CONTRASTING EFFECTS OF PHORBOL ESTER AND AGONIST-MEDIATED ACTIVATION OF PROTEIN-KINASE-C ON PHOSPHOINOSITIDE AND CA2+ SIGNALING IN A HUMAN NEUROBLASTOMA

The effects of protein kinase C (PKC) activation on muscarinic recepto r-mediated phosphoinositide and Ca2+ signalling were examined in the h uman neuroblastoma, SH-SY5Y, Carbachol evoked rapid transient elevatio ns of Ins(1,4,5)P-3 and intracellular [Ca2+] followed by lower sustain ed elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuat ed transient phases. Removal of the transplasmalemmal Ca2+ gradient co upled with depletion of intracellular Ca2+ stores with thapsigargin al so reduced carbachol-mediated Ins(1,4,5)P-3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P-3 responses to carbac hol thereby implicating both Ca2+- 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 )P-3 metabolism or phosphoinositide synthesis. Ln electroporated cells , PDBu inhibited Ins(1,4,5)P-3 accumulation mediated by carbachol or g uanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhi bitor, Re-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback: inhib ition by agonist-activated PKC. Muscarinic antagonist activity of Re-3 18220 complicated such assessment at low agonist concentrations. Carba chol or PDBu induced cytosol to membrane translocation of PKC alpha. T his was faster and possibly greater with PDBu, which may explain the l ack of feedback by agonist-activated PKC, These results indicate that, in SH-SY5Y cells? PDBu activation of PKC preferentially inhibits rapi d muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P hydrolysis. This is at least :partially t hrough inhibition of Gq-protein/phosphoinositidase C coupling. However , at least at high agonist concentrations, a major agonist-mediated PK C feedback is not present in these cells.