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Abstract
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.
Highlights
Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)1 is a minor membrane-associated phospholipid that is a substrate for enzymes involved in important cellular signal transduction pathways (1)
PtdIns(4,5)P2 can be regenerated by the sequential phosphorylation of PtdIns and phosphatidylinositol 4-phosphate (PtdIns(4)P) by phosphatidylinositol 4-kinase (PtdIns 4-kinase) and phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), respectively (6). This resynthesis is vital in maintaining an agonist-sensitive PtdIns(4,5)P2 pool, as the cellular content of PtdIns(4,5)P2 is small in comparison to the rate at which it may be consumed during receptor-mediated activation of phospholipase C (PLC) (7)
Incubation of SH-SY5Y human neuroblastoma cells with [3H]inositol resulted in marked changes in the absolute and relative amounts of radioactivity incorporated into the phosphoinositides over the first 20 h
Summary
Cell Culture—Experiments were performed on SH-SY5Y cells Where carbachol was removed to examine recovery, the agonist-containing buffer was aspirated, each well washed (2 ϫ 1 ml Krebs/HEPES), and the incubation continued in the presence of 200 l of buffer. After vortexing with 0.5 ml of a 1:1 (v/v) freshly prepared mixture of tri-n-octylamine and 1,1,2-trichlorotrifluoroethane, 20 l of 250 mM NaHCO3 was added to a 300-l aliquot of the aqueous phase This was applied to a Dowex (AG1-X8) formate column which was washed with 20 ml of water and 10 ml of 25 mM ammonium formate. Experiments examining recovery following termination of carbachol action by addition of atropine were performed as described above. For experiments in which cells were pretreated with wortmannin, this was added during the final 10 min of incubation and incorporated with agonist additions. Statistical comparisons were by Student’s two-tailed paired or unpaired t test or, where multiple comparisons were required, by one-way analysis of variance followed by Duncan’s multiple range test at p Ͻ 0.05 and p Ͻ 0.01
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