Ca2+ inhibition of beta-adrenergic receptor- and forskolin-stimulated cAMP accumulation in C6-2B rat glioma cells is independent of protein kinase C.
In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.
Department of Biochemistry and Molecular Biology, Georgetown University, School of Medicine, Washington, D.C. 20007.
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Debernardi
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Munshi
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Brooker
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D005910:Glioma; D006868:Hydrolysis; D007545:Isoproterenol / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D010716:Phosphatidylinositols; D011943:Receptors, Adrenergic, beta; D013729:Terpenes; D005576:Colforsin; D019284:Thapsigargin; D000242:Cyclic AMP; D011493:Protein Kinase C; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium