title: ATP-evoked inositol phosphates formation through activation of P2U purinergic receptors in cultured astrocytes: regulation by PKC subtypes alpha, delta, and theta.
abstract: ATP-induced phosphoinositide (PI) hydrolysis was studied in cultured astrocytes. To characterize the P2 purinergic receptor-mediated effects of ATP, the subtype-specific agonists 2-methylthio ATP (2-MeSATP), UTP, and alpha, beta-methylene ATP were compared. ATP, UTP, or 2-MeSATP induced a dose-dependent increase of inositol phosphates (IP) accumulation; alpha, beta-methylene ATP and adenosine had no effect. The order of potency was ATP > or = UTP >> 2-MeSATP. Cross-desensitization experiments indicated that ATP interacted with both P2U and P2Y receptors. P2U was the predominant P2 receptor in mediating PI hydrolysis in astrocytes. The effect of ATP, UTP, or 2-MeSATP was markedly inhibited by pretreatment of cells with pertussis toxin (PTX), indicating that both P2U and P2Y receptors coupled to phospholipase C through PTX-sensitive G protein. Short-term (10 min) treatment of cells with 1 microM TPA attenuated ATP, UTP, and 2-MeSATP-induced PI breakdown; however, long-term (24 h) pretreatment resulted in marked potentiation of both ATP and UTP, and restoration of 2-MeSATP responses. In a further analysis of the effect of TPA, 10 min and 1.5 h pretreatment attenuated ATP-and UTP-induced PI breakdown, but this inhibitory action was lost after 3 h of treatment. Both 6 and 24 h pretreatments resulted in a potentiation. Western blot analysis showed translocation of protein kinase C (PKC) alpha, -delta, and -theta from the cytosol to the membrane following 10 min and 1.5 h treatments, and restoration to basal levels in the membrane fraction was seen after 3 h of treatment. On the other hand, partial and complete down-regulation of these three isoforms was seen after 6 and 24 h of treatment, respectively. PKC eta was translocated but not down-regulated by TPA. These results suggested that PKC alpha, -delta, and -theta, not -eta may exert tonic inhibition on P2U receptor-mediated PI turnover in unstimulated astrocytes.
authors.affiliation: Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: C C
authors.initials: CC
authors.lastname: Chen
authors.forename: W C
authors.initials: WC
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D007295:Inositol Phosphates / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000187:drug effects*
chemical_list: D007295:Inositol Phosphates; D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate; D011493:Protein Kinase C