title: Contrasting effects of phorbol ester and agonist-mediated activation of protein kinase C on phosphoinositide and Ca2+ signalling in a human neuroblastoma.
abstract: The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhibitor, Ro-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback inhibition by agonist-activated PKC. Muscarinic antagonist activity of Ro-318220 complicated such assessment at low agonist concentrations. Carbachol or PDBu induced cytosol to membrane translocation of PKC alpha. This was faster and possibly greater with PDBu, which may explain the lack of feedback by agonist-activated PKC. These results indicate that, in SH-SY5Y cells, PDBu activation of PKC preferentially inhibits rapid muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P2 hydrolysis. This is at least partially through inhibition of Gq-protein/phosphoinositidase C coupling. However, at least at high agonist concentrations, a major agonist-mediated PKC feedback is not present in these cells.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, U.K.
authors.forename: G B
authors.initials: GB
authors.lastname: Willars
authors.forename: R A
authors.initials: RA
authors.lastname: Challiss
authors.forename: J A
authors.initials: JA
authors.lastname: Stuart
authors.forename: S R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D019832:N-Methylscopolamine; D009447:Neuroblastoma; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010716:Phosphatidylinositols; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D012602:Scopolamine Derivatives; D013729:Terpenes; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D008094:Lithium; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D002118:Calcium; D019832:N-Methylscopolamine