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VOL. 73 (4), 1127-1157, 2007 PHYSIOLOGICAL ROLE OF EXTRACELLULAR...

at the level of the number of responding cells but also in the
magnitude of the response to nucleotides (51). These in vitro
developmental changes were more significant in metabotropic
responses to pyrimidine nucleotides, UTP and UDP, which were
down and up regulated respectively, during the time in culture. These
changes correlated with changes in the mRNA expression levels for
P2Y4 and P2Y6 receptors.

P2Y1 receptor is present in almost all, if not all, granule neurons
when single cell microfluorimetric experiments are carried out, and
cells challenged with 2MeSATP, that is a full synthetic agonist of this
receptor. In most cells the calcium responses are completely
inhibited, or significantly reduced, in the presence of MRS2179, that
is the best antagonist of P2Y1 available. The mRNA expression and
the presence of the protein measured by western blot, for P2Y1
receptor, increases with time in culture, but in a more moderate way
compared with the P2Y4 and P2Y6 receptors.

In addition to P2Y1, the presence of P2Y12 and P2Y13 has been
recently reported in granule neurons, all of them being members of
the ADP responding nucleotide receptors. Thus the metabotropic
cascades activated by ADP require to be carefully studied to properly
asignate the function to one of the receptors families. Recently our
group has found that Glycogen synthase kinase-3 (GSK-3) a
multifaceted enzyme involved in development, neurogenesis, and
survival at the CNS was induced to phosphorylation and subsequent
inhibition of its catalytic activity by 2-methyl-thio-ADP (2MeSADP).
This compound could be acting through several P2Y-ADP receptors
expressed in granule neurons, as RT-PCR expression was found
for P2Y1, P2Y12 and P2Y13 receptors. As the effect on GSK-3
phosphorylation was sensitive to pertussis toxin and was unaffected
by specific antagonists of P2Y1 and P2Y12 receptors, such as
MRS2179 and 2-methyl-thio-AMP, respectively, the pharmacological
data fitted well with a Gi-coupled P2Y13 receptor. The signalling
cascade from P2Y13 after activation through 2MeSADP is relatively
complex as it was able to phosphorylate and activate extracellular
signal-regulated kinase (ERK)-1,2 and Akt proteins, but its effect on
GSK-3 phosphorylation was primarily dependent on the phosphatidyl
inositol-3 kinase (PI3-K)/Akt pathway, as it was abolished by the PI3-
K inhibitor wortmannin. GSK-3 inactivation by 2MeSADP in granule

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