Differential Regulation of Muscarinic Acetylcholine Receptor-sensitive Polyphosphoinositide Pools and Consequences for Signaling in Human Neuroblastoma Cells

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)P2turns 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. 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)P2turns 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. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 1The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; [Ca2+ ]i, intracellular [Ca2+]; [3H]GroPIns, glycerophosphoinositol; [3H]GroPIns(4)P, glycerophosphoinositol 4-phosphate; [3H]GroPIns(4,5)P2, glycerophosphoinositol 4,5-bisphosphate; InsPx, inositol mono- and polyphosphates; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PI3-K, phosphoinositide 3-kinase; PLC, phospholipase C; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns 4-kinase, phosphatidylinositol 4-kinase; PtdIns(4)P 5-kinase, phosphatidylinositol 4-phosphate 5-kinase.1The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; [Ca2+ ]i, intracellular [Ca2+]; [3H]GroPIns, glycerophosphoinositol; [3H]GroPIns(4)P, glycerophosphoinositol 4-phosphate; [3H]GroPIns(4,5)P2, glycerophosphoinositol 4,5-bisphosphate; InsPx, inositol mono- and polyphosphates; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PI3-K, phosphoinositide 3-kinase; PLC, phospholipase C; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns 4-kinase, phosphatidylinositol 4-kinase; PtdIns(4)P 5-kinase, phosphatidylinositol 4-phosphate 5-kinase.is a minor membrane-associated phospholipid that is a substrate for enzymes involved in important cellular signal transduction pathways (1Lee S.B. Rhee S.G. Curr. Opin. Cell Biol. 1995; 7: 183-189Google Scholar). Thus, PtdIns(4,5)P2 is a substrate for both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3-K) activities. The importance of the signaling pathway initiated by PI3-K and resulting in the generation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is rapidly emerging (2Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Google Scholar, 3Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Google Scholar). However, all estimates so far suggest that the PI3-K pathway utilizes only a small fraction of the PtdIns(4,5)P2 pool compared with that hydrolyzed by PLC in a signaling cascade (4Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Google Scholar, 5Batty I.H. Downes C.P. Biochem. J. 1996; 317: 347-351Google Scholar). The latter enzyme liberates both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and 1,2-diacylglycerol, which mobilize Ca2+ from intracellular stores and activate several isoforms of protein kinase C, respectively (6Berridge M.J. Nature. 1993; 361: 315-325Google Scholar). Both Ins(1,4,5)P3 and diacylglycerol are recycled to provide the substrates (myo-inositol and CMP-phosphatidate) necessary for phosphatidylinositol (PtdIns) resynthesis. PtdIns(4,5)P2 can then 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 (6Berridge M.J. Nature. 1993; 361: 315-325Google Scholar). This resynthesis is vital in maintaining an agonist-sensitive PtdIns(4,5)P2pool, 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 PLC (7Downes C.P. Hawkins P.T. Stephens L.R. Michell R.H. Drummond A.H. Downes C.P. Inositol Lipids and Cell Signalling. Academic Press, London1989: 3-39Google Scholar). Although it is possible that changes in substrate/product concentrations, particularly the dramatic changes that might occur in the immediate vicinity of activated PLC, may play a role in regulating PtdIns(4,5)P2 supply, the pathway is likely to possess more sophisticated regulatory features that enable supply to be matched to demand under agonist-stimulated conditions. In this context a number of potential regulatory mechanisms for PtdIns 4-kinase and/or PtdIns(4)P 5-kinase activities have been proposed (8Conway B.R. Withiam-Leitch M. Rubin R.P. Mol. 1993; Scholar, Biochem. Biophys. Scholar, J. 1995; Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. 1992; 267: Scholar, L.R. Jackson T.R. Hawkins P.T. Biochem. J. 1993; Scholar, Scholar, M. Mol. 1996; a of the mechanisms and of regulation (2Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Google Scholar). the of PtdIns(4,5)P2 occur to allow sustained phosphoinositide there is to indicate the extent to which resynthesis to regulatory of signaling by is for resynthesis of PtdIns(4,5)P2 is required during agonist-mediated and of this substrate can to the rapid receptor that is an of this signal transduction pathway 1993; Scholar, J. 1995; Scholar). of the human neuroblastoma cell have features of and have been used in from signal transduction to 1995; Scholar, Biochem. J. Scholar, J. 1995; Scholar, Mol. 1995; Scholar, J. Scholar). used this neuroblastoma to and regulatory of receptor-mediated phosphoinositide and Ca2+ signaling Biochem. J. Scholar, J. 1995; Scholar, Mol. 1995; Scholar, J. and have to be of receptor in cellular 1993; Scholar, J. 1995; Scholar). In the study we therefore, used to quantitatively the regulation of PtdIns(4,5)P2 and resynthesis during of which are of the J. Scholar). In addition we have to the of reduced PtdIns(4,5)P2 supply on signaling PLC and to agonist-mediated of PtdIns(4,5)P2 to the rapid of receptor-mediated phosphoinositide signaling. on a from J. and in with and at in and with and required at an and the at the of inositol mono- and with for in then and the cell and for in of and with was then and the with of carbachol was to the was and the in the of of In agonist was in by addition of of with an of on the was and of with of a of and of was to a of the This was to a which was then with of and of with of and by and the glycerophosphoinositol phosphates and as of PtdIns, PtdIns(4)P, and PtdIns(4,5)P2, from cell upon Mol. Scholar). removal of the for the of as of and then to of the was in a of and at to These in of and of and hydrolyzed by addition of of in at during a at and then and the and of the was that with of The was to by addition of and to a The [3H]GroPIns, [3H]GroPIns(4)P, and then as Mol. and by PtdIns(4,5)P2 was by of Ins(1,4,5)P3 by following a I.H. Biochem. J. Scholar). as from not with in of and to for during which at then on for and then to with and then to the that at The Ins(1,4,5)P3 which been from the PtdIns(4,5)P2 was as from assessed I.H. Biochem. J. to allow levels of to be to the of Cell in and the as following of carbachol by addition of as of also to the potential and of the Ins(1,4,5)P3 as for the of inositol phosphates and However, following of the carbachol and of the with was for the required in of and with carbachol for by the addition of an of of the was and for Ins(1,4,5)P3 by a Biochem. J. Scholar). The intracellular was in of with and in of the was and as with the of the of the of cellular was to the at 5 and the with for at was following of in of and at for to and in of at at the was every s as an of by the addition of of carbachol to a concentration of The was to as M. J. Biol. Chem. in the of to by the addition of to The of on receptor-mediated phosphoinositide signaling was as with the that the and and Although wortmannin is for to inhibit PI3-K M. J. Biol. Chem. this been to inhibit isoforms of PtdIns 4-kinase 1995; Scholar, 1996; Scholar). We therefore, used wortmannin in an to to the immediate of blocking the of PtdIns(4)P and PtdIns(4,5)P2 under basal and agonist-stimulated conditions. in which with this was during the of and with agonist In the of the of wortmannin on inositol phosphates and wortmannin was in a of from J. 1995; Scholar, Mol. 1995; Scholar, Mol. was from was from was from was from cell in for and Ins(1,4,5)P3 to a of for and PtdIns(4,5)P2 at the data are as with the number of in by a and are as M. by by of by at and of human neuroblastoma with [3H]inositol in changes in the and of the phosphoinositides over the not However, there no for under basal agonist-stimulated 5 not and the phosphoinositide to be in In all therefore for and under the fraction of the inositol phospholipid and only minor and addition of a concentration of carbachol that is for the receptor-mediated in there was a rapid and of We that this of is in the of a of inositol therefore the of both generation and to the s of with carbachol there was an in of basal levels that was sustained the of agonist In the cell there rapid and in the levels of and upon agonist to and of basal by s These reductions sustained the of agonist In the level of to only of basal levels over the this small of it is that the specific activities of the during this of and therefore the changes in are likely to the changes in The of carbachol to the of and of the phosphoinositides was at s following agonist for for the of and for the of The for agonist-stimulated changes in levels was indicating that carbachol was more at than The minor reductions in particularly at of carbachol, an of agonist potency for this The of from of and upon agonist following to a concentration of the agonist These data indicated in the of resynthesis of the polyphosphoinositides. Thus, of with no following removal of carbachol, there was a of s of a and The rate of of was compared with that of of s and s by of to of basal levels in s following carbachol for full to to basal levels with a of s of changes the indicated a dependence of the sustained on receptor This was by the of the to of following addition of a maximally effective concentration of carbachol reduced from to of basal over a this levels from to of However, the rate and extent of of was from to of basal over the The addition of during with carbachol no upon the small of not changes in changes in levels was indicated by the of PtdIns(4,5)P2 during carbachol and following addition of Thus, with carbachol for s in an in the of PtdIns(4,5)P2 which was s of addition These also the of Ins(1,4,5)P3 from the This the and of receptor activation in Biochem. J. Scholar, J. 1995; Scholar, Mol. 1995; Scholar, J. and the of Ins(1,4,5)P3 levels to basal by s following addition also by of changes in PtdIns(4,5)P2 and s following addition of in a the of to carbachol from to 5 no on the rate extent of of the inositol phosphates phosphoinositides not and of for with pertussis no on the extent of the rate and extent of of the inositol phosphates phosphoinositides following 5 to carbachol and data not of with carbachol in the of in an of that was rapid over the of of of the basal which was this there was a sustained over a of carbachol of of The sustained and of demonstrate an effective of inositol that the of inositol the Despite this the of the was in the and of not We therefore, to use to cellular levels of the and an of PtdIns 4-kinase and was of wortmannin to a in over a to of 5 this there was no of wortmannin on levels not to of basal levels 5 In there was a more dramatic in to of basal levels 5 The of wortmannin to changes over a was a and wortmannin was more at basal levels of than with of and for the of wortmannin on basal and levels of and of and with wortmannin for to the addition of carbachol for s are of wortmannin was s carbachol there was an immediate in the agonist-mediated of levels by 5 the in and levels levels in the of agonist and was no on the of levels not We also in an on the that this been to inhibit PI3-K not PtdIns 4-kinase J. Biol. Chem. and therefore, enable the of wortmannin on PtdIns 4-kinase and However, it that this also the PtdIns 4-kinase 1996; Scholar). the addition of in the of carbachol of to by wortmannin not The of wortmannin to changes in was and and wortmannin to be with to all for of and and for of and been to inhibit partially PtdIns 4-kinase not PtdIns(4)P 5-kinase from 1995; Scholar). This that to cellular levels of PtdIns(4,5)P2 in the study was dependent upon a reduced supply of PtdIns(4)P and not an of PtdIns(4)P 5-kinase. This was by in in which we that wortmannin reduced the from PtdIns(4)P not PtdIns(4,5)P2 over a under basal not of wortmannin to with carbachol the of Ins(1,4,5)P3 and the sustained of the of with carbachol also in a of of a rapid by a sustained of to carbachol the of and the sustained This sustained of is dependent upon the of Ca2+ an as which likely J. 1995; Scholar). of to also in Ca2+ and a sustained of of to that by carbachol, this was by wortmannin not Thus, a of Ca2+ does not the of wortmannin to inhibit Ca2+ signaling (and also phosphoinositide signaling a in the of addition of carbachol and no on the of Ins(1,4,5)P3 at s reduced the at s following agonist addition a to that in which a with carbachol for 5 we to of the Ins(1,4,5)P3 responses. This with and of less than which the of of PtdIns(4,5)P2 the was is in a in agonist potency than a in the and we therefore an of in which with a concentration of carbachol to the for the Ins(1,4,5)P3 in J. 1995; Scholar). Compared with with carbachol, a more in the of the Ins(1,4,5)P3 which than 5 to and the of PtdIns(4,5)P2 in which the carbachol was for than 5 not the that during sustained receptor the rate of PtdIns(4,5)P2 be to the rate of The rate of following removal of carbachol therefore an of the rate of PtdIns(4,5)P2 which during sustained receptor the rate we that the PtdIns(4,5)P2 during sustained receptor activation be over every This that of PtdIns(4,5)P2 PLC PtdIns(4,5)P2 is compared with following agonist removal of resulting in an of and that all of the PtdIns(4,5)P2 an agonist-sensitive pool. However, a fraction of PtdIns(4,5)P2 15% of is to by wortmannin during carbachol 5 and is to 5 This fraction may therefore, be to agonist-stimulated PLC and a agonist-insensitive pool. 15% of basal PtdIns(4,5)P2 an agonist-insensitive then the pool over every 5 s under sustained receptor number of have that the M3-muscarinic receptor which is for activation of PLC in J. a rapid, of agonist This is indicated by a of in in which inositol been with to all of phosphoinositide 1993; Scholar, J. 1995; Scholar, J. Scholar). In addition, rapid is in the of Ins(1,4,5)P3 following carbachol as in this and Biochem. J. Scholar, J. 1995; Scholar, Mol. 1995; Scholar, J. Scholar). be therefore, that the utilization of PtdIns(4,5)P2 during sustained receptor as that of a partially desensitized receptor. The rate of PtdIns(4,5)P2 utilization by upon agonist addition may be far than the rate of of PtdIns(4,5)P2 following addition of carbachol is than during the sustained This is in with the in the of over the of agonist and the sustained in J. Scholar, Scholar). However, that the of resynthesis is during the of we demonstrate that resynthesis of PtdIns(4,5)P2 is required for the s of PLC this and the and sustained is likely to be in of that PtdIns(4,5)P2 is hydrolyzed in the of and we have this basal turnover of wortmannin under basal an immediate in 5 no in for at 5 Thus, during this of was to of a PtdIns 4-kinase by there was to the levels of The rate of of therefore, provide an of the basal of This rate of that an of to the total cellular pool over every 5 under basal conditions. The rate of basal PtdIns(4,5)P2 utilization is less than the rate of during sustained receptor activation and at less than that upon agonist addition These that of PtdIns(4)P to PtdIns a PtdIns(4)P and of PtdIns(4)P by PLC are following addition of to an of basal and an therefore, of the in the of PtdIns(4,5)P2 following agonist a consequence of the potential of basal of and over basal of PtdIns(4,5)P2 during the immediate and sustained of are likely to be with and in in which basal PtdIns(4,5)P2 was assessed by the of in However, to the of may not occur under of the enzyme Biochem. resulting in an of basal is likely that the extent of The basal level of PtdIns(4,5)P2 was to with [3H]inositol at This to estimates of PtdIns(4,5)P2 levels and utilization in of cell number and protein content of a of of estimates to a cell cellular protein Thus, the basal level of PtdIns(4,5)P2 is of which in an agonist-sensitive pool. sustained receptor activation with a maximally effective concentration of carbachol, PtdIns(4,5)P2 levels to of which in an agonist-sensitive pool. sustained receptor PtdIns(4,5)P2 over at a rate of during the of receptor activation this rate is at estimates of the of protein A.H. J. Biol. Chem. we can estimate that the of of PLC to be maximally activated to this PtdIns(4,5)P2 is to be the substrate for both PLC and PI3-K activities. Despite the dependence upon PtdIns(4,5)P2 as the substrate for Ins(1,4,5)P3, and this a minor fraction of the cellular inositol phospholipid content under basal conditions. The that PtdIns(4,5)P2, and PtdIns(4)P, can of cellular (1Lee S.B. Rhee S.G. Curr. Opin. Cell Biol. 1995; 7: 183-189Google Scholar, M. Cantley L.C. J. Biol. Chem. Scholar, M. J. Biol. Chem. Scholar, Nature. might provide for the cellular levels of the polyphosphoinositides. of the cellular PtdIns(4,5)P2 it is that regulatory mechanisms supply and demand are to be particularly during agonist-stimulated PLC and/or PI3-K The study that and sustained of both PtdIns(4)P and PtdIns(4,5)P2 are by a maximally effective concentration of carbachol. In PtdIns(4,5)P2 is at and as this level can be by for under of agonist activation of M3-muscarinic The in the cellular levels of with the of Ins(1,4,5)P3 in and that of PtdIns(4)P to PtdIns(4,5)P2 is required to both the and sustained of signaling The sustained of the polyphosphoinositides are to the extent of agonist carbachol was more at PtdIns(4)P than This the of mechanisms that to the pool of PtdIns(4,5)P2 at the expense of protection is also indicated by the of of PtdIns(4)P and PtdIns(4,5)P2 following of receptor Thus, PtdIns(4,5)P2 not only to basal levels with a also by PtdIns(4)P protection of PtdIns(4,5)P2 is in with in Mol. Scholar, A.H. Biochem. J. Scholar, C.P. Biochem. J. and that substrate and are to be the only the supply of Thus, mechanisms that in with and the data indicate that mechanisms are of in of supply occur an activation of the for generation and/or a in the activities of the that to be involved in substrate L.R. Jackson T.R. Hawkins P.T. Biochem. J. 1993; Scholar). However, it that the in demand for PtdIns(4,5)P2 be by changes in activities as this a level of substrate L.R. Jackson T.R. Hawkins P.T. Biochem. J. 1993; Scholar). changes in and kinase activities may there is on the regulation of activities. a of evidence to regulation of both PtdIns 4-kinase and PtdIns(4)P 5-kinase (8Conway B.R. Withiam-Leitch M. Rubin R.P. Mol. 1993; Scholar, Biochem. Biophys. Scholar, J. 1995; Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. 1992; 267: Scholar, L.R. Jackson T.R. Hawkins P.T. Biochem. J. 1993; Scholar, Scholar, M. Mol. 1996; Scholar). to the study is the that a activation of for the regulation of PtdIns(4)P 5-kinase M. Mol. 1996; Scholar, M. Mol. 1996; Scholar). Thus, in PtdIns(4,5)P2 to basal levels in a pertussis toxin-sensitive following removal of and it been that this an phosphoinositide upon M. Mol. 1996; Scholar). are in to of the study in which we a of the Ins(1,4,5)P3 and a of of pertussis on the rate extent of of PtdIns(4,5)P2 following receptor the in cell a of to at levels in is However, the study that at levels of receptor protein pertussis toxin-sensitive play no role in regulating resynthesis of PtdIns(4,5)P2 the level of this substrate in indicating that this is not a the use of wortmannin we have that 15% basal of PtdIns(4,5)P2 is to agonist-stimulated have been in Mol. Scholar). The for the of this fraction is may to the cell J. Biol. Chem. Scholar, J. Biol. Chem. 1996; and this the potential to regulatory on the signaling the agonist-insensitive pool we that the agonist-sensitive pool of PtdIns(4,5)P2 over at a rate of times under sustained with a maximally effective concentration of carbachol. In the of resynthesis, the substrate pool be consumed in less than s of agonist This with a estimate for PtdIns(4,5)P2 turnover in of s and the critical dependence of Ins(1,4,5)P3 on PtdIns(4,5)P2 resynthesis. changes in the basal level of PtdIns(4,5)P2 therefore, have on phosphoinositide responses. of in the of receptor-mediated phosphoinositide and the critical dependence of this signaling pathway on a (and PtdIns 4-kinase as in 1995; Scholar, J. Biol. Chem. 1995; Scholar). indicated that phosphorylation of at the by the as potential pathway for the generation of PtdIns(4,5)P2 Cantley L.C. Nature. Scholar). this of also upon a the data suggest that this pathway not at all to the of PtdIns(4,5)P2 in under basal agonist-stimulated conditions. The of wortmannin to PtdIns(4,5)P2 during sustained receptor activation is also by a of This that the of PLC isoforms to PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 S.G. J. Biol. Chem. 1992; 267: the cellular pool of PtdIns does not provide a substrate for PLC during activation in The of the substrate of receptor-activated PLC is to in and the of as wortmannin to the phosphoinositide a to this the of wortmannin to the sustained during receptor that there is no Ca2+ a Ca2+ than a which been in J. 1992; Scholar, J. J. Biol. Chem. 1992; 267: Scholar). Although both wortmannin and are for to PI3-K, of this enzyme for the of the Thus, the of wortmannin on both basal and levels is at that have been to 1995; J. Biol. Chem. 1995; (and to inhibit of the PtdIns 4-kinase activities that are in cell C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Google the PtdIns 4-kinase J. Biol. Chem. 1996; Scholar, Cantley L.C. J. Biol. Chem. Scholar). Although we that wortmannin is not a specific of kinase M. J. Biol. Chem. Scholar, 1995; Scholar, J. Biol. Chem. 1995; the data in the study are with an of this enzyme and are in with in which been used to cellular levels of the polyphosphoinositides in during of Mol. Scholar). The study that may be used to phosphoinositide in an in cell this is not the in This is as a consequence of a intracellular pool of and a inositol is required to the to J. 1993; Scholar). the and to of PLC signaling by in and 1993; Scholar, J. 1995; Scholar, J. Scholar, it is that the of the inositol pool a on of phosphoinositide The data the of the agonist-sensitive PtdIns(4,5)P2 and the use of wortmannin that the immediate consequence of blocking the of PtdIns(4)P (and also therefore PtdIns(4,5)P2) is the failure of Ins(1,4,5)P3 generation and Ca2+ These data indicate that the rate of supply of PtdIns(4,5)P2 be and matched to demand to a full failure of agonist-mediated phosphoinositide signaling. a in the of PtdIns(4,5)P2 is a possible the rapid, of PLC that of agonist of of of J. 1995; Scholar, J. Scholar, Scholar). of to to However, we demonstrate that the sustained in the level of PtdIns(4,5)P2 is to the concentration of carbachol. This that at agonist concentrations, there is PtdIns(4,5)P2 for and that substrate supply is not for the of PLC to a of PtdIns(4,5)P2 and that this is at a rate to the extent of receptor The Ins(1,4,5)P3 to activation of in and of a rapid by a sustained Biochem. J. Scholar, J. 1995; Scholar, Mol. 1995; Scholar, J. Scholar). We have that phases of Ins(1,4,5)P3 represent and respectively 1993; Scholar). In the study we also demonstrate of the Ins(1,4,5)P3 a These indicate that with a concentration of carbachol in of the Ins(1,4,5)P3 upon with a concentration of carbachol. Although of the to a concentration of carbachol the of PtdIns(4,5)P2, of the to a concentration was Thus, there are there is PtdIns(4,5)P2 to a Ins(1,4,5)P3 and to a agonist concentration partially This that there are to the of PtdIns(4,5)P2 (and of the signal transduction that are involved in the We have that this may be to phosphorylation of the receptor in a to the phosphorylation and of the 1993; Scholar). The data indicate a critical dependence upon a and PtdIns 4-kinase for the of PtdIns(4,5)P2 and indicate that the immediate consequence of blocking the of PtdIns(4,5)P2 is the failure of receptor-mediated phosphoinositide and Ca2+ signaling. that enable the supply of PtdIns(4,5)P2 to be matched to and mechanisms are effective under of receptor activation in Thus, the critical dependence upon PtdIns(4,5)P2 data indicate that agonist-mediated of this substrate is to for receptor in The of PLC activation and extent of by receptor activation in this cell is with that by receptor in a of cellular I.H. Downes C.P. Biochem. J. 1996; 317: 347-351Google Scholar, 1993; Scholar, J. 1995; Scholar, J. Biol. Chem. suggesting that the are likely to have to PLC signaling We for We also to of of and of Cell and of for during the of this