Phosphoinositide Concentration Research Articles

Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.

Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity.

Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.

Decoding the role of phosphoinositides in phototropin signaling involved in chloroplast movements

In angiosperms, light-dependent chloroplast movements are exclusively mediated by UVA/blue light receptors - phototropins. The two photoreceptors of Arabidopsis thaliana, phot1 and phot2, have overlapping roles in the control of these movements. Experiments performed in different plant species point to the participation of phosphoinositides in blue light-controlled chloroplast relocations. Here, we report a summary of recent findings presenting the involvement of phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 3- and 4-phosphates in weak blue light-mediated (accumulation) and strong blue light-mediated (avoidance) responses of chloroplasts. The blue light-activated alterations in phosphoinositide concentration are partly responsible for cytosolic Ca2+ changes. Ca2+ influx from apoplast does not seem to be involved in the mechanism of movement responses. In summary, interplay between phosphoinositides and intracellular Ca2+ regulates chloroplast redistribution in response to blue light in higher plants.

Analysis of the Binding Kinetics of Phosphoinositide Binding Domains Utilizing Voltage Sensing Phosphatases and Total Internal Reflection Fluorescence Microscopy

Changes in phosphoinositide concentrations ([PI]) in cell membranes constitute important signaling events. Such changes are detected by proteins containing PI binding domains like the pleckstrin homology (PH) domains, e.g. PI(4,5)P2 binding PLCδ1-PH, or PH unrelated PI-binding domains, e.g. the PI(4,5)P2 binding tubby domain. These domains can be tagged with a fluorescent label, thereby creating a tool for the detection of [PI] changes in living cells. Since [PI] can change on a sub-second timescale, it is conceivable that the kinetics of PI (un-)binding of these domains become a limiting factor in the detection of such changes. Curiously, for PLCδ1-PH conflicting data on these kinetics have been reported, with dissociation time constants ranging from about 0.25 s to nearly 3 s.To investigate this problem, we used voltage sensing phosphatases (VSPs) to rapidly deplete PI(4,5)P2 while monitoring the dissociation of GFP-tagged PLCδ1-PH and tubby domains from the plasma membrane using total internal reflection microscopy (TIRF-M) imaging at 10 or 20 frames per second. These experiments revealed rapid dissociation of both domains. If Xl-VSP1 was used, the kinetics of PI(4,5)P2 break down were rate limiting for the observed dissociation. In contrast, PI(4,5)P2 break down by Ci-VSP was sufficiently rapid to reveal differences in the time course of dissociation between both domains, with observed time constants below 1 s.In conclusion, we find the dissociation rates of PLCδ1-PH and tubby domains to be fast enough to faithfully track changes in [PI(4,5)P2] on a sub-second timescale.This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.

Class I Phosphatidylinositol Transfer Proteins (PITPs) Promote PIP2 Synthesis and IP3 Production in Mouse Platelets

Abstract 363Phosphoinositides are the phosphorylated forms of phosphatidylinositol. Although they constitute only 1% of platelet membrane phospholipids, phosphoinositides are important contributors to platelet signaling. As an example the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or PIP2) has been shown to regulate integrin activation, actin assembly, and secretion in platelets. PtdIns(4,5)P2 contributes to these events by directly binding to signaling proteins, and also by serving as a substrate for PLC and PI3K to generate the second messengers Inositol(3,4,5)P3 (Ins(3,4,5)P3) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The phosphoinositide concentration in different cellular compartments is tightly regulated both in time and space, and their relative amounts of various phosphoinositides change within seconds of agonist stimulation. Given their importance in platelet biology, we sought to better understand the mechanism of phosphoinositide synthesis and function. Class I PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that bind to and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner. Although there are no studies on the function of PITPs in hematopoietic cells, the proteins are essential in yeast cells for the biosynthesis and metabolism of phosphoinositides. This suggests that PITPs may also be essential for phosphoinositide metabolism in platelets. There are two dominant Class I PITP family members, PITPα and PITPβ in murine platelets. We introduced conditional loss-of-function mutations into the murine PITPα and PITPβ genes These mice were crossed with transgenic mice expressing Cre recombinase under the control of the platelet specific promoter PF4, thereby generating mice lacking individual PITP isoforms only in platelets and megakaryocytes. Mice lacking either platelet PITP isoform have approximately one-third lower platelet counts than littermate controls, but otherwise have normal blood counts and appear phenotypically normal. We have also generated a few viable double knockout (PITPαfl/fl PITPβfl/fl PF4Cre+) mice and found that these mice produce some platelets even in the absence of both PITP isoforms. Next, we analyzed the concentration of individual phosphoinositides in platelets of the knockout mice by thin layer chromatography. Although loss of either PITP isoform did not affect PtdIns(4)P synthesis in resting or thrombin stimulated platelets, PtdIns(4,5)P2 synthesis was significantly inhibited in platelets lacking either PITPα (∼40% of normal) or PITPβ (∼50% of normal). This suggests that both PITPα and PITPβ are required for normal PtdIns(4,5)P2 synthesis in platelets. To test whether PITP isoforms are critical for second messenger formation, we analyzed the formation of Ins(3,4,5)P3 in response to thrombin stimulation. We found that the PITPα-null mutation causes an almost absence of Ins(3,4,5)P3 formation. The PITPβ-null mutation also caused a profound defect in Ins(3,4,5)P3 formation, albeit less than that seen following PITPα deletion. Nonetheless, both PITPα and PITPβ knockout platelets were able to increase their intracellular calcium concentration following thrombin stimulation to approximately 50% of normal. These data imply that a significant portion of the calcium response in platelets results from the influx of extracellular calcium and not by the Ins(3,4,5)P3–dependent release of calcium from intracellular stores. Further, platelets lacking either PITP isoform exhibited only a moderate (∼50%) defect in Akt phosphorylation – a PtdIns(3,4,5)P3 synthesis-dependent event. Although both Ins(3,4,5)P3 and PtdIns(3,4,5)P3 are derived from PtdIns(4,5)P2, it is remarkable that PITP enzymes are more critical for Ins(3,4,5)P3-synthesis than for PtdIns(3,4,5)P3-synthesis (as suggested by Akt phosphorylation). Finally, we performed pilot experiments of the ex vivo function of platelets lacking both PITP isoforms and found that double knockout platelets had a profound loss of agonist-induced platelet aggregation. Together, our data suggest that PITPα and PITPβ are both critical for the synthesis of essential platelet phosphoinositides and second messengers. Thus, these enzymes may represent novel therapeutic targets for the prevention and treatment of arterial occlusion. Disclosures:No relevant conflicts of interest to declare.

Probing the regulation of TASK potassium channels by PI(4,5)P2 with switchable phosphoinositide phosphatases

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of Gαq is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) as the cause for channel closure or to a direct inhibitory interaction of active Gαq with the channel. Here, we address the role of PI(4,5)P2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell.When expressed in CHO cells, TASK-1- and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P2-5'-phosphatase. Depletion of both PI(4,5)P2 and PI(4)P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P2 and PI(4)P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P2.

Controlling the Activity of a Phosphatase and Tensin Homolog (PTEN) by Membrane Potential

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

Quantitative Analysis of Binding Affinities of PI(4,5)P2 Sensor Domains in Living Cells by Using the Voltage-Controlled PI(4,5)P2-5’-Phosphatase, Ci-VSP

Dynamic changes in the phosphoinositide (PI) concentration in the cell membrane play an important role in the regulation of many cellular processes. Of particular interest is the PI phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a genuine signaling molecule that, e.g., controls the function of some ion channels. Plasma membrane PI concentration in living cells is often monitored with genetically encoded fluorescence labeled PI-binding probes such as the PI(4,5)P2 specific PLCδ1-PH-GFP and tubby-GFP probes. Knowledge of their PI(4,5)P2 affinities is essential in selecting the most suitable probes and for appropriate interpretation of experimental results. These affinities of PI(4,5)P2 probes apparently differ and can be modified by mutagenesis; however, a quantitative analysis of affinity in vivo is lacking. To address this issue we employed the voltage dependent PI(4,5)P2-5′-phosphatase Ci-VSP which alters plasma membrane [PI(4,5)P2] as a function of membrane voltage in a graded and reversible manner (Halaszovich et al., 2009, JBC 284:2106-13). We co-expressed Ci-VSP with various GFP-tagged PI(4,5)P2 probes and used total internal reflection microscopy (TIRF-M) to measure membrane association of the probe at different membrane voltages. Generally, the TIRF signal decreased upon depolarization, indicating the translocation of the probe from the cell membrane into the cytosol in response to the decrease in [PI(4,5)P2]. Voltage-dependent changes of TIRF signals allowed construction of fluorescence-voltage relations for each probe that can be used as surrogate PI(4,5)P2 binding curves. Comparison of these curves defined the rank order of PI(4,5)P2 affinities of the various probes. We conclude that Ci-VSP is a valuable tool for analyzing PI(4,5)P2 affinities in living cells, which we demonstrated by determining the affinities of fluorescence labeled PI(4,5)P2-probes. Supported by DFG grant OL 240/2 and SFB 593 to D.O.

Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.

Sensing voltage for function

The first protein to sense changes in membrane potential but not function as an ion transporter is now identified by Yoshimichi Murata, Hirohide Iwasaki, Mari Sasaki, Yasushi Okamura, and colleagues (National Institutes of Natural Sciences, Aichi, Japan). Activation of this sensor probably induces phosphorylation-based signaling events.The group discovered this sea squirt protein, Ci-VSP, based on its sequence homology with ion channels. But the homology was confined to four transmembrane segments that comprise a voltage sensor. This domain functions just like the voltage sensor domains of channel proteins. Ci-VSP also contains a cytoplasmic phosphatase domain, but lacks a pore domain to transport ions across the plasma membrane. Figure Ci-VSP's voltage sensor (blue) regulates a phosphatase domain (green), not a pore domain (pink), as in voltage-gated channels. Using an in vivo bioassay, Okamura's group showed that Ci-VSP changes cellular phosphoinositide concentrations in response to membrane potential changes. “These data provide the first evidence since the Hodgkin-Huxley age that a molecular function other than that of ion channels is regulated by membrane voltage,” he says. Ci-VSP is expressed in sperm and might function in sperm motility or morphology. The authors' next goal is to identify the natural substrate of Ci-VSP, which they suspect is PIP3, so that they can determine whether membrane hyperpolarization or depolarization activates the enzyme. Reference: Murata, Y., et al. 2005. Nature. 435:1239–1243. [PubMed]

Measurement and immunofluorescence of cellular phosphoinositides.

Phosphoinositides are a vitally important class of intracellular-signaling molecules that regulate cellular processes, including signaling through cell-surface receptors, remodeling of the cytoskeleton, vesicle-mediated protein trafficking, and various nuclear functions. Methods for the analysis of in vivo phosphoinositide concentration, such as the one described in this chapter enable quantification of all phosphoinositides from a population of cells. This method involves metabolic labeling of cells with myo<-[2-3H] inositol, followed by lipid extraction, and quantification by high-performance liquid chromatography (HPLC). It provides improved efficiency and reproducibility when analyzing yeast, plant cells, and is applicable to animal cells as well. In addition, a technique for determining the intracellular location of phosphoinositides is described. When quantification and localization techniques are used in parallel, an investigator can identify cell, and even subcellular concentration changes. The technique described in this chapter uses immunodetection with antiphosphoinositide antibodies to determine the localization and relative concentrations of phosphinositides in fixed cells. The availability of antibodies allows an investigator to perform immunofluorescence and potentially immunoelectron microscopy of phosphoinositide localization on particular cellular, organellar, or vesicular membranes.

PIP 2-PDZ Domain Binding Controls the Association of Syntenin with the Plasma Membrane

PDZ proteins organize multiprotein signaling complexes. According to current views, PDZ domains engage in protein-protein interactions. Here we show that the PDZ domains of several proteins bind phosphatidylinositol 4,5-bisphosphate (PIP 2). High-affinity binding of syntenin to PIP 2-containing lipid layers requires both PDZ domains of this protein. Competition and mutagenesis experiments reveal that the protein and the PIP 2 binding sites in the PDZ domains overlap. Overlay assays indicate that the two PDZ domains of syntenin cooperate in binding to cognate peptides and PIP 2. Experiments on living cells demonstrate PIP 2-dependent and peptide-dependent modes of plasma membrane association of the PDZ domains of syntenin. These observations suggest that local changes in phosphoinositide concentration control the association of PDZ proteins with their target receptors at the plasma membrane.

The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.

Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators

Conditions for Superoxide anion (O − 2) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O − 2 production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O − 2 production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O − 2 production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 μ m OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O − 2 production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.

Microwave induced stimulation of32Pi incorporation into phosphoinositides of rat brain synaptosomes

Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the 32Pi-incorporation into phosphoinositides. The extent of 32Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate 32Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP2, PIP and PI and increased PA labeling. Li+ also inhibited the microwave stimulated PIP2, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A23187, inhibited the basal and microwave stimulated 32Pi labeling of PIP and PIP2, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP2 labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.

Reduced phosphoinositide concentrations in anterior temporal cortex of Alzheimer-diseased brains.

Samples of brain anterior temporal cortex from 17 patients with Alzheimer's disease and 18 age-matched controls have been analysed for myo-inositol and the three phosphoinositides. There was significantly less phosphatidylinositol in the Alzheimer samples (1.36 mumol/g wet weight) than in the controls (2.28 mumol/g). The concentrations of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were also lower in Alzheimer cortex, but differences from the control group were not statistically significant. Free myo-inositol concentrations were 5.11 mumol/g (Alzheimer) and 4.44 mumol/g (control) and again the difference was not significant. The lack of phosphoinositides in Alzheimer temporal cortex may impair receptor function.

Incorporation of injected [32P] phosphate into the phosphoinositides of subcellular fractions from young rat brain.

1. Six subcellular fractions were prepared from the brain of 3-week-old rats after the injection of [(32)P]orthophosphate into the subarachnoidal space. The fractions were characterized by chemical and enzymic determinations and by electron microscopy. 2. The highest concentration of phosphoinositides was found in the microsomal fraction. 3. After an exchange period of 4hr. in vivo, the specific radioactivity of phosphatidylinositol was high in the nuclear, mitochondrial and supernatant fractions, and that of diphosphoinositide was high in the nuclear, microsomal and supernatant fractions. Triphosphoinositide specific activity was highest in the myelin fraction. 4. Specific activities (counts/100sec./mug. of P) were in the following order for all fractions except myelin: diphosphoinositide>phosphatidylinositol>triphosphoinositide. For myelin the order was: diphosphoinositide>triphosphoinositide>phosphatidylinositol. 5. Phosphatidylinositol extracted from a tissue fraction by acidified chloroform-methanol had roughly twice the specific activity of that extracted by neutral solvents. The same applied to diphosphoinositide.