Synthesis Of Polyphosphoinositides Research Articles

C-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides

The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II β was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate

TRPC3, 6 and 7 channels constitute a subgroup of non-selective, calcium-permeable cation channels within the TRP superfamily that are activated by products of phospholipase C-mediated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP 2). A number of ion channels, including other members of the TRP superfamily, are regulated directly by PIP 2. However, there is little information on the regulation of the TRPC channel subfamily by PIP 2. Pretreatment of TRPC7-expressing cells with a drug that blocks the synthesis of polyphosphoinositides inhibited the ability of the synthetic diacylglycerol, oleyl-acetyl glycerol, to activate TRPC7. In excised patches, TRPC7 channels were robustly activated by application of PIP 2 or ATP, but not by inositol 1,4,5-trisphosphate. Similar results were obtained with TRPC6 and TRPC3, although the effects of PIP 2 were somewhat less and with TRPC3 there was no significant effect of ATP. In the cell-attached configuration, TRPC7 channels could be activated by the synthetic diacylglycerol analog, oleyl-acetyl glycerol. However, this lipid mediator did not activate TRPC7 channels in excised patches. In addition, channel activation by PIP 2 in excised patches was significantly greater than that observed with oleyl-acetyl glycerol in the cell-attached configuration. These findings reveal complex regulation of TRPC channels by lipid mediators. The results also reveal for the first time direct activation by PIP 2 of members of the TRPC ion channel subfamily.

Subcellular Localization and Structural Function of Endogenous Phosphorylated Phosphatidylinositol 4-Kinase (PI4K92)

Anti-phosphopeptide antibodies were raised against phosphatidylinositol 4-kinase (PI4K92) phosphorylation sites (Suer, S., Sickmann, A., Meyer, H. E., Herberg, F. W., and Heilmeyer, L. M. Jr. (2001) Eur. J. Biochem. 268, 2099-2106). Characterization proved three of them (anti-pSer-294, anti-pSer-496, and anti-pThr-504 antibody) to be highly specific, recognizing solely PI4K92 phosphorylated at these sites, respectively. Indirect immunofluorescence reveals that PI4K92 phosphorylated on Ser-294 localizes exclusively at the Golgi. The enzyme phosphorylated on Ser-496 and Thr-504 is detected in nuclear speckles. Phosphorylation of Ser-294 on PI4K92 increases the lipid kinase activity and thus serves better in maintaining Golgi function and morphology (compare Hausser, A., Storz, P., Martens, S., Link, G., Toker, A., and Pfizenmaier, K. (2005) Nat. Cell Biol. 7, 880-886). Microinjection of anti-pSer-496, but not of anti-pSer-294 or anti-pThr-504 antibody, into the cytoplasm or into the nucleus of HS68 cells leads to development of hotspots, probably representing aggregated PI4K92, and in later stages, cells become apoptotic and finally die. The association of phosphorylated PI4K92 with nuclear speckles is dynamic and follows the morphological alteration of speckles upon inhibition of mRNA transcription with alpha-amanitin. Overexpressed PI4K92 phosphorylated on Ser-294 is not transported to the nucleus, and that phosphorylated on Ser-496 is found in the nucleus and mislocalized at the Golgi complex. We conclude that nuclear phosphatidylinositol 4-phosphate, and consequently, synthesis of polyphosphoinositides are required for a correct nuclear function.

Gibberellin Signaling Pathway:Role of Phospholipids in Barley Aleurone

The present report describes intracellular signaling events elicited by gibberellin (GA) in barley aleurone, where the phytohormone induces an increase on phosphoinositides turnover in a time-dependent manner. A biphasic behavior for PtdInsP was demonstrated by changes in [32P]PtdInsP levels, the earlier phase peaking at 5 min and the second phase at 25 min while PtdInsP2 turnover was rapid but transient, peaking only at 5 min. In contrast, the structural phospholipid PtdCho turnover was only affected after 40 min. In addition, GA stimulated the synthesis of all phospholipids. Incorporation of [32P]Pi in unstimulated aleurone showed that both synthesis and degradation of polyphosphoinositides were relatively faster than those of structural phospholipids. We discuss the possible reasons why the changes in synthesis and turnover of phosphoinositide are required for GA signaling pathway and why it may be a critical event in the control of secretion in aleurone.

Characterization of Recombinant Phosphatidylinositol 4-Kinase β Reveals Auto- and Heterophosphorylation of the Enzyme

Phosphatidylinositol (PI) 4-kinases catalyze the synthesis of PI 4-phosphate, an important intermediate for the synthesis of membrane polyphosphoinositides, regulators of multiple cellular functions. Two mammalian PI 4-kinases have been cloned, a 230-kDa enzyme (alpha-form) and a 110-kDa (beta-form), both of which are inhibited by >0.1 microm concentrations of the PI 3-kinase inhibitor, wortmannin (WT). In the present study, we created a glutathione S-transferase-PI4Kbeta fusion protein for expression in Escherichia coli. The purified protein was biologically active and phosphorylated PI in its 4-position with WT sensitivity and kinetic parameters that were identical to those of purified bovine brain PI4Kbeta. In addition to its lipid kinase activity, the enzyme exhibited autophosphorylation that was enhanced by Mn(2+) ions and inhibited by WT and another PI 3-kinase inhibitor, LY 294002. The recombinant protein was unable to transphosphorylate, but its isolated C-terminal catalytic domain still displayed autophosphorylation, suggesting that the autophosphorylation site resides within the C-terminal catalytic domain of the protein and is held in position by intramolecular interactions. Autophosphorylation inhibited subsequent lipid kinase activity, which was reversed upon dephosphorylation, by protein phosphatases, PP1 and PP2A(1), suggesting that it may represent a regulatory mechanism for the enzyme. Phosphorylation of endogenous or overexpressed PI4Kbeta was also observed in COS-7 cells; however, the in vivo phosphorylation of the expressed protein was only partially inhibited by WT and also occurred in a catalytically inactive form of the enzyme, indicating the presence of additional phosphorylation site(s). Successful bacterial expression of PI4Kbeta should aid research on the structure-function relationships of this protein as well as of other, structurally related enzymes.

Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases.

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 4-kinases is the first step in the synthesis of polyphosphoinositides, the lipid precursors of intracellular signaling molecules. We have recently identified a cytosolic PtdIns 4-kinase (cPI4K) in the bovine adrenal cortex that is distinguished from previously known PtdIns 4-kinases by its sensitivity to the PtdIns 3-kinase inhibitor wortmannin (WT). The present study has further characterized this soluble enzyme and compared its properties to those of the membrane-bound, type II PtdIns 4-kinase activity of the adrenal cortex and the type III enzyme of bovine brain. The enzymatic activity of adrenal cPI4K was inhibited not only by WT (IC50 approximately 50 nM) but also by LY-294002 (IC50 approximately 100 microM), another inhibitor of PtdIns 3-kinase, and neither compound affected type II PtdIns 4-kinase at concentrations that inhibited cPI4K. In contrast to the type II enzyme, cPI4K had a significantly higher Km for ATP, was relatively insensitive to inhibition by adenosine (Ki approximately 800 microM vs approximately 40 microM), had lower affinity for PtdIns, and was not inhibited by Ca2+ ions. These properties identify the WT-sensitive adrenal cPI4K as a type III PtdIns 4-kinase that is distinct from the tightly membrane-bound, Ca2+- and adenosine-sensitive, type II PtdIns 4-kinase. The type III PtdIns 4-kinase prepared from bovine brain exhibited similar kinetic parameters as the adrenal cPI4K, and was also inhibited by WT with an IC50 of 30-50 nM. Since WT inhibits the synthesis of agonist-regulated phosphoinositide pools in intact cells at micromolar concentrations, these findings indicated that type III rather than type II PtdIns 4-kinases are responsible for the maintenance of the precursor phospholipids required for intracellular signaling through the inositol phosphate/Ca2+ pathway.

Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle

Synthesis of polyphosphoinositides has been studied in transverse (T−) tubule and sarcoplasmic reticulum (SR) membrane fractions of frog skeletal muscle, following 32P-labeling with [γ- 32P]ATP. Purified SR and T-tubule fractions respectively synthesize 9.4 ± 0.8 and 71.9 ± 9.8 pmol PtdInsP/mg protein per min, indicating nearly 8-fold higher activity of PtdIns kinase in the T-tubules than in the SR. The activity of this enzyme in both membrane systems is maximum at pH 7 and p Ca 6. PtdInsP 2 is synthesized from the endogenous PtdInsP, only in T-tubule membranes by the action of PtdInsP kinase. This lipid is the most intensely 32P-labeled phosphoinositide (181.7 ± 9.2 pmol/mg per min) in these membranes. PtdIns kinase in the T-tubule and SR membranes, and PtdInsP kinase in the former are modulated by the free [Mg 2+]. Loss of radiolabel from transiently maximal 32P-incorporation in polyphosphoinositides in T-tubule membranes, concomitant with a decrease in the ATP concentration in the incubation buffer, shows the occurrence of phosphoinositidases in these membranes. Under the conditions used, no such activities were evident in SR membranes. Compound 48/80, a mixture of condensation products of N-methyl- p-methoxyphenethylamine with formaldehyde, known to block phosphoinositidase C and phospholipase A 2, causes a dose-dependent increase in the 32P-label of PtdInsP, in T-tubule membranes. The synthesis of lyso PtdIsnP 2, a deacylated form of PtdInsP 2 which occurs in nearly equal quantities in both T-tubule and SR membranes may result from a mechanism independent of phospholipase A 2.

Stimulation by d-glucose of the synthesis of polyphosphoinositides in pancreatic islets

d-glucose (16.7 mM) stimulates the synthesis of polyphosphoinositides in in intact pacreatic islets prelabelled with tritiated myo-inositol and incubated in the absence of extracellular Ca 2+. ATP (1.0 mM) exerts a comparable effect in sonicates of prelabelled normal or tumoral islet cells. In the acellular system, ATP fails to affect the generation of tritiated inositol phosphates in the absence of Ca 2+, but augments the Ca 2+-stimulated production of inositol mono-, bis- and triphosphates. The latter effect is not reproduced by α, ß-methylene ATP, suggesting that it is not attributable to a purinergic mechanism. Whether in the absence or presence of ATP, the Ca 2+-induced increment in inositol phosphates production coincides with a comparable decrease in tritiated polyphosphoinositides. It is proposed, therefore, that the increased production of inositol phosphates in intact islets stimulated by nutrient secretagogues is attributable, in part at least, to an accelerated generation of polyphosphoinositides, possibly resulting from a rise in cytosolic ATP concentration.

Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.

Enzymic synthesis of pyrene-labeled polyphosphoinositides and their behavior in organic solvents and phosphatidylcholine bilayers

A method is reported for the synthesis of pyrene-labeled analogues of phosphatidylinositol 4-phosphate (Pyr-PIP) and phosphatidylinositol 4,5-biphosphate (Pyr-PIP2) from sn-2-(pyrenyl-decanoyl)phosphatidylinositol (Pyr-PI) using partially purified PI and PIP kinase preparations. Phosphorylation of Pyr-PI and Pyr-PIP was extensive (more than 50%) provided that the ATP concentration was high and that stabilizing agents such as sucrose and polyethylene glycol were present in the incubation medium. Pyr-PIP and Pyr-PIP2 were isolated by chromatography on immobilized neomycin. The identity of the products was established by thin-layer chromatography, UV-absorption spectroscopy, and spectrofluorometry. The pyrene excimer/monomer fluorescence technique revealed that, in contrast to Pyr-PI, Pyr-PIP and Pyr-PIP2 formed clusters in organic solvents. By use of the same technique for model membranes, it was shown that in phosphatidylcholine bilayers the collision frequency of the three fluorescent phosphoinositides decreased in the order PI greater than PIP greater than PIP2. Addition of Ca2+ at concentrations above 0.1 mM increased the collision frequency of Pyr-PIP2 and, to a much lesser extent, Pyr-PIP; Ca2+ had no effect on Pyr-PI.

Cloning and expression of bovine brain inositol monophosphatase.

Inositol monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated. All contain an open reading frame encoding a 277-amino acid protein. No significant sequence homology was found with any known protein. The open reading frame was inserted into a bacterial expression vector in order to confirm the presumed identity of the protein. The expressed protein reacted with an anti-inositol monophosphatase monoclonal antibody. In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to Km values for substrate and Li+ sensitivity of inositol 1-phosphate hydrolysis.

Lipid phosphorylation in isolated rat liver nuclei Synthesis of polyphosphoinositides at subnuclear level

Isolated rat liver nuclei and subnuclear fractions synthesize polyphosphoinositides in vitro in a mode dependent on the presence of nuclear membrane, detergent and exogenous substrates. The nuclear membrane is not essential as a source of lipid kinases, since the addition of erogenous phosphatidylinositol or phosphatidylinositol monophosphate to reaction mixtures lacking membranes restores the synthesis of phosphatidylinositol mono- and bisphosphate, respectively. Inositide phosphorylation is best accomplished by high-salt extracted nuclei and pre-detergent lamina. These data suggest that the nucleus, and especially the nuclear periphery, is a cell compartment in which polyphosphoinositide synthesis occurs; this might be related to the progression of phosphatidylinositol metabolism-dependent signals to the genetic apparatus.

Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation.

Previous work demonstrated the existence of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in rat liver nuclei, with the suggestion that these activities are in the nuclear membrane [Smith & Wells (1983) J. Biol. Chem. 258, 9368-9373]. Here we show that highly purified nuclei from Friend cells, washed free of nuclear membrane by Triton, can incorporate radiolabel from [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells, being barely detectable in growing cells and much greater after dimethyl sulphoxide-induced differentiation; this difference is mostly due to different amounts of phosphatidylinositol phosphate in the isolated nuclei. We suggest that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.

Effect of phenobarbital on metabolism of polyphosphoinositides of rat brain synaptosomes

Synthesis and degradation of polyphosphoinositides in a rat brain synaptosome preparation were depressed by phenobarbital. Phosphatidylinositol-4-phosphate kinase (PIP-kinase), the enzyme which synthesizes phosphatidylinositol-4, 5-bisphosphate (PIP 2) was most strongly affected (50% inhibition at 3 mM phenobarbital); phosphatidylinositol (PI-kinase) followed (50% at 15 mM). The phosphoesterases were less sensitive: PIP-monoesterase (50% at 39 mM), PIP 2-monoesterase (at 47 mM), and, least inhibited, PIP-diesterase (50% at 65 mM) and PIP 2-diesterase (at 68 mM). Phenobarbital by inhibiting PIP-kinase may reduce the membrane concentration of PIP 2 and thus dampen the stimulus-response which leads to the hydrolysis of PIP 2 and the formation of the second messenger, inositol-1,4,5-triphosphate (IP 3), involved in mobilization of intracellular Ca 2+.

Interleukin 2 rapidly stimulates synthesis and breakdown of polyphosphoinositides in interleukin 2-dependent, murine T-cell lines.

Several T-cell functions are controlled by the regulatory peptide interleukin 2 (IL-2). Binding of IL-2 with specific receptors has been well documented, but the molecular mechanism by which IL-2/IL-2 receptor interaction is transduced is not known. We have found that treatment of IL-2-dependent T-cell lines with IL-2 is followed by a rapid stimulation of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H]inositol. Increased incorporation of the metabolic precursor into phosphatidylinositol and phosphatidylinositol 4-monophosphate, together with the appearance of radiolabeled phosphatidylinositol 4,5-bisphosphate, occurred within minutes of treatment with IL-2 of factor-dependent CT6 cells. Analysis of labeled water-soluble compounds from prelabeled cells indicated a rapid (within 1 min) stimulation of inositol phospholipid hydrolysis following IL-2 treatment. Increased recovery of [3H] inositol phosphates and appearance of [3H]inositol trisphosphate were observed after treatment with IL-2 of CT6 cells, as well as of a second IL-2-dependent cell line, CTB6. These findings suggests that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain IL-2 signals are transduced.

Breakdown and synthesis of polyphosphoinositides in fMetLeuPhe-stimulated neutrophils

The interconversions of the inositol-containing lipids (PI, PI-P and PI-P 2) and their products (DG, inositol phosphates and PA) in human and rabbit neutrophils stimulated with fMetLeuPhe and PMA have been examined. PMA causes only the phosphorylation of PI to PI-P whereas fMetLeuPhe causes phosphorylation of both PI and PI-P yielding PI-P 2 and the hydrolysis of all three lipids. While the predominant reaction is breakdown of PI to PA catalysed by phospholipase D, approx. 2% of PI is converted to polyphosphoinositides and then broken down by the phospholipase C route yielding inositol phosphates and DG. The latter reaction occurs without detectable lag and is a function of receptor occupancy. The amount of inositol trisphosphate thus formed would be sufficient to release Ca 2+ from intracellular stores.

Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.