Abstract
Cyclic AMP-dependent induction of differentiation by activation of the beta-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the beta-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5'-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in non-stimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeSADP-dependent inhibition of GFAP synthesis. The P2Y(12) and P2Y(13) receptor antagonist AR-C69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloro-methylene ATP] decreased PKB phosphorylation to the level in non-stimulated cells, whereas the P2Y(13) antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and P(1),P(3)-di(adenosine-5') tetraphosphate (Ap(4)A) did not alter the 2MeSADP-induced phosphorylation of PKB, showing that enhanced PKB activity and subsequent phosphorylation of glycogen synthase kinase-3 is due to stimulation of the P2Y(12) receptor. In addition, experiments in the presence of pertussis toxin and phosphatidylinositol 3-kinase (PI 3-K) activity assays demonstrated that the P2Y(12) receptor-mediated increase in PKB phosphorylation is G(i) protein- and PI 3-K-dependent. The presented data demonstrated that a cAMP-dependent inhibition of PKB induces differentiation of C6 glioma cells and that inhibition of adenylate cyclase and reactivation of the PI 3-K/PKB pathway by the P2Y(12) receptor reverses differentiation into enhanced proliferation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Diphosphate / analogs & derivatives*
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Adenosine Diphosphate / pharmacology
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Adenosine Monophosphate / analogs & derivatives*
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Adenosine Monophosphate / pharmacology
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Adenylyl Cyclase Inhibitors
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Adrenergic beta-Agonists / pharmacology
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Animals
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Cell Differentiation / drug effects
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Cell Differentiation / physiology*
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Cell Division / drug effects
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Cell Line, Tumor
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Cyclic AMP / metabolism*
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Enzyme Inhibitors / pharmacology
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GTP-Binding Protein alpha Subunits, Gi-Go / drug effects
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GTP-Binding Protein alpha Subunits, Gi-Go / metabolism
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Glial Fibrillary Acidic Protein / biosynthesis
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Glioma / drug therapy
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Glioma / metabolism*
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Membrane Proteins / agonists
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Membrane Proteins / antagonists & inhibitors
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Membrane Proteins / metabolism*
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Pertussis Toxin / pharmacology
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphorylation / drug effects
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Protein Serine-Threonine Kinases*
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Proto-Oncogene Proteins / antagonists & inhibitors*
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Purinergic P2 Receptor Agonists
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Purinergic P2 Receptor Antagonists
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Rats
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Receptors, Adrenergic, beta / drug effects
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Receptors, Adrenergic, beta / metabolism*
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Receptors, Purinergic P2 / metabolism*
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Receptors, Purinergic P2Y12
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Thionucleotides / pharmacology
Substances
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Adenylyl Cyclase Inhibitors
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Adrenergic beta-Agonists
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Enzyme Inhibitors
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Glial Fibrillary Acidic Protein
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Membrane Proteins
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P2ry12 protein, rat
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P2ry13 protein, rat
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Proto-Oncogene Proteins
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Purinergic P2 Receptor Agonists
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Purinergic P2 Receptor Antagonists
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Receptors, Adrenergic, beta
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Receptors, Purinergic P2
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Receptors, Purinergic P2Y12
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Thionucleotides
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methylthio-ADP
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Adenosine Monophosphate
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Adenosine Diphosphate
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cangrelor
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Cyclic AMP
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Pertussis Toxin
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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GTP-Binding Protein alpha Subunits, Gi-Go