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Nucleotide effects are mediated trough two subclasses
of specific cell surface receptors, ionotropic P2X and
metabotropic P2Y receptors (4). Seven distinct P2X
subtypes (P2X1-7) have been cloned from mammalian
species, showing a topology that includes two
transmembrane spanning regions, a large extracellular loop
and intracellular N and C termini. These proteins assemble
into ATP activated channels either as homomers or
heteromers, each functional receptor containing at least
three monomers. P2X receptor activation results in Na+
and Ca2+ influx across the cell membrane, which leads to
depolarization. Membrane depolarization subsequently
activates voltage-operated calcium channels, thus causing
accumulation of Ca2+ ions in the cytoplasm. Although the
detailed signalling mechanism have not been established
for most P2X receptor subtypes, it is well known that
cytoplasmic Ca2+ elevation triggers a variety of
intracellular events, in part, trough activation of mitogen-
activated protein kinases (MAPKs), protein kinase C
(PKC) and calmodulin (5-7).
The P2Y receptors are classical 7-transmembrane
domain metabotropic receptors coupled to G proteins.
Currently, eight P2Y receptors subtypes (P2Y1, 2, 4, 6, 11, 12,
13, 14) have been cloned. P2Y1, 11, 12, 13 receptors are
selectively activated by adenine nucleotides, whereas P2Y4
and P2Y6 are stimulated by pyrimidines. The P2Y2
receptor responds equally well to purine and pyrimidine
triphosphates (ATP and UTP) and P2Y14 is a sugar-
nucleotide (UDP-glucose) responding receptor. Most
members of the P2Y family (P2Y1, 2, 4, 6, 11) are functionally
coupled to Gq/11 proteins and stimulate phosphoinositide-
specific phospholipase C, resulting in the formation of
inositol-(1,4,5)-trisphosphate and diacylglycerol with
subsequent mobilization of Ca2+ from internal stores.
P2Y11 receptor additionally stimulates adenylate cyclase,
whereas P2Y12, P2Y13 and P2Y14 promote Gi/0 inhibition of
adenylate cyclase activity. The stimulation of several P2Y
receptors is also commonly associated with the activation
of MAPKs (in particular extracellular signal-regulated
protein kinase 1/2, ERK1/2). Other classes of protein, such
as phospholipases A2 and D, PKC or phosphatidylinositol
3-kinase, can be also activated according to the cell
context and the particular P2Y subtype (5, 8, 9).
Several neuroblastoma cell lines, each originating from
a single cell that has been isolated from a spontaneously
occurring or induced tumor of the nervous system, are now
available and can be maintained in culture infinitely.
Neuroblastoma derived cell lines have been used as an in
vitro model for the study of a variety of neurotransmitter
receptors including those for nucleotides. Indeed, the P2Y2
receptor was originally cloned from the mouse
neuroblastoma/rat glioma hybrid line NG108-15 (10).
Further studies have revealed the presence in these cells of
the P2X7 receptor and metabotropic receptors other than
the P2Y2 subtype (11, 12). Functional P2X7 receptors can
be also detected in different human neuroblastoma lines
(13, 14) as well as in the mouse Neuro-2a neuroblastoma
cells (6, 15). In addition, a range of cell lines of both
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Javier Gualix et al.
mouse and human neuroblastoma origin have been shown
to functionally express pyrimidine selective P2Y receptors
(16-18).
SK-N-MC human neuroblastoma are known to
constitutively express adrenergic a2C, ß1 and ß3 (19, 20),
dopamine D1 (21), neuropeptide Y1 (22) and muscarinic
M1 receptors (23). The regulation and signalling pathways
activated by these receptors have been extensively
investigated using SK-N-MC cells as a model (24-28). SK-
N-MC cell line also served to analyze the integration or
cross-communication of signals upon activation of
different types of neurotransmitter receptors (29). More
recently, SK-N-MC has been used as a parental line with
which generate cellular lines that stably overexpress
proteins of interest in the study of distinct
neurodegenerative disorders, such as the amyloid
precursor protein APP (30), carboxyl-terminal fragments
of the APP protein (31, 32), or different mutated forms of
the Parkin protein (33).
Earlier experiments showed that ATP and other
nucleotides are able to induce response (phosphoinositide
hydrolysis and formation of inositol phosphates) in the
SK-N-MC cells, thus indicating the presence of not fully
characterized P2 receptors in this neuroblastoma cell line
(23).
In the present study we have analyzed the presence of
functional P2 receptors in the human neuroblastoma SK-
N-MC cells. If such receptors were present in these cells,
SK-N-MC cell line could serve as a useful model to
analyze several aspects of the signalling mediated through
nucleotide receptors in neural tissues. This includes the
regulation and transduction pathways activated by P2
receptors, their interaction with other neurotransmitter
receptors or their putative relationship with proteins
involved in neurodegenerative processes
2. MATERIALS AND METHODS
2.1. Materials
2-methylthyo-adenosine-5'-diphosphate (2-MeSADP),
a,ß-methylene-adenosine 5'-triphosphate (a,ß-meATP),
ADP, adenosine-5'-0-(2-thiodiphosphate) (ADPßS), ATP,
2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate
(BzATP), CTP, pyridoxalphosphate-6-azophenyl-2',4'-
disulphonic acid (PPADS), 2',3'-O-(2,4,6-
trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP),
UDP, UTP and ivermectin were purchased from Sigma (St
Louis, Missouri, USA). N6-methyl-2'-deoxyadenosine-
3',5'-bisphosphate (MRS2179) was purchased from Tocris
Cookson (Bristol, UK). Products for cell culture,
immunocytochemical and western-blot studies and
fluorescent probes are included in the specific methods
section. Other analytical grade reagents were purchased
from Merck (Darmstadt, Germany).
2.2. Cell culture
The SK-N-MC human neuroblastoma cell line (HTB-
10) was purchased from the American Type Culture
@Real Academia Nacional de Farmacia. Spain