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intracelular Ca2+ concentration were measured when cells were Javier Gualix et al.
challenged with 10µM ADP (A), 10 µM 2-MeSADP (B) OR 10 A), or the P2X antagonist, TNP-ATP (100 µM, B). Traces
µM AdßS (C), both in the absence and in the presence of the represent the mean of the responses obtained in 128 (A) or 94 (B)
individual cells. Horizontal bars above traces indicate duration of
selective P2Y1 antagonist, MRS 2179 (10 µM). Traces represent drug applications.
the mean of the responses measured in 156 (A), 140 (B) or 144
(C) individual cells. Horizontal bars above traces indicate To further characterize the P2X receptor that is
duration of drug applications. responsible for the MRS2179 resistant component of the
ATP response, we analyze the effect of several P2X
We also tested the ability of ATP (100 µM) to induce agonists on the SK-N-MC cells. No cells showed
[Ca2+]i increases in the SK-N-MC cells. Almost all SK-N- responses to either 100 µM a,ß-meATP or 100 µM BzATP
MC cells (96.8 ± 0.4% of total cells, n = 882) showed (Fig 5A). However, responses to CTP (300 µM) were
responses when challenged with this nucleotide. Thus, present in a 67.6 ± 10.1% of the analysed cells (n = 396,
ATP was used in some of our assays as an indicator of cell Fig 5B). Moreover, both the response to CTP (300 µM)
functionality (see Figures 5A and B). Responses to 100 and the MRS2179-resistant component of the ATP (100
µM ATP were partially blocked by MRS2179 (10 µM), the µM) response were potentiated in the presence of 5 µM
residual response observed in the presence of the ivermectin (Fig 5B and C), a positive allosteric modulator
antagonist being a 13.3 % of the original one (Fig 4A). of the P2X4 receptor (34, 35).
The P2X antagonist TNP-ATP (100 µM) also acted as a
partial inhibitor of ATP responses, reducing by 50.2 % the Figure 5. Intracellular calcium increments evoked by
amplitude of the calcium transients elicited by the agonist different purinergic agonists and effect of the P2X4
(Fig 4B). When SK-N-MC cells were treated with both potentiator ivermectin in the SK-N-MC cells. (A) Effect of
antagonists, MRS2179 (10 µM) and TNP-ATP (100 µM), 100 µM BzATP or 100 µM a,ß-meATP on the intracellular Ca2+
responses to 100 µM ATP were completely blocked concentration in the SK-N-MC cells. At the end of the
(results not shown). This is indicating that ATP is exerting experiments, cells were challenged with 100 µM ATP to test their
its effects through the interaction with two different P2
receptors: a P2Y1 receptor, sensitive to MRS2179, and a @Real Academia Nacional de Farmacia. Spain
P2X receptor which is blocked by TNP-ATP.

A ATP MRS2179
ATP
1,35
F340/F380 1,30
1,25
1,20 50 100 300 350 400 450
1,15
1,10 Time (s)
1,05
1,00
0,95
0,90

0

B ATP TNP-ATP
ATP
1,10

1,05

F340/F380 1,00

0,95

0,90

0,85 50 100 300 350 400 450
0
Time (s)

Figure 4. ATP induces calcium responses in the SK-N-MC
cells that are inhibited by MRS2179 and TNP-ATP. Changes
in the intracellular Ca2+ concentration were measured when cells

were stimulated with 100 µM ATP applied alone or in the
presence of the selective P2Y1 antagonist, MRS 2179 (10 µM,

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