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Functional characterization of P2Y1 and P2X4 receptors in human neuroblastoma SK-N-MC cells
functionality. Traces represent the mean of the responses
obtained in 108 individual cells. (B) SK-N-MC cells were
stimulated with 300 µM CTP both in the absence and in the
presence of the P2X4 positive modulator ivermectin (5 µM). At
the end of the experiments, cells were challenged with 100 µM
ATP to test their functionality. Traces represent the mean of the presence of functional P2Y6 receptors in the SK-N-MC
responses obtained in 135 individual cells that showed responses
to CTP. (C) SK-N-MC cells were stimulated with 100 µM ATP,
both in the absence and in the presence of the P2Y1 antagonist
MRS2179 (10 µM), to selectively analyze the MRS2179-resistant
component of the ATP response. Afterwards, cells were
challenged with 100 µM ATP in the presence of MRS2179 (10
µM) and ivermectin (5 µM), in order to analyze the effect of the
positive modulator of the P2X4 receptor on the MRS2179-
resistant component of the ATP response. Traces are the mean of
the responses measured in 140 individual cells. Horizontal bars
above traces indicate duration of drug applications in all cases. TNP-ATP. P2Y11 receptor, which is sensitive to ATP (36,
4. DISCUSSION
The limited biological material and the cellular
heterogeneity of primary neuronal cultures frequently
represent a disadvantage for the in vitro study of the
nucleotide mediated signalling in the nervous system. The
use of homogeneous cultured neuroblastoma cell lines that
constitutively express P2 receptors allows avoiding these
inconveniences, so these lines could constitute a reliable
and convenient model with which analyze the signal
transduction pathways and intracellular events associated
with the activation of nucleotide receptors in neural
tissues.
In the present study we have investigated the presence
of functional P2 receptors in human neuroblastoma SK-N-
MC cells by a combination of immunological and calcium
measurement experiments. Immunological assays showed
the expression of P2Y1, P2Y6, P2Y11 and P2Y13 receptors
in the SK-N-MC cell line. P2X1, P2X4, P2X5, P2X6 and
P2X7 subunits can also be detected in the SK-N-MC cells
by means of immunocytochemical and western blot assays.
Despite the abundant expression of P2 receptors in the
SK-N-MC cells only a few range of P2X or P2Y agonists
are able to elicit calcium responses. A similar feature can
be also observed in other neuroblastoma cell types. For
instance, murine N2a cells express a wide range of P2X
subunits, including P2X1, P2X3, P2X4 and P2X7.
However, of these, the P2X7 receptor was the only
functional receptor (6).
The finding that ADP, 2-MeSADP and ADPßS induce
calcium increases that are inhibited by the subtype-
selective P2Y1-antagonist MRS2179 (36, 37), strongly
support the presence of a functional P2Y1 receptor in the
SK-N-MC cell line. ADP and its derivatives also activate
the P2Y13 receptor (36, 38, 39), which can be
immunologically detected in SK-N-MC cells. However if
the calcium transient induced by these compounds were, at
least in part, due to their interaction with a P2Y13 receptor,
a residual response should be observed in the presence of
MRS2179 that, at concentrations up to 100 µM, had no
significant effect on the P2Y13 receptor (36, 38). This is
not the case: responses to ADP and ADP derivatives are
almost completely blocked by MRS2179, thus indicating
@Real Academia Nacional de Farmacia. Spain
that these agonists increase the intracellular calcium
concentration due to their exclusive interaction with a
P2Y1 receptor in the SK-N-MC cells. On the other hand,
the lack of effect of UDP, a selective agonist at the
pyrimidinergic P2Y6 receptor (36, 37), precludes the
cells, in spite of our data showing their robust expression.
Regarding ATP, this nucleotide was also able to induce
calcium transients in the SK-N-MC cells. The partial
inhibition that both the specific P2Y1 antagonists
MRS2179 and the P2X antagonist TNP-ATP (40) exert on
the ATP responses indicates that this nucleotide is
activating at least two different receptors in the SK-N-MC
cells: the previously mentioned PY1 receptor, which is
blocked by MRS2179, and a P2X receptor, inhibited by
37), can be also detected at the protein level in the SK-N-
MC cells. However, if ATP were activating also this
receptor, a residual response to the agonist should be
observed in the combined presence of MRS2179 and TNP-
ATP. This is not the case: ATP responses in SK-N-MC
cells are completely blocked by means of a combination of
MRS2179 and TNP-ATP. Additionally, the lack of effect
of BzATP and UTP, both potent agonists at the P2Y11
receptor (36, 41), also precludes the presence of functional
P2Y11 receptors in the SK-N-MC cell line.
We tried to establish the identity of the P2X receptor
that is responsible for the MRS2179-resistant component
of the ATP response. SK-N-MC cells did not respond to
a,ß-meATP or BzATP but increased their [Ca2+]i when
challenged with CTP. Although P2X1 and P2X6 subunits
can be immunodetected in the SK-N-MC cells, the
presence of functional P2X1 or P2X6 channels can be
ruled out due the lack of action of a,ß-meATP, a potent
agonist of both receptors (42-44). a,ß-meATP is a weak or
inactive agonist at the P2X5 and P2X7 receptors but, if
these channels were functional in the SK-N-MC cells,
responses to BzATP should be obtained, as both receptors
are activated by this nucleotide analog (42, 45, 46). The
pattern of agonist activity observed in the SK-N-MC cells
is, however, consistent with the presence of a functional
P2X4 receptor. P2X4 channels have been proved to be
nearly insensitive to methylene-substituted ATP analogs,
whereas they can be activated by CTP, this compound
being a less potent agonist than ATP (42, 47). An unusual
property of the P2X4 receptor that differentiates it from all
other homomeric P2X channels is its potentiation by
ivermectin. This antiparasitic agent is a positive allosteric
modulator that specifically augments the response of P2X4
receptor to its agonists (34, 35). SK-N-MC cells showed
responses to CTP that increased in the presence of
ivermectin, this being an additional evidence favouring the
presence of a functional P2X4 receptor in the SK-N-MC
cell line. Moreover, the residual responses to ATP in the
presence of MRS2179 were also potentiated by ivermectin,
which could indicate that the P2X4 receptor is responsible
for the MRS2179-resistant component of the ATP
response in the SK-N-MC cells.
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