Pathways for phosphoinositide synthesis.

In eukaryotic cells, phosphatidylinositol can be phosphorylated on the inositol ring by a series of kinases to produce at least seven distinct phosphoinositides. These lipids have been implicated in a variety of cellular processes, including calcium regulation, actin rearrangement, vesicle trafficking, cell survival and mitogenesis. The phosphorylated lipids can act as precursors of second messengers or act directly to recruit specific signaling proteins to the membrane. A number of the kinases responsible for producing these lipids have been purified and their cDNA clones have been isolated. The most well characterized of these enzymes are the phosphoinositide 3-kinases. However, progress has also been made in the characterization of phosphatidylinositol 4-kinases and phosphatidylinositol-4-phosphate 5-kinases. In addition, new pathways involving phosphatidylinositol-5-phosphate 4-kinases, phosphatidylinositol-3-phosphate 5-kinases and phosphatidylinositol-3-phosphate 4-kinases have recently been described. The various enzymes and pathways involved in the synthesis of cellular phosphoinositides will be discussed.     

Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography

Recent advances in research on the physiological roles of phosphoinositides in eukaryotic organisms indicate a need to distinguish molecular phosphoinositide species on the basis of their characteristic head groups as well as their glycerolipid moieties. Accurate identification of phosphoinositide species in biological samples poses an analytical challenge, because structurally similar inositol phosphate head groups must be resolved, as must lipid-associated fatty acids. Although intact phosphoinositide species have been successfully analyzed, such analyses employ state-of-the-art liquid chromatography/mass spectrometry and require expensive equipment not accessible to many researchers. Described here is a cost-efficient and reliable alternative developed by adaptation of a combination of classic methods for lipid analysis, thin-layer chromatography and gas chromatography.     

Elevating PI3P drives select downstream membrane trafficking pathways

Phosphoinositide signaling lipids are essential for several cellular processes. The requirement for a phosphoinositide is conventionally studied by depleting the corresponding lipid kinase. However, there are very few reports on the impact of elevating phosphoinositides. That phosphoinositides are dynamically elevated in response to stimuli suggests that, in addition to being required, phosphoinositides drive downstream pathways. To test this hypothesis, we elevated the levels of phosphatidylinositol-3-phosphate (PI3P) by generating hyperactive alleles of the yeast phosphatidylinositol 3-kinase, Vps34. We find that hyperactive Vps34 drives certain pathways, including phosphatidylinositol-3,5-bisphosphate synthesis and retrograde transport from the vacuole. This demonstrates that PI3P is rate limiting in some pathways. Interestingly, hyperactive Vps34 does not affect endosomal sorting complexes required for transport (ESCRT) function. Thus, elevating PI3P does not always increase the rate of PI3P-dependent pathways. Elevating PI3P can also delay a pathway. Elevating PI3P slowed late steps in autophagy, in part by delaying the disassembly of autophagy proteins from mature autophagosomes as well as delaying fusion of autophagosomes with the vacuole. This latter defect is likely due to a more general defect in vacuole fusion, as assessed by changes in vacuole morphology. These studies suggest that stimulus-induced elevation of phosphoinositides provides a way for these stimuli to selectively regulate downstream processes.     

The Role of Phosphoinositides in Synapse Function

Since the discovery of phosphatidylinositol-3-kinase, scientific interest in the biological functions of phosphoinositides has greatly increased. Currently, seven phosphoinositides have been identified. These phosphoinositides are specifically localized to organelle membranes, their site of action. Phosphoinositides can regulate neuronal function by specifically recruiting downstream proteins that have phosphoinositide-binding domains. To date, it is well accepted that phosphoinositides play important roles in a broad spectrum of neuronal functions from regulating neural development to modulating synapse function. This review will provide an overview of the function and distribution of phosphoinositides at synapses.     

Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling

The C-terminal Eps15 homology domain (EHD) 1/receptor-mediated endocytosis-1 protein regulates recycling of proteins and lipids from the recycling compartment to the plasma membrane. Recent studies have provided insight into the mode by which EHD1-associated tubular membranes are generated and the mechanisms by which EHD1 functions. Despite these advances, the physiological function of these striking EHD1-associated tubular membranes remains unknown. Nuclear magnetic resonance spectroscopy demonstrated that the Eps15 homology (EH) domain of EHD1 binds to phosphoinositides, including phosphatidylinositol-4-phosphate. Herein, we identify phosphatidylinositol-4-phosphate as an essential component of EHD1-associated tubules in vivo. Indeed, an EHD1 EH domain mutant (K483E) that associates exclusively with punctate membranes displayed decreased binding to phosphatidylinositol-4-phosphate and other phosphoinositides. Moreover, we provide evidence that although the tubular membranes to which EHD1 associates may be stabilized and/or enhanced by EHD1 expression, these membranes are, at least in part, pre-existing structures. Finally, to underscore the function of EHD1-containing tubules in vivo, we used a small interfering RNA (siRNA)/rescue assay. On transfection, wild-type, tubule-associated, siRNA-resistant EHD1 rescued transferrin and β1 integrin recycling defects observed in EHD1-depleted cells, whereas expression of the EHD1 K483E mutant did not. We propose that phosphatidylinositol-4-phosphate is an essential component of EHD1-associated tubules that also contain phosphatidylinositol-(4,5)-bisphosphate and that these structures are required for efficient recycling to the plasma membrane.     

Separation of phosphatidylinositols and other phospholipids by two-step one-dimensional thin-layer chromatography

A quick and efficient thin-layer chromatographic procedure is described for the separation of phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol, phosphatidylcholine, phosphatidic acid, and phosphatidylethanolamine. The method involves development of the phospholipids successively in two different solvent systems but in the same direction. The method is simple, reproducible, and gives good resolution of the six different phospholipids tested. About 8-10 32P-labeled phospholipids isolated from rat hepatocytes were separated by this method; the six mentioned above were identified. Thus, the technique has potential application for both qualitative and quantitative analysis of phospholipid mixtures, such as the phosphoinositides, in cell or tissue extracts.     

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [³²P]orthophosphate or myo-[³H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of ³²P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.     

Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells.

Quantitative changes in the total mass and the molecular species of 1,2-diacyl-sn-glycerol (DAG) and phosphatidic acid (PA) formed upon muscarinic receptor activation were studied in cultured human SK-N-SH neuroblastoma cells. DAG was isolated from the total lipid extracts of carbachol (CCh)-stimulated and unstimulated cells and after benzoylation, was subjected to reverse phase high performance liquid chromatography to separate the component species. The molecular species of DAG were identified by analyzing the fatty acid composition of each separated fraction by gas chromatography, and their total and individual masses were quantified from the known amount of an internal standard, 1,2-distearoyl-sn-glycerol, added during the extraction of the lipid. Relatively high basal levels of DAG (1.5 nmol/mg protein) are present in these cells, and addition of CCh elicited a 50-60% increase in the total amounts of DAG within 5 min. The increase was biphasic: an initial major peak at 5 min was followed by a sustained increase that persisted for at least 30 min. An increase in DAG was elicited by both full and partial muscarinic agonists and was blocked by atropine. The presence of extracellular Ca2+ was necessary for muscarinic receptor-activated formation of DAG. To determine the source of the DAG, the molecular species of the major phospholipids present in SK-N-SH cells were also analyzed. The phospholipids were first enzymatically hydrolyzed to DAGs which were then analyzed as described above. A number of unusual fatty acids, the major one being 20:3 (n-9), were present in these lipids especially in the phosphoinositides and also in the DAG formed after CCh stimulation. Within 5 s of CCh stimulation there were transient increases in the DAG species representative of phosphoinositides. By 5 min the newly formed molecular species of DAG resembled a mixture of phosphoinositides and phosphatidylcholine (PC). Quantitative comparison of the molecular species compositions of phosphoinositides, PC, and newly formed DAGs indicated that at time periods up to 10 min, approximately 30% of the DAG originated from the phosphoinositides and the rest from PC. At longer intervals (greater than 20 min), most (85%) of DAGs originated from PC. Activation of muscarinic receptors in SK-N-SH cells also elicited an increase in PA (200% in 5 min). A quantitative molecular species analysis, using 1,2-distearoyl-sn-glycerol-3-P as internal standard, was performed by enzymatic (alkaline phosphatase) hydrolysis of PA to DAG and subsequent analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a phosphoinositide-specific phospholipase C from bovine brain

A soluble phosphoinositide-specific phospholipase C (PLC) was purified 58,000-fold from bovine brain. The enzyme, one of six distinct PLC activities detected in brain, accounted for approximately 15% of the soluble phosphatidylinositol-4,5-bisphosphate-phospholipase C (PIP2-PLC) activity in this tissue. The purification scheme included hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on phosphatidylinositol-Sepharose (PI-Sepharose). The enzyme was specifically eluted from the PI-Sepharose with PI, calcium, and detergent. The purified PLC had an estimated molecular weight of 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaved as a monomeric protein during sedimentation on glycerol gradients. The enzyme required calcium for activity, exhibited a pH optimum of 6.5, and cleaved only phosphoinositides. The rates of PIP2 and phosphatidyl-4-monophosphate hydrolysis exceeded the rate of PI hydrolysis under all conditions tested. These properties are consistent with a potential role for this PLC in the early events involved in cellular calcium mobilization.     

The phosphoinositide kinase PIKfyve/Fab1p regulates terminal lysosome maturation in Caenorhabditis elegans

Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.     

Chemical synthesis of all-trans-beta-retinoyl phosphat.

all-trans-beta-Retinoic acid is phosphorylated to retinoyl phosphate by bis(triethylamine) phosphate with yields of 10-15%. The product is soluble in methanol and is eluted from DEAE-cellulose acetate at a concentration of 0.1M-ammonium acetate in 99% (v/v) methanol. Its phosphate/retinoic acid molar ratio is 1. Retinoyl phosphate has an absorption maximum at 360nm in methanol, whereas retinoic acid has a maximum at 350 nm. The compound is hydrolysed at pH2 and pH13 for 20 min at 37 degrees C, but is relatively stable under the same conditions at pH4, 6, 8 and 10. Retinoyl phosphate (RF 0.1) can be separated from retinyl phosphate (RF 0.2) by chromatography on thin layers of silica gel in chloroform/methanol/water (60:25:4, by vol.).     

Phosphoinositide metabolism in sections of the cortex of the large hemisphere of the brain in anoxia]

Depolarization (an increased concentration of KCL in the medium) has been investigated for its effect on the content and turnover rate of phospholipid phosphorus from the rat brain cortex slice under normal oxygen supply and under anoxia. It is shown that anoxia results in a small increase of phosphatidyl-inositol-4.5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) content and in the depression of the turnover rate of all the phosphoinositides. Depolarization leads to a decrease in PIP2 concentration with a simultaneous increase in their turnover rate, these results being more expressed under anoxia. The development of depolarization by the 5th min. of anoxia in vivo leads, most probably, to the enhanced PIP2 breakdown, that is to a progressive decrease in their content.     

Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry

An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)₂(C18:1)₂ versus (C18:2)₃(C18:0)₁. These compounds were then analyzed by HPLC-MS³ to examine specific diac ylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined.     

一种简便的肌醇磷脂微量分析方法

分析磷脂酰肌醇循环(PI cycle)的磷脂组分常采用双向薄层层析法．建立了一个简单快速的单向薄层层析分离肌醇磷脂方法．首先采用不同的有机溶剂体系分别提取非多磷酸肌醇磷脂和多磷酸肌醇磷脂,然后用不同的层析展开体系,对两部分磷脂进行单向薄层层析分离．采用无载体 ³²P标记实验对该方法分离效果进行了观察．此法适用于同位素标记和非标记样品中肌醇磷脂组分的比较分析及多磷酸肌醇磷脂的提取、纯化和定量．     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

Fusion,Leakage and Surface Hydrophobicity of Vesicles Containing Phosphoinositides: Influence of Steric and Electrostatic Effects

Calcium and lanthanum ion-induced fusion of lipid vesicles containing phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) and its associated membrane properties, e.g., surface dielectric constant and vesicle leakage, were studied by fluorescence methods. The presence of poly-phosphorylated phosphoinositides (PPI) in lipid vesicles enhanced fusion, depending on the PPI phosphorylation level and the PPI concentration, as determined by the lipid mixing assay. This correlation held even at physiologically relevant small concentrations of PPI in vesicle membranes. However, the presence of nonphosphorylated PI inhibited fusion due to the steric effect of the inositol ring. The cation threshold concentration for the lipid mixing of vesicles made of mixtures of phosphatidylserine (PS) with PI increased with increasing PI contents. For all vesicle systems studied, a decrease in vesicle surface dielectric constant and an increase in vesicle leakage accompanied fusion. The presence of the nonphosphorylated inositol ring in PI did not interfere with the changes in the surface dielectric constant caused by fusogenic cations. Therefore, we deduce that the reduction of the surface dielectric constant is a necessary condition for membrane fusion to occur but it does not correlate with membrane fusion when interacting membranes are blocked for close approach as by the nonphosphorylated inositol ring.     

Total synthesis of chain-length-uniform dolichyl phosphates and their fitness to accept hexoses in the enzymatic formation of lipoglycans

Dolichols of defined uniform chain length (C35, C45, and C55) and geometry were prepared by total synthesis according to the following principle: (E,E)-Farnesol, activated as its 4-tolyl sulfone, is condensed with 8-chloroneryl benzyl ether, the sulfonyl group removed and the ether linkage cleaved by lithium/triethylamine. The resulting elongated prenol is converted again to its corresponding 4-toly/sulfone; at this stage isomers are removed by chromatography. After several cycles of this C10-elongation sequence the synthesis is completed in the same way but using 8-chlorocitronellyl benzyl ether as building block to introduce the saturated alpha-isoprene unit. The dolichols obtained were chemically phosphorylated (POCl3/Et3N). Both, the alcohols and their phosphate esters, are characterized spectroscopically. 1H- and 13C-NMR data are recorded for qualitative and stereochemical comparison with natural dolichols. The authentic dolichyl phosphates (Dol-7-P, Dol-9-P, and Dol-11-P) were assayed relative to the natural dolichyl phosphate mixture from pig liver as acceptors for transglycosylation from nucleoside diphosphate sugars (glucose, mannose) by standardized membrane vesicle preparations from plants (Volvox) and animals (liver). Even the shortest chain dolichyl 7-phosphate has full activity in this lipoglycan-forming reaction.     

Insulin and epidermal growth factor do not affect phosphoinositide metabolism in rat liver plasma membranes and hepatocytes

Recent studies with viral oncogene tyrosine kinases have suggested that these kinases may phosphorylate phosphoinositides and diacylglycerol. Since the receptors for insulin and epidermal growth factor (EGF) also possess tyrosine kinase activity, we have investigated possible effects of insulin and EGF on phosphoinositide metabolism in rat liver plasma membranes and rat hepatocytes. In plasma membranes prepared from rats injected 18 h prior with [3H]myo-inositol or incubated with [gamma-32P]ATP, phosphatidylinositol-4-P and phosphatidylinositol-4,5-P2 were formed, but there were no effects of either insulin or EGF although these agents stimulated protein tyrosine phosphorylation. In hepatocytes incubated with [3H]myo-inositol, label was incorporated into phosphatidylinositol, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2, but there was no effect of insulin. Incubation of hepatocytes with [3H]myo-inositol plus insulin or EGF for 2 h also did not alter the formation of [3H]myo-inositol-1,4,5-P3 from [3H]phosphatidylinositol-4,5-P2 induced by vasopressin. These findings suggest that the tyrosine kinase activity of liver insulin and EGF receptors is not important in phosphoinositide formation.     

Early effects of luteinizing hormone on mitochondrial phosphoinositides in ovarian follicles

It is not clear if luteinizing hormone (LH) stimulates breakdown as well as synthesis of phosphoinositides in ovarian tissue. Possibly, LH stimulation results in hydrolysis of ovarian phosphoinositides in discrete subcellular compartments while increasing their synthesis at other sites. To investigate this hypothesis, we determined the effects of LH on phosphoinositide metabolism in whole homogenates and mitochondria of ovarian follicles. Medium (3-7 mm) follicles from porcine ovaries were preincubated for 2 h in phosphate (PO4)-free medium with 32PO4, and incubated without or with LH (1 microgram/ml). Phosphatidylinositol (PI) and related compounds, phosphatidic acid (PA), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), accounted for 40% of the radiolabeled phospholipids in whole homogenates and over 60% in mitochondria from preincubated follicles. After 5 min, LH caused a significant decrease in radiolabeling of PIP2 and PIP in mitochondria, but not in whole homogenates. Luteinizing hormone increased radiolabeling of PIP2, PIP, PI and PA within 10 min in whole homogenates, and within 20 to 30 min in mitochondria. This delayed increase in radiolabeling of mitochondrial phosphoinositides after LH treatment was accompanied by decreases in PIP2, PIP and PI radiolabeling in whole homogenates. Follicles also were preincubated for 4 h with [3H]inositol, then for 15 min with 10 mM LiCl (an inhibitor of inositol phosphate hydrolysis). Inositol phosphate accumulation in 30 min was 2.7 times higher in homogenates of LH-treated follicles then in untreated follicles. Also, LH significantly decreased inositol bisphosphate, but did not change inositol trisphosphate accumulation. Accumulation of inositol phosphates in mitochondria was not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.