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.     

Activation of inositol phospholipid breakdown in HL60 cells by P2-purinergic receptors for extracellular ATP. Evidence for mediation by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms

The mechanisms whereby P2-purinergic receptors for extracellular ATP are coupled to the inositol phospholipid-signaling system were studied in the HL60 human promyelocytic leukemia cell line. Brief pretreatment of either undifferentiated or differentiated HL60 cells with various activators of protein kinase C Ca2+/phospholipid-dependent enzyme (e.g. phorbol myristate acetate) produced a 50-fold decrease in the potency of extracellular ATP to induce mobilization of intracellular Ca2+. The ATP-induced increase in rate of inositol trisphosphate (InsP3) accumulation in these 4-beta-phorbol 12-myristate-13-acetate-treated cells was characterized by a 40% decrease in the maximal rate of InsP3 accumulation. Incubation of the cells with NaF also induced mobilization of the same Ca2+ stores released in response to extracellular ATP; this provided indirect evidence that the transmembrane signaling actions of P2-purinergic receptors may be mediated by GTP-binding regulatory proteins. This latter possibility was further supported by the finding that treatment of either undifferentiated or differentiated HL60 cells with pertussis toxin produced a significant, but partial, inhibition of ATP-induced signaling actions. These included: 1) a 60-70% decrease in the maximum rate of InsP3 accumulation, and 2) a 1.5 log unit increase in the half-maximally effective [ATP] required for mobilization of intracellular Ca2+. In cells treated with both pertussis toxin and 4-beta-phorbol 12-myristate-13-acetate, there was an 80% decrease in maximal rate of ATP-induced InsP3 accumulation and near-complete inhibition of ATP-induced Ca2+ mobilization. Significantly, the residual, pertussis toxin-insensitive portion of ATP-induced signaling was observed in the same samples of differentiated HL60 cells wherein pertussis toxin treatment produced complete abolition of InsP3 accumulation and Ca2+ mobilization in response to occupation of chemotactic peptide receptors. These results indicate that the activation of inositol phospholipid breakdown by P2-purinergic receptors in HL60 cells may be mediated by both pertussis toxin-sensitive and toxin-insensitive mechanisms; this suggests that these myeloid progenitor cells may express two distinct types of GTP-binding proteins coupled to phospholipase C.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.     

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.     

Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation at 5 microM. Carbachol increased the accumulation of [3H]inositol phosphates only in the presence of added guanine nucleotide. Calcium increased [3H]InsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal [3H]InsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous [3H]InsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited [3H] InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated [3H]InsP3 formation although calcium-stimulated [3H]InsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated [3H]InsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of [3H]InsP3 results from inhibition of the G protein interaction with phospholipase C.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells

Absolute concentrations of inositol phosphate isomers (InsP(s] were quantified in the myeloid cell line HL-60 using the metal-dye detection technique. Stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) led to distinct alterations in at least seven different inositol phosphate species. Whereas the intracellular concentrations of the tetrakisphosphate isomers (InsP4(s] were found below the micromolar range, inositol 1,3,4,5,6-pentakis- and hexakisphosphate levels were about two orders of magnitude higher (36 and 54 +/- 2 microM (mean +/- S.D.), respectively). The three InsP4(s) showed distinct kinetic pattern upon receptor activation, the transient elevation of inositol 1,3,4,5-tetrakisphosphate being faster both in onset and in redecrease than inositol 1,3,4,6-tetrakisphosphate. Whereas the two latter isomers reached maximally 2.75 and 2.9 +/- 0.2 microM, respectively, 1 min after stimulation, inositol 3,4,5,6-tetrakisphosphate remained elevated (3.5 +/- 0.4 microM) up to 5 min after fMLP. Unexpected changes in highly phosphorylated InsP(s) were observed, notably a rise in inositol 1,3,4,5,6-pentakisphosphate and in inositol hexakisphosphate to 52 +/- 3 and 60 +/- 1 microM, respectively. In terms of mass, the increases in highly phosphorylated inositols are by far highest among all InsP(s). Combining radiotracer method with mass determination it was observed that the specific radioactivity of various InsP(s) was different and changed markedly upon fMLP stimulation, in spite of a prolonged labeling period leading to apparent isotopic steady state. The data presented demonstrate agonist-induced elevations of highly phosphorylated InsP(s) and suggest that inositol 1,4,5-trisphosphate, product of receptor-activated phospholipase C, is metabolized rather via phosphorylation than only by dephosphorylation pathways.     

Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.

After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP6 alone (up to 250 microM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100-200% higher than 'stimulus only' controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1-2 min preincubation with InsP6 and the effect is dose-dependent (maximum at approx. 100 microM InsP6). As others have shown FMLP stimulation of superoxide anion production has no external Ca2+ dependence but the presence of low levels of Ca2+ and Mg2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP6 in mammalian cells in the 30-100 microM range suggest that the local release of this inositol polyphosphate from damaged or effect cells could have a physiologically important modulatory role on neutrophil functions.     

A specific increase in inositol 1,4,5-trisphosphate 3-kinase B expression upon differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) are of great hope for regenerative medicine due to their dual pluripotency and self-renewal properties. We report a comparison of inositol phosphate (InsP(s)) production in undifferentiated, differentiated hESCs and in two cancer cell lines, Ntera2 cells, a human embryonal carcinoma cell (hECC) line and HeLa cells. To evaluate the potential impact of InsP(s) in differentiation, hESCs were spontaneously differentiated in culture for two weeks. The distribution of the different InsP(s) was affected upon differentiation: the level of highly phosphorylated InsP(s) was decreased. In contrast, the total level of phosphoinositides (PI) was increased. Using real time quantitative PCR (qPCR), the mRNA expression of several enzymes of the metabolism of InsP(s) was determined: a specific increase in inositol 1,4,5-trisphosphate 3-kinase A and B (ITPKA and ITPKB) was observed upon hESCs spontaneous differentiation. Ins(1,4,5)P(3) 3-kinase activity, undetectable in undifferentiated hESCs, increased upon differentiation. The same observation was made by Western blotting using an antibody directed against human ITPKB. This is the first report showing the potential implication of soluble InsP(s) in hESCs and possible function of isoenzymes of the inositol trisphosphate 3-kinase family in differentiation.     

Phorbol esters inhibit inositol phosphate and diacylglycerol formation in proliferating HL60 cells. Relationship to differentiation

Phorbol 12-myristate 13-acetate (PMA) induces the differentiation of the human promyelocytic cell line, HL60, towards adherent macrophage-like cells within 2 days. We have examined the early effects of PMA on inositol phosphates and on diacylglycerol production, two second messengers derived from inositol lipids. In proliferating HL60 cells, PMA induced a time- and concentration-dependent decrease in inositol phosphate levels. Maximal effects were seen after 1 h at 10 nM PMA. PMA also induced the translocation of protein kinase C from the cytosol to the membrane. Comparison between the differentiating effects of several phorbol esters and of 1-oleoyl-2-acetylglycerol with their ability to inhibit inositol phosphate formation suggests that the two effects are correlated.     

Histamine-induced phosphoinositide metabolism in cultured human umbilical vein endothelial cells. Association with thromboxane and prostacyclin release

Histamine stimulation of cultured human umbilical vein endothelial cells induced dose- and time-dependent increases in glycerophosphoinositol (GroPIns), inositol-1-phosphate (InsP), inositolbisphosphate (InsP2) and inositoltrisphosphate (InsP3) in addition to release of thromboxane A2 and prostacyclin. Increases in InsP2 and InsP3 were immediate while increases in GroPIns and InsP occurred only after 1 min. Thromboxane A2 and prostacyclin release paralleled GroPIns and InsP production. The data indicate that, in endothelial cells, histamine evokes early hydrolysis of polyphosphoinositides, and that subsequent mobilization of arachidonic acid for thromboxane and prostacyclin synthesis involves both deacylation and phosphodiesteratic cleavage of phosphatidylinositol.     

Expression level of inositol trisphosphate and inositol tetrakisphosphate receptors and their influence on Ca2+ release in permeabilized HL-60 and T15 cells

To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL-60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP.     

Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells

Insulin and insulinlike growth factors I and II (IGF-I and IGF-II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I-labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF-I or -II binding occurred with induced HL60 cell differentiation. Insulin and IGF-I demonstrated cross competition for receptor binding and down-regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF-I and insulin increased HL60 cell proliferation, as assessed by 3H-thymidine uptake, IGF-I greater than insulin. IGF-I binding and mitogenic effects were blocked by the monoclonal anti-IGF-I receptor antibody IR3, indicating that IGF-I-induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti-IGI-I and anti-insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.     

Metabolism of inositol phosphates in the protozoan Paramecium. Characterization of a novel inositol-hexakisphosphate-dephosphorylating enzyme.

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[3H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 microM, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)P5. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50-85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal-transduction mechanisms is unlikely. In homogenates prepared from myo-[3H]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InsP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 microM were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1.     

Dynamic changes in neutrophil cytoskeleton during priming and subsequent surface stimulated functions.

The naturally occurring inositol polyphosphate, inositol hexakisphosphate (InsP6, phytic acid) primes human neutrophils for enhanced responses to stimulatory agonists. Unlike other primers InsP6 has no effect at high doses (up to 500 microM) on the neutrophil's basal rate of superoxide production (O2-). Pretreatment of the cells for 2 min with InsP6 (100 microM) substantially enhances fMet-Leu-Phe (FMLP) (10(-7) M)-induced respiratory burst. In investigations of the dynamic aspect of neutrophil actin during priming and stimulation with FMLP the content of F-actin has been measured with and NBD (nitrobenz-2-oxa-1,3-diazole)-phallacidin assay. The induced assembly of F-actin is rapid (peaks at approximately 45 s) followed by depolymerization. In contrast, priming with InsP6, with or without FMLP stimulation, results in a sustained assembly of F-actin as visualized with a rhodamine-phalloidin staining technique. These changes in macromolecular status and distribution of F-actin during InsP6 priming occur in the absence of any other demonstrable functional responses and thus InsP6 may be a useful tool to follow early events in neutrophil functions or to monitor the presence of unprimed and preprimed subpopulations in the circulation. Since InsP6 is present in high concentrations in neutrophils (up to 100 microM), its release from damaged or effete cells may have a modulatory role on neutrophil functions.     

Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase.

The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.     

Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate.

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras-induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein-stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3-kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.     

Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells

Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.