In this study we have quantitatively assessed the basal turnover of
phosphatidylinositol 4,5-bisphosphate (
PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in
phosphoinositides in the human
neuroblastoma cell line, SH-SY5Y. We demonstrate that the
polyphosphoinositides represent a minor fraction of the total cellular
phosphoinositide pool and that in addition to rapid, sustained increases in [3H]
inositol phosphates dependent upon the extent of receptor activation by
carbachol, there are equally rapid and sustained reductions in the levels of
polyphosphoinositides. Compared with
phosphatidylinositol 4-phosphate (
PtdIns(4)P),
PtdIns(4,5)P2 was reduced with less potency by
carbachol and recovered faster following agonist removal suggesting protection of
PtdIns(4,5)P2 at the expense of
PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a
pertussis toxin-sensitive
G-protein regulation of
PtdIns(4)P 5-kinase. Using
wortmannin to inhibit
PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of
PtdIns(4)P (and therefore
PtdIns(4,5)P2) is a failure of agonist-mediated
phosphoinositide and Ca2+ signaling. The use of
wortmannin also indicated that
PtdIns is not a substrate for receptor-activated
phospholipase C and that 15% of the basal level of
PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of
PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of
carbachol. Immediately following agonist addition,
PtdIns(4,5)P2 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 data indicate that resynthesis of
PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of
phosphoinositide and Ca2+ signaling on
PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.