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DAVID LEÓN NAVARRO Y COLS. AN. R. ACAD. NAC. FARM.

Additionally, we investigated if calcium entry after glutamate
receptor activation was able to induce the phosphorylation of
CaMKII. Results showed that L-Glu (100 µM) was able to induce an
important increase in CaMKII phosphorylation in granule neurons,
at both experimental times, 1 and 5 min (219 ± 33% and 387 ± 19%,
respectively) (Figure 5A and 5B). As a control, immunostaining
studies —with the antibody that recognizes both non-phosphorylated
and phosphorylated forms of CaMKII— showed no differences in
the labelling intensity between control and L-Glu-stimulated cells, as
was expected for total enzyme detection (Figure 5A).

Then, we used the selective CaMKII antagonists, KN-62 (10 µM)
and KN-93 (10 µM). The effective inhibitory concentration values
of both antagonists have been reported to be 1 µM (29, 30) with no
significant effect observed at concentrations 100 µM on activities
of other kinases, such as MLCK, PKC, or cAMP-dependent protein
kinase. As it can be observed in the Figures 5C and 5D, when
ATP-mediated responses were measured five minutes after L-Glu
(100 µM) exposition in granule neurons pre-treated with KN-62 and
KN-93, the inhibitory effects of L-Glu on ATP-induced currents were
abolished. The pooled average data compared with the first ATP-
induced current were: KN-62, 92 ± 3% (p > 0.05, n = 67 neurons);
KN-93, 87 ± 5% (p > 0.05, n = 45 neurons). However, neither KN-62
nor KN-93 was able to abolish the inhibitory effect of L-Glu on ATP-
mediated responses measured immediately after L-Glu exposition.

4. DISCUSSION

The present study confirms the existence of a negative cross-talk
between nucleotide receptors activated by ATP and glutamate
receptors on granule neurons growth in culture. The relevance of
these results relies not only because it demonstrates a new type of
interaction between receptors with different structure and function
but also because it allows to speculate the presence of a similar
interaction between both group of receptors in other brain regions,
such as hippocampus and cortex, where ATP-containing synaptic
vesicles can coexist with L-Glu-containing ones and, therefore,
release of both neurotransmitters can occur simultaneously (31).

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