title: P2U-purinergic receptors on C6-2B rat glioma cells: modulation of cytosolic Ca2+ and cAMP levels by protein kinase C.
abstract: The activation of P2-purinergic receptors on C6-2B rat glioma cells caused a transient increase in cytosolic-free Ca2+ concentration ([Ca2+]i) as detected by Fura 2 fluorescence ratio imaging of single cells. These purinergic receptors are of the P2U subtype because UTP and ATP were equipotent and substantially more potent than the P2X- and P2Y-selective agonists alpha,beta-methylene ATP and 2-methylthio ATP, respectively. There was homologous desensitization of the Ca2+ responses between UTP and ATP but no heterologous desensitization between these nucleotides and another Ca(2+)-mobilizing receptor agonist, alpha-thrombin. The UTP-induced peak [Ca2+]i rise was insensitive to chelation of extracellular Ca2+ with EGTA. However, the response was abolished after either depletion of intracellular Ca2+ stores with the microsomal Ca(2+)-ATPase inhibitor thapsigargin or blockade of Ca2+ release from intracellular stores with the muscle relaxant dantrolene. The activation of P2U-purinergic receptors and thrombin receptors increased the formation of total inositol phosphates (IPs) and inhibited cAMP accumulation elicited with either the beta-adrenergic receptor agonist (-)-isoproterenol, or forskolin, a direct activator of adenylyl cyclase. UTP- and alpha-thrombin-induced changes in the levels of IPs, cytosolic Ca2+, and agonist-elicited cAMP accumulation were dramatically inhibited (> 80%) by acute treatment of the cells with the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate but not with the inactive ester 4 alpha-phorbol 12,13-didecanoate. We conclude that in C6-2B cells, the increase in [Ca2+]i after activation of P2U-purinergic receptors is primarily a result of IPs-mediated release of Ca2+ from intracellular stores with secondary influx of Ca2+ by capacitative mechanisms. Also, the inhibition by UTP and alpha-thrombin of agonist-elicited cAMP accumulation is mediated through an increase in [Ca2+]i.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007.
authors.forename: R
authors.initials: R
authors.lastname: Munshi
authors.forename: M A
authors.initials: MA
authors.lastname: DeBernardi
authors.forename: G
authors.initials: G
authors.lastname: Brooker
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D007295:Inositol Phosphates / Q000096:biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D007295:Inositol Phosphates; D011983:Receptors, Purinergic; D000242:Cyclic AMP; D011493:Protein Kinase C; D002118:Calcium; D014544:Uridine Triphosphate