Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells,tissues and biological fluids

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.     

Inhibitory action of spermidine on formyl-methionyl-leucyl-phenylalanine stimulated inositol phosphate production in human neutrophils

The effect of the polyamine, spermidine, on formyl-methionyl-leucyl-phenylalanine stimulated hydrolysis of polyphosphoinositides was examined in purified human neutrophils by measurement of inositol phosphate production from radioactively labelled inositol. Spermidine inhibited formyl-methionyl-leucyl-phenylalanine stimulated inositol phosphate production by neutrophil in a dose dependent manner. Inhibition of formyl-methionyl-leucyl-phenylalanine stimulated inositol phosphate accumulation by spermidine was maximal at 10 microM and the IC50 value for this effect was 4.2 microM spermidine. This action of spermidine, thought to be mediated by a membrane component other than phospholipase C, may reflect a control mechanism modulating the response of the polyphosphoinositide system.     

Effect of the inositol polyphosphate InsP(6) on DNA-PK-dependent phosphorylation

Inositol hexakisphosphate (InsP(6)) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP(6) stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP(6) binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP(6)-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP(6) for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP(6)-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP(6)-binding subunit of DNA-PK resulted in loss of InsP(6)-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP(6)-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP(6) binding failed to restore InsP(6)-dependent phosphorylation. While additional protein kinases may participate in InsP(6)-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP(6) by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase-related protein kinase DNA-PK.     

Priming effect of calcium ionophores on phorbol ester-induced respiratory burst in mouse peritoneal neutrophils.

The abilities of three calcium ionophores (A23187, 4-bromo-A23187, and ionomycin) to modulate the respiratory burst of neutrophils induced by phorbol ester and to increase the concentration of free intracellular Ca2+ ([Ca2+]i) were compared. The production of reactive oxygen species (ROS) was determined by luminol-dependent chemiluminescence and [Ca2+]i was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 microM) and ionomycin (0.001-0.5 microM) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burst induced by phorbol ester. The integral response (total production of ROS over 6 min) had a bell-shaped dependence on the concentration of ionomycin and A23187 with increase and decrease at low and high concentrations of the ionophores, respectively. The maximal effect was found at 0.5 microM ionomycin and 2 microM A23187, these concentrations resulting in transient increases in [Ca2+]i to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophores had no effect in calcium-free media, though they increased [Ca2+]i to approximately 400 nM through the mobilization of intracellular Ca2+. In cells with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with phorbol ester amplified twofold the production of ROS. The inhibition of phospholipase A2 with 4-bromophenacyl bromide significantly decreased the production of ROS. Thus, the entrance of Ca2+ and generation of arachidonic acid under the influence of phospholipase A2 are necessary for the ionophore-induced priming of production of ROS during cell activation with phorbol esters.     

Increased levels of inositol hexakisphosphate (InsP6) protect HEK293 cells from tumor necrosis factor (alpha)- and Fas-induced apoptosis

The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor alpha (TNF(alpha))-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFalpha-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFalpha-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFalpha-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis.     

The signaling role of inositol hexakisphosphate kinases (IP6Ks)

The past ten years have seen a contained explosion of interest in inositol pyrophosphates. The early cloning of the IP6Ks and the more recent identification of the PP-IP5Ks have allowed the development of essential experimental tools to investigate the physiological role of inositol pyrophosphates. However, for this exciting field of research to gain momentum, simpler and more reliable research protocols need to be further developed. The ability to resolve and quantify inositol pyrophosphates using gel electrophoresis (Losito et al., 2009) has dramatically altered the way we are studying this class of molecules, opening new avenues for research. The use of this technology to resolve, detect and characterize inositol pyrophosphates extracted from cells certainly represents one desirable aim. The most crucial objective, however, is to obtain definite proof of the new mechanism of post-translational modification by identifying with biophysical methods the presence in vivo of pyrophosphorylated serines. This will hopefully precipitate the development of new ways to detect this modification, for example through the production of antibodies that specifically recognize pyrophosphorylated serines.     

Temperature influence on stimulated PMN respiratory burst.

Body temperature can modulate the pathogenesis of infectious, metabolic and autoimmune diseases. This effect has been attributed to several hypothesized mechanisms. Body temperature could play an important role in influencing some cellular functions of human white blood cells. In this work we examined the temperature effect on the respiratory burst in human neutrophils. Human polymorphonuclear leucocytes (PMN) were obtained from heparinized venous blood by dextran sedimentation and erythrocyte lysis with NH4Cl (0.87%). Granulocytes were stimulated with opsonized zymosan (OZ), formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate (PMA), and monosodium urate (MSU) crystals at different temperatures (26, 37, 39, 40, 42 degrees C). The technique of luminol dependent chemiluminescence (CL) was used as indicator of oxygen free radicals (OFR) release by stimulated cells. OFR production from PMN stimulated with OZ, PMA, FMLP was higher at 37 degrees C than at 26, 39, 40, 42 degrees C (p < 0.001 OZ stimulated PMN at 40-42 degrees C; p < 0.05 PMA stimulated PMN at 42 degrees C. Significantly different from 37 degrees C value). OFR release from PMN stimulated with MSU crystals was significantly increased at 39 degrees C compared to 37 degrees C value (p < 0.001). This effect could not only be attributed to temperature influence on neutrophil activity. The specific polymorphonuclear leukocyte response to the microcrystals and the temperature influence on chemical and physical characteristics of the crystals may play an important role. We are now studying the temperature effect on activity of PMN exposed to others crystals.     

The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.     

Effect of magnetic resonance imaging on human respiratory burst of neutrophils

It is known that low intensity magnetic fields increase superoxide anion production during the respiratory burst of rat peritoneal neutrophils in vitro. We investigated whether the high intensity magnetic fields (1.5 T) during magnetic resonance imaging can influence the human neutrophil function under in vivo conditions. Blood samples were obtained from 12 patients immediately before and after magnetic resonance imaging (mean time 27.6(+/-11.4 min)). The induced respiratory burst was investigated by the intracellular oxidative transformation of dihydrorhodamine 123 to the fluorescent dye rhodamine 123 via flow cytometry. The respiratory burst was induced either with phorbol 12-myristate 13-acetate, Escherichia coli, N-formyl-methionyl-leucylphenylalanine or priming with tumor necrosis factor followed by FMLP stimulation. There was no significant difference between the respiratory burst before and after magnetic resonance imaging, irrespective of the stimulating agent. Short time exposure to a high intensity magnetic field during magnetic resonance imaging seems not to influence the production of radical species in living neutrophils.     

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) primes the respiratory burst and stimulates protein biosynthesis in human neutrophils

Pre-treatment of human neutrophils with rGM-CSF resulted in a 3-fold increase in the rate of fMet-Leu-Phe stimulated reactive oxidant generation, as assessed by luminol- and lucigenin-chemiluminescence and O2- secretion. When blood-stream neutrophils were incubated in RPMI 1640 medium supplemented with [35S]methionine, both fMet-Leu-Phe (0.1 microM) and gamma-interferon (100 U/ml) stimulated a 3-4-fold increased incorporation of label into TCA-precipitable material. Similarly, rGM-CSF (50 U/ml) also stimulated protein biosynthesis in bloodstream neutrophils, and newly labelled polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Two classes of polypeptides were visualised on these gels: the relative rate of labelling of one class changed very little upon rGM-CSF treatment whereas the relative rate of labelling of a second group increased 3-12-fold.     

Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.     

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils

In the present study, we have examined the potential ability of 5'-AMP-activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or with 5'-AMP significantly attenuated both phorbol 12-myristate 13-acetate (PMA) and formyl methionyl leucyl phenylalanine-stimulated superoxide anion O2- release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA-dependent H2O2 release, and induced the phosphorylation of c-jun N-terminal kinase 1 (p46), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5'-AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.     

The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme

A superoxide-forming oxidase from activated human neutrophil membranes was solubilized by two slightly different methods, then purified by "dye-affinity" chromatography. Kinetic studies of the purified preparations gave Vmax values of 5-10 mumol of O-2/min/mg of protein, and Km values for NADH and NADPH that were in reasonable agreement with values determined previously using particulate and crude solubilized preparations of the respiratory burst oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed prominent bands at 67, 48, and 32 kDa, together with some minor contaminants, whereas gel electrophoresis under non-denaturing conditions gave a single major band that when eluted and re-electrophoresed in the presence of sodium dodecyl sulfate showed bands at 67, 48, 32 kDa. We believe that all three bands represent oxidase components. The flavin content of the purified enzyme was 20.4 +/- 2.0 S.E. pmol of FAD/microgram of protein, whereas heme averaged 0.1 +/- 0.02 pmol/microgram and ubiquinone could not be detected. Assuming that the enzyme is composed of one 67-kDa subunit, one 48-kDa subunit, and one 32-kDa subunit (i.e. that its molecular mass is approximately 150 kDa), it can be calculated to have a turnover number of 700-1500 min-1, in agreement with a value reported previously for oxidase in a particulate O-2-forming system (Cross, A. R., Parkinson, J. F., and Jones, O. T. G. (1985) Biochem. J. 226, 881-884), and to contain the following quantities of redox carriers (mol/mol): FAD, 3.0; heme, 0.015; ubiquinone, less than 0.06. It remains to be determined whether this preparation represents the complete respiratory burst oxidase or is only the pyridine nucleotide dehydrogenating component of a more complex enzyme.     

ACh and 5-HT induced changes in the concentration of cytosolic inositol trisphosphate (InsP3) and inositol bisphosphate (InsP2) in the ABRM of Mytilus edulis L.

1. For determination of the phosphoinositides and inositol phosphates present in anterior byssus retractor muscle (ABRM) of Mytilus edulis fiber bundles of this muscle were incubated with [3H]-inositol. Close-to-equilibrium labelling was achieved after 14-17 hr of incubation. 2. The phosphoinositides formed during incubation were identified as phosphatidylinositolphosphates by thin layer chromatography and as glycerophosphoryl esters by anion-exchange chromatography after deacylation. Besides PtdIns, PtdInsP and PtdInsP2 two labelled products are formed, which could not be identified. 3. Inositol phosphates were separated by anion-exchange chromatography. InsP, InsP2 and InsP3 are present, while InsP4 seemed to be absent. 4. Incubation of pre-labelled fibers with ACh induces the accumulation of InsP3 and InsP2 immediately. While 5-Ht accomplishes the accumulation after a lag time of 25 sec. The concentration of cytosolic InsP does not change.     

Priming effects of mammalian tachykinins on human neutrophils.

The undecapeptide substance P (SP) is known to activate different cell types involved in inflammatory and immune processes. By evaluating primed stimulation of human neutrophils, we now demonstrate that SP (10 nM-0.1 mM) dose-dependently enhances superoxide anion production from cells stimulated by the phospholipid mediator Platelet Activating Factor (PAF). We also provide evidence that neurokinin A (NKA), which is released, as well as SP, from C fibers of sensory nerves, potentiates PAF-evoked superoxide anion generation, while neurokinin B (NKB) is ineffective.     

Intrapopulational heterogeneity of human neutrophils based on chemokinetic and respiratory burst reactions

The chemokinetic test (ameboid motility) and enhancement of oxygen-dependent metabolism of neutrophils (the NBT test) were considered in human blood stimulated with Staph. aureus allergen. There were three variants of stimulated cells: 1) neutrophils developing ameboid motility (13,5 +/- 1,7% cells), 2) neutrophils with activated oxygen-dependent metabolism (11.5 +/- 0.6%), 3) neutrophils positive in both the tests (2.6 +/- 0.8%). Unstimulated cells accounted for 71.6 +/- 4.1%. Considerable differences were recorded for each variant. The data obtained are regarded as evidence of dissimilar capability of neutrophils of the realization of effector functions.