P(2U)-PURINERGIC RECEPTORS ON C6-2B RAT GLIOMA-CELLS - MODULATION OF CYTOSOLIC CA2-KINASE-C( AND CAMP LEVELS BY PROTEIN)

The activation of P2-purinergic receptors on C6-2B rat glioma cells ca used a transient increase in cytosolic-free Ca2+ concentration ([Ca2+] i) as detected by Fura 2 fluorescence ratio imaging of single cells. T hese purinergic receptors are of the P2U subtype because UTP and ATP w ere equipotent and substantially more potent than the P2x- and P2Y-sel ective agonists alpha,beta-methylene ATP and 2-methylthio ATP, respect ively. There was homologous desensitization of the Ca2+ responses betw een UTP and ATP but no heterologous desensitization between these nucl eotides and another Ca2+-mobilizing receptor agonist, alpha-thrombin. The UTP-induced peak [Ca2+]i rise was insensitive to chelation of extr acellular Ca2+ with EGTA. However, the response was abolished after ei ther depletion of intracellular Ca2+ stores with the microsomal Ca2+-A TPase inhibitor thapsigargin or blockade of Ca2+ release from intracel lular stores with the muscle relaxant dantrolene. The activation Of P2 U-purinergic receptors and thrombin receptors increased the formation of total inositol phosphates (IPs) and inhibited cAMP accumulation eli cited with either the beta-adrenergic receptor agonist (-)-isoproteren ol, or forskolin, a direct activator of adenylyl cyclase. UTP- and alp ha-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 4b eta-phorbol 12-myristate 13-acetate but not with the inactive ester 4a lpha-phorbol 12,13-didecanoate. We conclude that in C6-2B cells, the i ncrease in [Ca2+]i after activation Of P2U-purinergic receptors is pri marily a result of IPs-mediated release of Ca2+ from intracellular sto res with secondary influx of Ca2+ by capacitative mechanisms. Also, th e inhibition by UTP and alpha-thrombin of agonist-elicited cAMP accumu lation is mediated through an increase in [Ca2+]i.