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M.ª TERESA MIRAS-PORTUGAL Y COLS. ANAL. REAL ACAD. NAC. FARM.

neurons resulted in nuclear translocation of its substrate ß-catenin,
which functions as a transcriptional regulator, this effect being lost

with wortmaninn (78). Figure 7 summarises the signalling cascade

from P2Y13 to GSK-3 and nuclear ß-catenin. Now, further work is
necessary to understand the physiological meaning of this signalling

pathway in CNS development, maintenance and senescence.

PRESENCE AND FUNCTION OF P2 RECEPTORS IN
CULTURED ASTROCYTES

Cultured astrocytes from rat cerebellum express almost all the
P2Y receptors described so far, and there are also evidence for the
presence and peculiar functionality of some P2X receptors.

The mRNA expression was studied by PCR and presence of
P2Y1,2,4,6,11,12,13 metabotropic nucleotide receptors was detected. At the
same time and if specific antibodies were available, studies of
immunocytochemistry or western blot were accomplished. Not
always the presence of mRNA corresponded with protein detection
by western blot or immunocytochemistry, or later on with functional
responses from astrocytes. For example, P2Y1 receptor correlates
well the mRNA expression, protein detection and cellular responses,
but that is not the case for P2Y12 receptor that in spite of mRNA
presence, there was no specific labelling of astrocytes with anti-
P2Y12 receptor antibody, whereas a specific staining was observed,
as positive control in platelets.

Studies of the P2X subunits expression was carried out by RT-
PCR techniques. In these assays high levels of mRNA codifying for
P2X1, P2X3, P2X4 and P2X7 subunits were detected. In contrast, P2X2,
P2X5 and P2X6 subunits appeared to be nearly absent in astrocytes
cultures. In most cases the mRNA detection does not correlate with
the presence of the protein. In fact, by immunocytochemistry using
specific antibodies only P2X4 and P2X7 subunits were detected. These
data are relevant because it is possible that P2X7 and P2X4 form
functional hetero-oligomers (37) in cerebellar astrocytes.

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