Inositol Metabolites Research Articles

Aggregated beta amyloid peptide 1-40 decreases Ca2+- and cholinergic receptor-mediated phosphoinositide degradation by alteration of membrane and cytosolic phospholipase C in brain cortex.

The effects of full-length amyloid beta protein, A(beta) (1-40), on phosphoinositide-specific phospholipase C (PLC) were investigated in synaptic plasma membranes (SPM) and cytosol prepared from the cerebral cortex of adult rats. Moreover, the role of A(beta) (1-40) on the activation of lipid peroxidation was evaluated. The activity of phospholipase C (PLC) acting on phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2) was determined using exogenous labeled substrates. The subcellular fractions were the source of enzyme(s). The radioactivity of lipid messengers derived from degradation of [14C- arachidonoyl] PI was also determined. The stable aggregated form of beta-amyloid peptide (1-40) at 25 microM concentration exerted reproducible effects. The aggregated form of A(beta) (1-40) inhibited Ca(2+)-regulated PI and PIP2 degradation by SPM and cytosolic enzymes. Aggregated A(beta) also decreased significantly the level of diacylglycerol, the product of PLC. This additionally supports the inhibitory effect of A(beta) on membrane-bound and cytosolic PLC. Moreover, A(beta) (1-40) significantly decreased the basal activity of the PIP2-PLC in SPM and the enzyme activity regulated through cholinergic receptors. However, in spite of the lower enzyme activity, the percentage distribution of inositol (1,4,5) P3 radioactivity (IP3) in the total pool of inositol metabolites was not significantly changed. The aggregated neurotoxic fragment, A(beta) (25-35), mimicked the effect of full-length A(beta) (1-40). A(beta) (1-40) enhanced the level of malondialdehyde indicating an activation of free radical stimulated membrane lipid peroxidation that may be involved in alteration of phospholipase(s) activity. Our results indicated that aggregated A(beta) (1-40) alters Ca(2+)-dependent phosphoinositide degradation affecting synaptic plasma membrane and cytosolic phospholipase(s) activity. Moreover, this peptide significantly decreased the phosphoinositide-dependent signal transduction mediated by cholinergic receptors. The effect of aggregated A(beta) (1-40) is more pronounced than that of the neurotoxic fragment A(beta) (25-35). Our study suggests that the deposition of aggregated A(beta) may alter phosphoinositide signaling in brain.

Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein alpha (PI-TPalpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPalpha. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPalpha. Overexpression of PI-TPalpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13-14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G(1) phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar. Upon equilibrium labeling of the cells with myo-[(3)H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4, 5-bisphosphate were the same as those in control cells. The addition of PI-TPalpha to a total lysate of myo-[(3)H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca(2+) further increased this formation. Based on these observations, we propose that PI-TPalpha is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.

Multiple Inositol Polyphosphate Phosphatase: Evolution as a Distinct Group within the Histidine Phosphatase Family and Chromosomal Localization of the Human and Mouse Genes to Chromosomes 10q23 and 19

Multiple inositol polyphosphate phosphatase is the only enzyme known to hydrolyze the abundant metabolites inositol pentakisphosphate and inositol hexakisphosphate. We have previously demonstrated that the chick homolog of multiple inositol polyphosphate phosphatase, designated HiPER1, has a role in growth plate chondrocyte differentiation. The relationship of these enzymes to intracellular signaling is obscure, and as part of our investigation we have examined the murine ((MMU)Minpp1) and human ((HSA)MINPP1) homologs. Northern blot analysis demonstrated expression of ((MMU)Minpp1in a variety of mouse tissues, comparable to the expression of other mammalian homologs, but less restricted than the expression ofHiPER1in chick. A purified (MMU)Minpp1 fusion protein cleaved phosphate from inositol (1,3,4,5)-tetrakisphosphate andpara-nitrophenyl phosphate. When the presumptive active site histidine was altered to alanine by site-directed mutagenesis, enzyme activity was abolished, confirming the classification of (MMU)Minpp1 as a histidine phosphatase. The amino acid sequences of the murine and human MINPP proteins share >80% identity with the rat enzyme and >56% identity with HiPER1, with conservation of the C-terminal consensus sequence that retains proteins in the endoplasmic reticulum. The intron/exon structure of the mammalian (MMU)Minpp1and (HSA)MINPP1genes is also conserved compared to the chickHiPER1gene. Sequence analysis of plant and fruit fly MINPP homologs supports the hypothesis that the MINPP enzymes constitute a distinct evolutionary group within the histidine phosphatase family. We have mapped (HSA)MINPP1to human chromosome 10q23 by fluorescencein situhybridization, YAC screening, and radiation hybrid mapping. This assignment places (HSA)MINPP1in a region of chromosome 10 that is frequently mutated in human cancers and places (HSA)MINPP1proximal to the tumor suppressorPTEN,which maps to 10q23.3. Using a radiation hybrid panel, we localized (MMU)Minpp1to a region of mouse chromosome 19 that includes the murine homolog ofPten.The evolutionary conservation of this novel enzyme within the inositol polyphosphate pathway suggests a significant role for multiple inositol polyphosphate phosphatase throughout higher eukaryotes.

Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae.

We recently demonstrated that the S. cerevisiae INP51 locus (YIL002c) encodes an inositol polyphosphate 5-phosphatase. Here we describe two related yeast loci, INP52 (YNL106c) and INP53 (YOR109w). Like Inp51p, the primary structures of Inp52p and Inp53p resemble the mammalian synaptic vesicle-associated protein, synaptojanin, and contain a carboxy-terminal catalytic domain and an amino-terminal SAC1-like segment. Inp51p (108 kD), Inp52p (136 kD) and Inp53p (124 kD) are membrane-associated. Single null mutants (inp51, inp52, or inp53) are viable. Both inp51 inp52 and inp52 inp53 double mutants display compromised cell growth, whereas an inp51 inp53 double mutant does not. An inp51 inp52 inp53 triple mutant is inviable on standard medium, but can grow weakly on media supplemented with an osmotic stabilizer (1 M sorbitol). An inp51 mutation, and to a lesser degree an inp52 mutation, confers cold-resistant growth in a strain background that cannot grow at temperatures below 15 degrees. Analysis of inositol metabolites in vivo showed measurable accumulation of phosphatidylinositol 4,5-bisphosphate in the inp51 mutant. Electron microscopy revealed plasma membrane invaginations and cell wall thickening in double mutants and the triple mutant grown in sorbitol-containing medium. A fluorescent dye that detects endocytic and vacuolar membranes suggests that the vacuole is highly fragmented in inp51 inp52 double mutants. Our observations indicate that Inp51p, Inp52p, and Inp53p have distinct functions and that substrates and/or products of inositol polyphosphate 5-phosphatases may have roles in vesicle trafficking, membrane structure, and/or cell wall formation.

Inositol phosphates and inositol phospholipids: how big is the iceberg?

Multiple inositol polyphosphate phosphatase is the only enzyme known to hydrolyze the abundant metabolites inositol pentakisphosphate and inositol hexakisphosphate. We have previously demonstrated that the chick homolog of multiple inositol polyphosphate phosphatase, designated HiPER1, has a role in growth plate chondrocyte differentiation. The relationship of these enzymes to intracellular signaling is obscure, and as part of our investigation we have examined the murine ((MMU)Minpp1) and human ((HSA)MINPP1) homologs. Northern blot analysis demonstrated expression of ((MMU)Minpp1in a variety of mouse tissues, comparable to the expression of other mammalian homologs, but less restricted than the expression ofHiPER1in chick. A purified (MMU)Minpp1 fusion protein cleaved phosphate from inositol (1,3,4,5)-tetrakisphosphate andpara-nitrophenyl phosphate. When the presumptive active site histidine was altered to alanine by site-directed mutagenesis, enzyme activity was abolished, confirming the classification of (MMU)Minpp1 as a histidine phosphatase. The amino acid sequences of the murine and human MINPP proteins share >80% identity with the rat enzyme and >56% identity with HiPER1, with conservation of the C-terminal consensus sequence that retains proteins in the endoplasmic reticulum. The intron/exon structure of the mammalian (MMU)Minpp1and (HSA)MINPP1genes is also conserved compared to the chickHiPER1gene. Sequence analysis of plant and fruit fly MINPP homologs supports the hypothesis that the MINPP enzymes constitute a distinct evolutionary group within the histidine phosphatase family. We have mapped (HSA)MINPP1to human chromosome 10q23 by fluorescencein situhybridization, YAC screening, and radiation hybrid mapping. This assignment places (HSA)MINPP1in a region of chromosome 10 that is frequently mutated in human cancers and places (HSA)MINPP1proximal to the tumor suppressorPTEN,which maps to 10q23.3. Using a radiation hybrid panel, we localized (MMU)Minpp1to a region of mouse chromosome 19 that includes the murine homolog ofPten.The evolutionary conservation of this novel enzyme within the inositol polyphosphate pathway suggests a significant role for multiple inositol polyphosphate phosphatase throughout higher eukaryotes.

Overexpression of D-myo-inositol-3-phosphate synthase leads to elevated levels of inositol in Arabidopsis.

In this paper, we report on the generation of transgenic Arabidopsis plants containing elevated levels of the gene product encoding the enzyme catalysing the first committed step in inositol biosynthesis, D-myo-inositol-3-phosphate (Ins3P) synthase. These plants exhibit both an increase in Ins3P synthase activity and an increase in the level of free inositol of over four-fold compared to wild-type plants. Despite these changes, we could detect no significant difference in phenotype in the transgenic plants for a number of characteristics linked with putative functions of inositol and inositol-derived metabolites. Our results indicate that the proposed engineering of inositol metabolism to generate specific plant phenotypes (e.g. salt tolerance) may require the manipulation of several genes, and that Ins3P synthase activity can be manipulated to increase the pool size of free inositol.

Non-muscarinic- and non-adrenergic-mediated effects of lindane on phosphoinositide hydrolysis in rat brain cortex slices.

The influence of lindane upon phosphatidylinositol hydrolysis in rat brain cortex slices has been investigated using anion-exchange chromatography in order to separate the water-soluble inositol metabolites. Acetylcholine, noradrenaline, and lindane induce the accumulation of myo-[2-3H]inositol as the water-soluble inositol metabolites. However, the cholinergic muscarinic antagonist atropine inhibited the stimulatory response of carbachol, but practically unmodified the effect that lindane has on inositol phosphate production. Also, prazosin anti-alpha 1 adrenoreceptors blocked noradrenaline-induced phosphoinositide hydrolysis, but had no effect on lindane-induced increase of inositol phosphate levels. The results suggest that lindane does not exert a general effect on the receptor-stimulated formation of inositol phosphates by both muscarinic and alpha 1-adrenergic agonists.

ATP a potent regulator of inositol phospholipids-phospholipase C and lipid mediators in brain cortex.

Adenosine-5'trisphosphate (ATP) is stored and co-released with various neurotransmitters but it may also act as a fast excitatory neurotransmitter trough the activation of purinoreceptor(s). In this study the effect of ATP on phospholipase C (PLC) degrading labelled PtdIns(4,5)P2 and PtdIns in brain cortex slices, brain homogenate and subcellular fractions was investigated. It was found that ATP added into brain slices activated significantly and specifically PtdIns(4,5)P2 degradation and this process was inhibited by theophylline. Moreover, ATP maintained a higher level of inositol(1,4,5)P3 radioactivity in total water-soluble inositol metabolites. However, ATP added directly for the assay of PLC into brain homogenate or subcellular fractions inhibits phosphoinositide degradation in a receptor-independent manner and suppresses conversion of Ins(1,4,5)P3 into Ins(1,4)P2. Our results indicate that ATP acting extracellularly through a purinergic receptor(s) activates PtdIns(4,5)P2 degradation and release of Ins(1,4,5)P3. ATP acting directly on PLC inhibits in a receptor-independent manner phosphoinositide degradation, and protects against liberation of lipid-derived second messengers.

Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta. Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M coeluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [3H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.

Enantioselective synthesis of inositols as intermediates for the preparation of deoxy-inositol phosphates from D-galactose

Optically pure 6-deoxy-inososes, 6-deoxy-inositols and 6-deoxy-inositol-1,4,5-trisphosphates were synthesized from D-galactose by the carbohydrate-inosose Ferrier rearrangement. 6-Deoxy-inositolphosphates exhibit a tight binding to the Ins-P 3-receptor making such compounds an interesting tool for studying the intracellular signalling. It is now well established that receptor stimulated hydrolysis of inositol phospholipids is a common mechanism for transmembrane signalling when cells respond to external stimuli such as hormones, neurotransmitters, antigens, light, growth factors and insulin 1. It was also shown that phosphatidylinositol-4,5-bisphosphate [(Ptd)Ins(4,5)P 2] is a major inositol lipid hydrolysed by activated phospholipase C, resulting into the simultaneous generation of two “second messengers”, D- myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P 3] and diacylglycerol (DG) 2. Ins(1,4,5)P 3 triggers the mobilization of Ca ++ from intracellular stores and DG stimulates protein phosphorylation via the activation of protein kinase C 3,4. In addition (Ptd)Ins(4,5)P 2 contains a high percentage of arachidonic acid in the sn-2 position, which is released for lipoxygenase and cyclooxygenase pathways. These “second messengers” and their metabolites control and modulate vital physiological processes by their independent, additive and synergestic effects. 5 Therefore, it is conceivable that inhibitors of the key enzymes of the phosphoinositide cascade could be of medicinal interest and could be also useful tools to elucidate the individual role of the inositol metabolites in regulation of cell functions. 6,7 In view of difficulties in the isolation of inositol phosphate metabolites from natural sources and the need for structural analogues, several synthetic studies have been reported 8. However, these have employed mostly the optically inactive myo inositol as a logical and cheap starting material. The crucial role of the phosphate esters at positions 1, 3, 4, and 5 of the myo inositol nucleus in the “second messengers” Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 is well established. For analogue synthesis, modifications of the centers C-1, C-3, C-4 and C-5 or alteration of the hydroxyl functions at neighboring carbons (C-2 or C-6), not involved at first glance in cellular processes, seemed justified. With these considerations in mind we have initiated a synthetic program aimed to proxide access to the hitherto unknown partially protected 6-deoxy-cyclitols 8 and 9, appropriate precursors of a variety of chiral deoxy-inositol phosphates 9. Our approach to the deoxy-inositols 8 and 9 has been envisaged from the chiral deoxy-inososes 6 and 7 which could be obtained by mercury(II) mediated carbohydrate-inosose Ferrier rearrangement 10 from hex-5- ene-pyranoside 5. Olefin 5 was readily prepared in a four steps sequence from methyl-β-D-galactopyranoside 1 in 60% overall yield 11. Treatment of 1 with 1,1-dimethoxycyclohexane in DMF in presence of sulfuric acid afforded acetal 2 in 90% yield. The latter was selectively brominated with triphenylphosphine-carbontetrabromide, leading to 3 (m.p. 122–123°C). Benzylation of 3 by a phase transfer process (powdered KOH, benzyltriethylammonium chloride, benzyl bromide in CH 2Cl 2) furnished benzyl ether 4 (m.p. 94–95°C,[ α] rmD 20 + 46). Access to olefin 5 (m.p. 61–62°C, [ α] rmD 20 − 55) was achieved by two methods. Initially the bromo compound 4 was dehydrobrominated with sodium hydride in DMF (3h, 100°C) giving 5 in 90% yield 12. An alternative route for the

Decreased phospholipase C-beta immunoreactivity, phosphoinositide metabolism, and protein kinase C activation in senescent F-344 rat brain.

Phosphoinositide metabolism, phospholipase C immunoreactivity, and protein kinase C translocation were measured in brain slices of 6- and 24-month-old F-344 rats. Basal phosphoinositide labeling and accumulation of [3H]inositol phosphates were reduced in the 24-month-old rats. The cholinergic agonist, carbachol, induced lower net accumulations of inositol phosphates in striatal, hippocampal, and cortical slices of the aged rats. The dose-response curve for carbachol showed a significantly lower maximal response in the striatum of senescent rats, whereas the time course of [3H]inositol incorporation into inositol metabolites and the accumulation of free [3H]inositol in tissues from young and old animals were not different. Quantitative analyses showed marked reductions in endogenous brain levels of the phosphoinositides and in phospholipase C-beta 1 immunoreactivity, but no marked reductions in endogenous brain levels of the phosphoinositides and in phospholipase C-beta 1 immunoreactivity, but no changes in phospholipase C-gamma levels in aged animals. Moreover, basal protein kinase C activity and carbachol-mediated translocation of the enzyme were significantly reduced in the cerebral cortex of the senescent animals. These findings imply that aging is associated with alterations in the brain content, metabolism, and activity of phosphoinositide-derived second messengers in the F-344 rat brain.

The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form.

Intracellular concentrations of inositol, glycerophosphoinositol and inositol pentakisphosphate increase during haemopoietic cell differentiation

We have analysed the levels of soluble inositol metabolites in HL60 cells as they differentiate towards neutrophils in response to a combination of all- trans-retinoic acid and granulocyte colony-stimulating factor and towards monocytes in response to 1α-25-dihydroxyvitamin D 3. In both cases, differentiation was accompanied by increases in intracellular inositol (Ins), glycerophosphoinositol (Gro PIns) and inositol pentakisphosphate (Ins P 5) concentrations. [Gro PIns] reached a peak early in the differentiation of both neutrophils and monocytes and subsequently fell to about double the starting level as the cells acquired mature characteristics, and [Ins P 5] rose later. Similarly, neutrophils derived in culture by the spontaneous differentiation of myeloid blast cells contained increased levels of Ins, Gro PIns and Ins P 5 when compared to their parental blast cells. We have also compared the inositol metabolites present in two pairs of cell lines which are representative of immature and mature B and T lymphocytes. The mature cells again contained the higher levels of Gro PIns and Ins P 5. We have previously demonstrated increases in Ins, Gro PIns and Ins(1,3,4,5,6) P 5 levels during the differentiation of HL60 cells towards neutrophils in response to DMSO and of Gro PIns during the monocytoid differentiation of normal primitive myeloid blast cells in response to PMA. These observations suggest that deacylation of phosphatidylinositol by a phospholipase A/lysophospholipase pathway, forming Gro PIns and probably also regulatory arachidonate metabolites, has some role in haemopoietic cell differentiation. The reasons why Ins(1,3,4,5,6) P 5 and Ins accumulate during haemopoietic differentiation remain unknown.

Characterization of phosphoinositide hydrolysis products induced by hexachlorocyclohexane isomers in rat brain cortex

Water-soluble inositol metabolites were separated by anion-exchange chromatography in order to determine whether or not gamma-hexachlorocyclohexane (gamma-HCH, lindane) and related compounds affect phosphatidylinositol hydrolysis in rat brain cortex slices. Hydrolysis was increased by delta- and gamma-HCH, while alpha- and beta-HCH were inactive. Muscarinic receptor stimulation of rat cortical slices with carbachol increases inositol phosphates formation. The combined effect of carbachol and the hexachlorocyclohexane isomers together were approximately equal to the sum of the effect of each one separately. The results suggest that lindane stimulates phosphoinositide phospholipase C and/or inhibits the phosphatases implicated in dephosphorylation of inositol phosphates.

Analysis of inositol metabolites produced by Saccharomyces cerevisiae in response to glucose stimulation.

When cultures of Saccharomyces cerevisiae are grown to stationary phase in medium containing [3H] inositol, significant amounts of radioactivity can be detected in phosphatidylinositol, phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. Addition of glucose to such cultures results in the generation of [3H]glycerophosphoinositol, [3H]glycerophosphoinositol 4-phosphate, and [3H]glycerophosphoinositol 4,5-bisphosphate in the extracellular medium. We found no evidence, however, for the stimulated formation of other inositol polyphosphates. This result suggests that glucose does not stimulate the "phospholipase C" signalling pathway established in higher eukaryotic cells but, in contrast, stimulates specific phospholipases A or B. A variety of cell division cycle (cdc) mutants have been studied to investigate the relationship between cell cycle progression and inositol metabolism in S. cerevisiae. Mutants which are defective for completion of cell cycle "START" (i.e. commitment to mitosis) show reduced formation of glycerophosphoinositol 4-phosphate and glycerophosphoinositol 4,5-bisphosphate in response to glucose. In contrast, cdc mutants which are defective in post-"START" processes show a larger glucose response than wild type cells. These results suggest that deacylation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate may be coordinated with cell cycle control in S. cerevisiae.

Phosphoinositide metabolism in a polyoma-BK-virus-transformed pancreatic islet cell line: evidence for constitutively activated phospholipase C.

We have characterized the phosphoinositide metabolism in a polyoma-BK-virus-transformed rat pancreatic islet cell line which has highly malignant characteristics, expresses viral T-antigen and has lost insulin-secreting capacity. After incorporation with [3H]inositol to isotopic equilibrium, all inositol metabolites were analyzed. When compared with normal pancreatic islets, increased levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), inositol 1,3,4-trisphosphates and inositol tetrakisphosphate (Ins-P4), and decreased levels of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) were found. The Ins-1,4,5-P3/PIP2 ratio increased, whereas the PIP2/PIP ratio was not altered after the transformation. In the pancreatic islet cell line there was a stable accumulation of inositol phosphates at 3.3 mM glucose. Glucose, KCl, cholecystokinin (CCK) and carbachol with and without LiCl were all without effect on the accumulation of inositol phosphates. Somatostatin inhibited the accumulation of inositol phosphates but a Ca(2+)-free/EDTA solution did not. Preincubation with cholera toxin or pertussis toxin inhibited the accumulation of inositol phosphates at 3.3 mM glucose except for Ins-P4, whereas no effect was observed on the phosphoinositides. NaF stimulated the accumulation of inositol phosphates, with a concomitant decrease in the phosphoinositides, whereas neomycin was without effect on the inositol phosphates. In normal pancreatic islets, pertussis toxin inhibited the CCK-induced increase in Ins-1,4,5-P3, whereas no effect was seen at 3.3 mM glucose. Finally, pertussis toxin inhibited the CCK-induced increase in the Ins-1,4,5-P3/PIP2 ratio in normal pancreatic islets. The same inhibition was also found in the pancreatic islet cell line at 3.3 mM glucose. We conclude that in the transformed pancreatic islet cell line the phosphoinositide hydrolysis is constitutively activated at the level of phospholipase C, with a substantial loss of regulatory control.

Phase resetting of the Neurospora crassa circadian oscillator: effects of inositol depletion on sensitivity to light.

The input pathway between the blue-light photoreceptor and the circadian oscillator of Neurospora crassa has not yet been identified. To test the hypothesis that an inositol phospholipid signaling system might be involved in blue-light signal transduction, phase resetting by light was assayed in the inositol-requiring inl strain under conditions of inositol depletion. Phase-resetting curves and dose-response curves indicated that cultures maintained on low inositol (25 microM) were several orders of magnitude more sensitive to light than those maintained on high inositol (250 microM). This difference in light sensitivity was a property of inositol auxotrophy and was not seen in the wild type or in an inositol-independent inl+ revertant. Phase resetting by temperature was not affected by inositol depletion, indicating that the effect on light resetting is specific to the light input pathway and is not the result of a change in the amplitude of the oscillator itself. The results indicate an indirect role for inositol metabolites in the light input pathway--one that is not likely to involve direct participation of an inositol phospholipid signal transduction mechanism.

Changes in cyclic strain increase inositol trisphosphate and diacylglycerol in endothelial cells.

The purpose of this study was to assess the effect of cyclic strain on phosphatidylinositol turnover in cultured bovine aortic endothelial cells (EC). Confluent EC grown on flexible membrane bottoms were deformed by vacuum to 24% maximum strain and subjected to two cyclic strain regimens. In the first set of experiments, EC were subjected to deformation at a frequency of 60 cycles/min for either 0 (stationary control), 1, 5, 10, 25, 50, or 100 cycles of stretch. In the second set of experiments, EC were preconditioned by deforming the membranes at 60 cycles/min for 24 h. The cycling frequency was then acutely increased to 100 cycles/min for 0, 1, 5, 10, 25, or 100 cycles. Inositol phosphate and diacylglycerol concentrations were determined at the end of each regimen. The results demonstrate that either the initiation of pulsatile stretch or an acute change in cyclic stretch frequency stimulates a sequential and transient generation of inositol trisphosphate, its metabolites inositol biphosphate and monophosphate, and diacylglycerol. Thus EC may respond to the initiation and change in cyclic stretch frequency by a signal transduction pathway involving inositol lipid metabolism.

Levels of inositol metabolites within normal myeloid blast cells and changes during their differentiation towards monocytes.

A homogeneous population of undifferentiated myeloid blast cells was purified from human fetal liver by rosette sedimentation of erythroblasts and macrophages, after coating these cells with monoclonal antibodies, followed by a cell elutriation step. The undifferentiated blast cells were maintained in culture, in a serum-free medium containing 1 mg l-1 inositol, by the presence of a high concentration of interleukin-3 (100 U ml-1). This allowed equilibrium labelling of cells with [2-3H]myo-inositol and analysis of the concentrations of inositol metabolites. The myeloid blast cells contained high concentrations of an unidentified inositol metabolite, possibly sn-glycero-3-phospho-1-inositol (GroPIns, 22 microM), inositol monophosphate (InsP, 16 microM), an unidentified inositol bisphosphate (InsP2, 9.4 microM), inositol pentakisphosphate (InsP5, 37 microM) and inositol hexakisphosphate (InsP6, 31 microM). These high concentrations are similar to those reported in the promyeloid cell line, HL60. Treatment of the blast cells with 10 nM phorbol myristate acetate (PMA) resulted in rapid differentiation of 48% of the cells towards monocytes. Notable changes in the levels of inositol metabolites included an increase in the putative GroPIns peak (to 73 microM) and decreases in the concentrations of InsP4 (from 4 microM to 1 microM) and InsP5 (to 21 microM). These changes in response to PMA, with the exception of the rise in the putative GroPIns, are similar to those reported in HL60 cells undergoing monocyte differentiation. These observations suggest that the abundant inositol polyphosphates may have an as yet unknown role in myeloid differentiation.

Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes

HL60 cells were adapted to grow in a serum-free medium containing 1 mg l-1 inositol, in which they differentiated normally towards neutrophils (in 0.9% by volume dimethylsulphoxide) and towards monocytes (in 10 nM phorbol myristate acetate). Cells that had been equilibrium-labelled with [2-3H]myo-inositol contained a complex pattern of inositol metabolites, several of which were at relatively high concentrations. These included InsP5 and InsP6, which were present at concentrations of about 25 microM and 60 microM, respectively. Striking and different changes occurred in the levels of some of the inositol polyphosphates as the cells differentiated towards either neutrophils or monocytes. Most notable were a large but gradual accumulation of Ins(1,3,4,5,6)P5 as HL60 cells decreased in size and acquired neutrophil characteristics, and much more rapid and sequential declines in InsP4, InsP5 and InsP6 as the cells started to take on monocyte character. There was a marked accumulation of free inositol and of phosphatidylinositol in the cells during neutrophil differentiation, probably caused at least in part by an increased rate of inositol uptake providing an increased intracellular inositol supply. The same accumulation of Ins(1,3,4,5,6)P5 occurred during neutrophil differentiation, whether it was induced by dimethylsulphoxide or by a combination of retinoic acid and a T-lymphocyte cell line-derived differentiation factor. Ins(1,4,5)P3, a physiological intracellular mediator of Ca2+ release from membrane stores, did not change in concentration during these differentiation processes. These observations suggest that some of the more abundant cellular inositol polyphosphates play some important, but not yet understood, role either in the processes of haemopoietic differentiation or in the expression of differentiated cell character in myeloid cells.