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P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε
Author(s) -
Chen ChingChow,
Chen WeiChyuan
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.69041409.x
Subject(s) - pertussis toxin , protein kinase c , phospholipase c , receptor , inositol , cytosol , p2y receptor , inositol phosphate , gtp' , biology , stimulation , g protein , kinase , chromosomal translocation , microbiology and biotechnology , chemistry , biochemistry , endocrinology , enzyme , purinergic receptor , gene
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 G q protein. Short‐term (10 min) treatment of cells with 1 µ M 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) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ 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 PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA‐induced translocation of PKCε 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 PKCε. Phosphoinositide hydrolysis induced by AlF 4 − , but not Ca 2+ 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 PKCε in the P2Y receptor/G q protein/phospholipase Cβ pathway might be the G q protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor‐mediated phosphoinositide turnover.