Incubation of Neuro 2A mouse
neuroblastoma cells with
UTP and
UDP results in a concentration-dependent increase in the accumulation of
inositol phosphates with equal potency and maximal effect;
ATP,
ADP, and
2-methylthioadenosine 5'-triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of
UTP and
ATP were not affected by pretreatment of cells with
pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to
pertussis toxin-insensitive
Gq protein. Short-term (10 min) treatment of cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the inhibition of the
UTP and
ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5-6 h) and was not seen after long-term (24 h) treatment. Western blot analysis showed the expression of
protein kinase C (PKC) alpha, epsilon, theta, and zeta in Neuro 2A cells. Translocation of PKC alpha, epsilon, and theta from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down-regulation of both membrane PKC alpha and theta were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA-induced translocation of
PKC epsilon was maintained after 3-6 h of treatment, and almost complete down-regulation occurred only after a 24-h treatment. The observed TPA-induced inhibition of
UTP- or
ATP-stimulated
phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of
PKC epsilon.
Phosphoinositide hydrolysis induced by
AlF4-, but not Ca2+
ionophores, was inhibited by a 10-min treatment with TPA. This was not seen after a 24-h treatment, indicating that the site of action of
PKC epsilon in the P2Y receptor/
Gq protein/
phospholipase C beta pathway might be the
Gq protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal
PKC epsilon in the regulation of the receptor-mediated
phosphoinositide turnover.