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VOL. 76 (1), 3-22, 2010 CROSS-TALK BETWEEN GLUTAMATE AND NUCLEOTIDE...

3.2. Activation of glutamate receptors decreases
3.2. the ATP-induced intracellular calcium increase

Using doses of ATP to which 50% of the cells responded, we
investigated by microfluorimetric experiments whether the calcium
entrance mediated by glutamate receptor could modify the [Ca2+]i
increase induced by ATP. To perform this study granule neurons
were stimulated first with 200 µM ATP, followed by a second double
pulse of 100 µM L-Glu and 200 µM ATP, and afterwards by 200 µM
ATP in a third pulse. Before each ATP pulse, the preparation was
always washed with the Locke’s solution for five minutes. As it
is shown in Figure 3A, the mean calcium increase induced by the
first application of 200 µM ATP was 209 ± 38 nM (n = 33 neurons),
once Grynkiewicz’s equation (20) had been applied. Similarly,
when granule neurons were challenged with 100 µM L-Glu, the
mean calcium increase observed was 210 ± 34 nM (n = 33 neurons).
However, when L-Glu (100 µM) was applied immediately before
ATP (200 µM), ATP-calcium transients were significantly reduced
to a value that was 60 ± 3% of their initial value (p < 0.001, n = 33
neurons). In these neurons, ATP-mediated responses remained
depressed 5 min after L-Glu (100 µM) application (by 73 ± 3%; p <
0.001, n = 33 neurons).

In order to investigate the possible inhibitory effect of L-Glu on
metabotropic P2Y receptor activation, ATP (200 µM) was assayed
in a virtually Ca2+-free medium ([Ca2+] ~ 50 nM) by using a Ca2+
chelator mixture of 5 mM EGTA/5.5 mM Tris (21). As Figure 3B
shows, in the presence of EGTA, individual granule neurons
responded to ATP which confirmed the presence of functional P2Y
receptor, accordingly with previous results (4). When ATP (EGTA)-
mediated responses were analysed after L-Glu stimulation, a
significant reduction could be detected in both times assayed (0 min,
76 ± 6%, p < 0.001, n = 34 neurons; 5 min, 78 ± 4%, p < 0.01, n = 34).

The effect of L-Glu on ATP-mediated responses was concentration
dependent. Thus, when granule neurons were challenged with L-Glu
10 µM, the second peak of calcium induced by ATP declined to
a value that was 72 ± 5% of their initial peak amplitude (p < 0.01,
n = 126 neurons). However, ATP-mediated response was fully
recovered five minutes after granule neurons were stimulated with

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