DIFFERENTIAL REGULATION OF MUSCARINIC ACETYLCHOLINE RECEPTOR-SENSITIVE POLYPHOSPHOINOSITIDE POOLS AND CONSEQUENCES FOR SIGNALING IN HUMAN NEUROBLASTOMA-CELLS

In this study we have quantitatively assessed the basal turnover of ph osphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) and M-3-muscarini c receptor-mediated changes in phosphoinositides in the human neurobla stoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositide s represent a minor fraction of the total cellular phosphoinositide po ol and that in addition to rapid, sustained increases in [H-3]inositol phosphates dependent upon the extent of receptor activation by carbac hol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P-2 was reduced with less potency by carbach ol and recovered faster following agonist removal suggesting protectio n of PtdIns(4,5)P-2 at the expense of PtdIns(4)P and indicating specif ic regulatory mechanism(s). This does not involve a pertussis toxin-se nsitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns I-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIn s(4,5)P-2) is a failure of agonist-mediated phosphoinositide and Ca2signaling. The use of wortmannin also indicated that PtdIns is not a s ubstrate for receptor-activated phospholipase C and that 15% of the ba sal level of PtdIns(4,5)P-2 is in an agonist-insensitive pool. We esti mate that the agonist-sensitive pool of PtdIns(4,5)P-2 turns over ever y 5 s (0.23 fmol/cell/min) during sustained receptor activation by a m aximally effective concentration of carbachol, Immediately following a gonist addition, PtdIns(4,5)P-2 is consumed >3 times faster (0.76 fmol /cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These da ta indicate that resynthesis of PtdIns(4,5)P-2 is required to allow fu ll early and sustained phases of receptor signaling Despite the critic al dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P-2 resynthesis, we find no evidence that this rate resynthesis is limiti ng for agonist-mediated responses.