
receptor activation are suggested to enhance epider-
mal growth factor and fibroblast growth factor-2-induced
neurosphere cell proliferation. In neurospheres, P2Y
1
re-
ceptors had the highest impact on Ca
⫹2
signaling with
strong participation of P2Y
2
receptors. The presence of a
specific P2Y
1
inhibitor or knockdown of P2Y
1
receptor
gene expression in neurospheres resulted in reduction of
both ATP-induced calcium transients and cell proliferation.
These data indicate a crucial function for P2Y1 receptor in
neural progenitor cells.
P19 cells passing all developmental stages from em-
bryonic cells to mature neurons and expressing along their
differentiation neuronal marker proteins identical to those
of developing neurons in vivo, are an adequate model for
studying purinergic receptor expression and activity, as
they endogenously express almost every purinergic recep-
tor subtype. As P19 cell cultures can be easily expanded,
they have advantages over the use of primary cultures in
large-scale assays.
Using the P19 cell line as an in vitro model for neuronal
differentiation, we have found that P2X
2
, P2X
6
, P2Y
2
and
P2Y
6
receptor gene and protein expression increases dur-
ing ongoing differentiation while P2X
3
, P2X
4
, P2Y
1
and
P2Y
4
receptor expression decreases at the same time.
P2X receptor expression was high in undifferentiated P19
cells, but decreased when cells differentiated to neural
progenitor cells and increased again during final matura-
tion of P19 neurons. The in vitro–observed pattern of
purinergic receptor expression during neuronal differenti-
ation is largely in agreement with that found in specified
regions and cell types in developing rat brain (Cheung et
al., 2005; Xiang and Burnstock, 2005).
Undifferentiated and neuronal-differentiated P19 cells
express functional P2X and P2Y receptors as determined
by single-cell calcium imaging. In undifferentiated cells
ionotropic P2X receptors were the major component con-
tributing to the ATP-induced calcium response. Neuronal-
differentiated P19 cells responded with higher elevations in
Fig. 5. Elevation of [Ca
2⫹
]
i
in embryonal and P19 neurons on day 8 of
differentiation induced by activation of P2X and P2Y receptors. Cells
were loaded with the calcium-sensitive dye Fluo-3 AM. [Ca
2⫹
]
i
was
monitored by single-cell calcium imaging using confocal microscopy.
Following stimulation with 100
M ATP and subsequent recording of
changes in [Ca
2⫹
]
i
, cells were washed with culture medium, and a time
interval of at least 5 min was kept between measurements to allow for
receptor resensitization. Arrows indicate time points of agonist appli-
cation. Application of 100
M ATP supposed to result in maximal
purinergic receptor activation evoked a rapid and transient increase in
[Ca
2⫹
]
i
in undifferentiated cells (Und) and differentiated cells on day 8.
Upper panels: Embryonic and neuronal differentiated P19 cells were
stimulated either by ATP alone or by 100
M ATP in the presence of
1 mM EGTA. Lower panels indicate values of [Ca
2⫹
]
i
increases ob-
tained in the presence of ATP or ATP and EGTA. The data shown are
mean values⫾S.E.M. of seven independent experiments consisting of
about 8 cells for each experiment, resulting in a total of 70–72 cells. **
P⬍0.0001 compared with control data.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–1181 1177
Fig. 6. Effects of the presence of purinergic receptor antagonists on ATP-, CCh- and NMDA-induced calcium flux in neuronal-differentiated P19 cells.
P19 cells were differentiated from day 3 on with 1
M concentrations of suramin, PPADS or reactive blue-2 (RB-2). Control experiments were set up
in the absence of purinergic receptor antagonists. On day 8 of differentiation, cells were loaded with Fluo-3 AM and calcium imaging was performed
by confocal microscopy. Following imaging variations in [Ca
2⫹
]
i
of an unstimulated cell population, agonist was added and cell stimulation was
recorded for further 3 min. (A) Representative images of calcium responses induced by 50
M ATP, 1.5 mM CCh or 100
M NMDA in the presence
or absence of PPADS, RB-2 or suramin. (⫺) Unstimulated cells. (⫹) Maximal stimulation observed following receptor stimulation with the respective
ligand. At the end of each experiment, 4-Br-A23187 (4-Br-A) was added to verify cell viability and maximal Ca
2⫹
responses. Panels (B) to (D) represent
results obtained in five independent experiments analyzing each 39–51 cells. (B) 50
M ATP; (C): 1.5 mM CCh or (D): 100
M NMDA in the absence
of inhibitor (1), in the presence of 1
M suramin (2), 1
M PPADS (3), or 1
M RB-2 (4). Free cytosolic calcium concentrations [Ca
2⫹
]
i
were calculated
from relative fluorescence values as detailed in the Experimental Procedures section. Error bars indicate mean⫾S.E.M.; * P⬍0.05 compared with
control data, ** P⬍0.0001 compared with control data. Transm., transmission.
R. R. Resende et al. / Neuroscience 146 (2007) 1169–11811178
[Ca
2⫹
]
i
upon ATP application when undifferentiated cells
did. However, in contrast to undifferentiated cells, in neu-
ronal-differentiated P19 cells more than 50% of the ATP-
induced calcium mobilization was due to metabotropic P2Y
receptor activation. In agreement with our data, a change
in the profile of expression of metabotropic and ionotropic
purinergic receptors was also observed during neuronal
differentiation of PC12 pheochromocytoma cells. Neuro-
nal-differentiated PC12 cells were more susceptive to in-
hibition by PPADS than undifferentiated PC12 cells were,
leading to the conclusion that P2X receptors including the
P2X
4
subtype were expressed in the undifferentiated stage
and P2Y receptor expression increased when PC12 cells
became neurons (Arslan et al., 2000).
Based on purinergic receptor pharmacology, reviewed
by von Kugelgen (2006), Kennedy (1990), and Lambrecht
(2000), the inhibition of P2Y
1
and possibly P2X
2
receptor
activity along the differentiation of P19 neural precursor
cells to neurons resulted in loss of NMDA receptor-induced
calcium response, whereas inhibition of P2Y
2
and possibly
P2X
2
receptors led to inhibition of cholinergic receptor
responses. As shown elsewhere (Ralevic and Burnstock,
1998), PPADS is active only on P2X
2
but not on P2X
4
receptors and exerts its inhibitory action by reducing tran-
sient Ca
2⫹
-signals. As P2X
2
mRNA and protein levels
increased during neuronal differentiation, we believe that
inhibition of P2X
2
receptors mostly contributed to the ab-
sence of NMDA-induced [Ca
2⫹
]
i
-response in differentiated
neurons. It is also possible that other purinergic receptor
subunits could associate with each other forming active
heteromeric P2X receptors, resulting in pharmacological
analysis to differ from structural analysis. For instance,
P2X
2
and P2X
3
subunit associations have been shown to
produce functional ATP-gated channels (Lewis et al.,
1995; Cook et al., 1997). The formation of heteromeric
channels of all possible combinations, except combining
P2X
4
and P2X
7
subtypes, were observed following cell
transfection with recombinant receptor subunits. Neverthe-
less, the predominant encountered P2X receptor is formed
by homomeric subunits (Torres et al., 1999). PPADS at the
concentration used is a partial inhibitor of the P2Y
1
recep-
tor with an IC
50
in the low micromolar range. For the
remaining P2Y receptors, IC
50
values larger than 100
M
were determined for PPADS-induced receptor inhibition
(von Kugelgen and Wetter, 2000). Suramin at a concen-
tration of 1
M is a partial antagonist for P2Y
1
, P2Y
2
and
P2Y
11
receptors (von Kugelgen and Wetter, 2000; Ralevic
and Burnstock, 1998). The inhibition obtained in the pres-
ence of 1
M reactive blue-2 could in principle be due to
P2Y
6
receptor blockade. However, the presence of
PPADS which at 1
M concentration is not an inhibitor of
P2Y
6
receptors (von Kugelgen and Wetter, 2000) also
resulted in inhibition of the CCh-induced [Ca
2⫹
]
i
-response
in differentiated P19 cells, excluding it from the inhibited
receptor group. Thus, the data obtained at the used an-
tagonist concentrations, indicate the involvement of P2Y
1
,
P2Y
2
and possibly P2X
2
receptors in the development of
functional cholinergic and glutamate-NMDA receptors in
differentiated P19 cells. Further investigations on detailed
pharmacology of these receptors were, however, beyond
the scope of the present study. However, final evidence for
the participation of P2Y
1
, P2Y
2
and P2X
2
receptors could
be obtained in in vitro differentiation experiments during
which expression of these receptors is specifically down-
regulated as a consequence of RNA interference
(RNA
i
). The RNA
i
approach could be used to knock
down the gene expression of the participating purinergic
receptors individually or in combination. Further possi-
bilities in studying the functions of P2Y
1
, P2Y
2
and P2X
2
receptors lie in the development of DNA- or RNA aptam-
ers as purinergic receptor subtype-specific inhibitors.
It is well established that neurotransmitters through
activation of their respective receptors trigger a specific
program of calcium transients inside the cell as prerequi-
sites for the progress of neuronal differentiation (Gu and
Spitzer, 1995). As demonstrated elsewhere, P2Y
1
recep-
tors were present in undifferentiated mesenchymal cells
and disappeared during later development, suggesting an
important role for these receptors in embryonic cells and
during the differentiation process (Meyer et al., 1999). In
human hematopoietic stem cells, only P2Y
1
and P2Y
2
receptors subtypes were expressed (Lemoli et al., 2004).
Scemes et al. (2003) demonstrated a role for P2Y
1
recep-
tors in migration of neural progenitor cells, whereas Arthur
et al. (2005) showed that P2Y
2
receptor was involved
in enhanced neuronal differentiation, corroborating our
results.
Our data suggest that ionotropic and metabotropic pu-
rinergic receptors participate in the regulation of cellular
processes during neuronal differentiation events, and
thereby are involved in the expression pattern and activity
of other metabotropic and ionotropic receptors which are
specific for the differentiation state of the cells. The obser-
vation that NMDA and acetylcholine receptor activity is af-
fected when cells were differentiated in the presence of in-
hibitors of purinergic receptor activity indicates that in the
presence of these inhibitors no functional synapses have
been formed. In this regard our data are in agreement with
the hypothesis that a network of ion channels and metabo-
tropic receptors is necessary for triggering differentiation to a
correct neuronal phenotype. This observation is also valid for
other systems. For instance, the inhibition of kinin-B2 recep-
tors by their specific inhibitor HOE-140, D-Arg-L-Arg-L-Pro-
L-Hyp-Gly-L-(2-thienyl)Ala-L-Ser-D-1,2,3,4-tetrahydro-3-
isoquinolinecarbonyl-L-(2
␣
,3

,7a

)-octahydro-1H-indole-2-
carbonyl-L-Arg during neuronal differentiation of P19 cells led
to a decrease in gene expression and activity of muscarinic
and possibly nicotinic acetylcholine receptors in P19 neurons
(Martins et al., 2005).
Intrinsic modulation of activity between purinergic,
muscarinergic and NMDA receptors was shown in the
developing retina of embryonic chicken and in adult hip-
pocampal neurons (Sakaki et al., 1996; Fujii et al., 2002;
Fujii, 2004; Wirkner et al., 2006). Regulatory pathways
between NMDA and purinergic receptors were noticed in
the presence of the NMDA antagonist D,L-2-amino-5-phos-
phonovalerate resulting in inhibited purinergic receptor ac-
tivity in CA1 neurons (Fujii et al., 2002; Fujii, 2004). In V
R. R. Resende et al. / Neuroscience 146 (2007) 1169–1181 1179
pyramidal neurons of the rat prefrontal cortex, NMDA-
induced receptor responses were increased in the pres-
ence of ATP and UTP, suggesting that purinergic PY or
P2X receptors contribute to the positive modulation of
NMDA receptor activity (Wirkner et al., 2006). Our data
suggest that the synergism of receptor function exists be-
tween purinergic and NMDA and cholinergic receptors.
The inhibition of P2Y
1
and possibly P2X
2
receptors in P19
neural progenitor cells resulted in P19 neurons with no
NMDA receptor activity and blockade of P2Y
2
and possibly
P2X
2
receptor in P19 neurons with inhibited cholinergic
receptor activity.
CONCLUSION
In summary, we have shown that the purinergic receptor
signaling is involved in earlier stages of cellular fate deter-
mination. Both, undifferentiated and neuronal-differenti-
ated P19 cells express functional P2X and P2Y receptors.
Purinergic receptor expression pattern during in vivo neu-
ronal development is similar that observed during the
course of neuronal differentiation of P19 cells making this
cell line an excellent model for studying purinergic recep-
tors in developmental processes. In summary, our data
suggest that ATP-induced calcium mobilization through
activation of purinergic P2X and P2Y receptors partici-
pates in differentiation of P19 neural progenitor to neurons,
where blockade of purinergic P2X
2
and P2Y
1
receptor
activity affects NMDA receptor function and metabotropic
P2Y
2
and the P2X
2
receptor is necessary for maintaining
cholinergic receptors in differentiated neurons.
Acknowledgments—H.U. and L.R.G.B. are grateful for grant sup-
port from FAPESP (Fundação de Amparo à Pesquisa do Estado
de São Paulo) and CNPq (Conselho Nacional de Desenvolvi-
mento Científico), Brazil. R.R.R’s. and K.N.G.’s Ph.D. theses are
supported by CAPES (Coordenação de Aperfeiçoamento de Pes-
soal de Nível Superior), Brazil. P.M.’s Ph.D. research was sup-
ported by a fellowship from FAPESP. The Center for Applied
Toxinology (CAT-CEPID), Butantan Institute, São Paulo, Brazil, is
acknowledged for performing DNA sequencing. We wish to thank
Prof. H. C. Schaller, Center for Molecular Neurobiology, University
of Hamburg, Germany, for giving us the P19 cell line. Adilson da
Silva Alves, Department of Physiology and Biophysics, Instituto de
Ciências Biomédicas, Universidade de São Paulo, São Paulo,
Brazil, is acknowledged for helping us with immunohistochemistry
procedures.
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(Accepted 12 February 2007)
(Available online 5 April 2007)
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