1. We recently reported that the activation by
UDP of rat P2Y6
nucleotide receptors expressed in 1321N1
astrocytoma cells protected them from
TNFalpha-induced apoptosis by suppressing activation of
caspase 3 and 8. This study aims to characterize the involvement of intracellular signaling pathways, including
kinases involved in the antiapoptotic effect of
UDP. 2. Cell death was induced in 1321N1
astrocytoma cells permanently expressing the rat
P2Y6 receptor by exposure to
TNFalpha in the presence of
cycloheximide. The apoptotic fraction was analyzed using flow cytometry. 3. The activation of
P2Y6 receptors by
UDP both protected the astrocytes from
TNF-alpha induced apoptosis and activated
protein kinase C (PKC) isotypes. The
phorbol ester PMA also activated PKC and protected the cells from
TNFalpha-induced cell death. The alpha- and epsilon-isotypes of PKC were both activated in a persistent fashion upon 5-min exposure to either
UDP (10 microM) or the
phorbol ester PMA (100 nM). The PKCzeta isotype was markedly activated upon
UDP treatment. 4. The addition of PKC inhibitors,
GF109203X or Gö6976, partially antagonized the protective effect of
UDP and reduced the
UDP-induced phosphorylation of extracellular signal-regulated
protein kinases (Erk). The inhibitors of Erk, PD98,059 or
U0126, antagonized
UDP-induced protection. 5. The antiapoptotic
protein, Akt, was not affected by
P2Y6 receptor activation. Incubation of the astrocytes with
calcium modifiers
BAPTA-AM or
dantrolene, did not affect the
UDP-induced protection from apoptosis. 6. The addition of
phospholipase C (PLC) inhibitors,
D609 or
U73122, partially antagonized both
UDP-induced protection and PKC activation.