Cell Calcium 43 (2008) 107–121
Mechanism of acetylcholine-induced calcium signaling during
neuronal 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 Bioqu´ ımica, Instituto de Qu´ ımica, Universidade de S˜ ao Paulo, Av. Prof. Lineu Prestes 748, 05508-900 S˜ ao Paulo, SP, Brazil
b
Departamento de Fisiologia e Biof´ ısica, Instituto de Ciˆ encias Biom´ edicas, Universidade de S˜ ao Paulo, Paulo, 05508-900 S˜ ao Paulo, SP, Brazil
Received 26 September 2006; received in revised form 12 April 2007; accepted 13 April 2007
Available online 26 July 2007
Abstract
Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal
development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma
embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity of
2
–
7
,
2
,
4
nAChR and M1–M5
mAChR subtypes during neuronal differentiation. Nicotinic
3
and
2
mRNA transcription was induced by addition of retinoic acid to
P19 cells. Gene expression of
2
,
4
–
7
,
4
nAChR subunits decreased during initial differentiation and increased again when P19 cells
underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca
2+
flux was observed in embryonic and neuronal-
differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The
nAChR-induced elevation of intracellular calcium ([Ca
2+
]
i
) response in undifferentiated cells was due to Ca
2+
influx. In differentiated P19
neurons the nAChR-induced [Ca
2+
]
i
response was reduced following pretreatment with ryanodine, while the mAChR-induced response was
unaffected indicating the contribution of Ca
2+
release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca
2+
responses.
The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca
2+
waves during initial
neuronal differentiation.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Neuronal differentiation; P19 embryonal carcinoma cells; Acetylcholine receptors; Ryanodine receptors; IP
3
receptors; Ca
2+
signaling
1. Introduction
The development of the nervous system is one of the most
important morphogenetic events occurring in the embryo.
This process is accompanied by cell proliferation and dif-
ferentiation as well as by tissue organization into a specific
architecture. Although the specific molecular pathways that
drive these events remain unresolved, it is widely believed
that proliferation and differentiation programs of the neural
∗
Corresponding author. Tel.: +55 11 3091 3810x223;
fax: +55 11 3815 5579.
E-mail address: henning@iq.usp.br (H. Ulrich).
1
Present address: Department of Biological Sciences, Columbia Univer-
sity, Amsterdam Av. 1212, New York, NY 10027, USA.
progenitors require the interaction of extrinsic and intrinsic
signals. While the function of growth factors in controlling
neuronal differentiation is well documented, there also is
increasing persuasive evidence for a role of neurotransmit-
ters and their respective receptors in this process [1,2]. Many
neurotransmitters are already present in the brain prior to
axonogenesis and synaptogenesis, raising the possibility that
they may mediate non-classical signaling. One such neuro-
transmitter is acetylcholine (ACh), whose biological actions
are mediated by both nicotinic (nAChRs) and muscarinic
receptors (mAChRs). Neuronal nAChRs are heterogeneous,
with at least six (
2
–
7
) and three (
2
–
4
) subunits
(reviewed in [3]). Nicotinic receptor subunits are among the
first membrane proteins to appear during central nervous sys-
tem (CNS) development, and their initial expression does not
0143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2007.04.007