P2Y receptor linked to phospholipase C: stimulation of neuro 2A cells by UTP and ATP and possible regulation by protein kinase C subtype epsilon.
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.
Chen
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CC
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, R.O.C.
Chen
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WC
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D006868:Hydrolysis; D007527:Isoenzymes / Q000502:physiology*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009711:Nucleotides / Q000494:pharmacology; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D011983:Receptors, Purinergic / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007527:Isoenzymes; D009711:Nucleotides; D010716:Phosphatidylinositols; D011983:Receptors, Purinergic; D010566:Virulence Factors, Bordetella; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D014544:Uridine Triphosphate