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ATP‐evoked inositol phosphates formation through activation of P 2U purinergic receptors in cultured astrocytes: Regulation by PKC subtypes α, δ, and θ
Author(s) -
Chen Ching C.,
Chen Wei C.
Publication year - 1996
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/(sici)1098-1136(199605)17:1<63::aid-glia6>3.0.co;2-8
Subject(s) - biology , purinergic receptor , phospholipase c , p2y receptor , adenosine , pertussis toxin , receptor , inositol , adenosine triphosphate , gtp' , protein kinase c , inositol phosphate , medicine , endocrinology , g protein , biochemistry , kinase , enzyme
ATP‐induced phosphoinositide (PI) hydrolysis was studied in cultured astrocytes. To characterize the P 2 purinergic receptor‐mediated effects of ATP, the sub‐type‐specific agonists 2‐methylthio ATP (2‐MeSATP), UTP, and α,β‐methylene ATP were compared. ATP, UTP, or 2‐MeSATP induced a dose‐dependent increase of inositol phosphates (IP) accumulation; α,β‐methylene ATP and adenosine had no effect. The order of potency was ATP ≥ UTP ≫ 2‐MeSATP. Cross‐desensitization experiments indicated that ATP interacted with both P 2U and P 2Y receptors. P 2U was the predominant P 2 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 P 2U and P 2Y receptors coupled to phospholipase C through PTX‐sensitive G protein. Short‐term (10 min) treatment of cells with 1 μM 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)α, −δ, and −θ 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. PKCη was translocated but not down‐regulated by TPA. These results suggested that PKCα, −δ, and −θ, but not −η may exert tonic inhibition on P 2U receptor‐mediated PI turnover in unstimulated astrocytes. © 1996 Wiley‐Liss, Inc.