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PubMed1989

Extracellular ATP stimulates poly(inositol phospholipid) hydrolysis and eicosanoid synthesis in mouse peritoneal macrophages in culture.

The effects of extracellular ATP on inositol phospholipid breakdown and synthesis of eicosanoids were studied in mouse peritoneal macrophages. Addition of ATP to intact cells labelled with [3H]inositol stimulated a rapid (within 10 s) formation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. In parallel there was also a substantial accumulation of inositol 1,3,4-trisphosphate and the monophosphate and bisphosphate derivatives of inositol. Within 10 s after the addition of 30 microM ATP there was a twofold increase in inositol trisphosphate (InsP3), which declined over 2 min. The ED50 for ATP-stimulated generation of InsP3 was approximately 12 microM. ADP and GTP showed only weak effects on InsP3 formation, while AMP and adenosine were completely ineffective at 30 microM. Furthermore, the rank order of potency of ATP analogues was ATP greater than ATP[S] greater than AdoPP[NH]P = AdoPP[CH2]P greater than AdoP[CH2]PP thus, indicating the presence of a P2y-purinergic receptor. Cells labelled with [3H]arachidonic acid showed a 50% increase of label in 1,2-diacylglycerol after 15 s upon stimulation with ATP. In parallel to the stimulation of inositol phospholipid hydrolysis, ATP also caused a marked synthesis of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in mouse peritoneal macrophages. The rank order of potency of ATP analogues was identical with that of InsP3 generation. The effect on eicosanoid synthesis could be mimicked by the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ATP-induced activation of P2y-purinergic receptors in mouse peritoneal macrophages triggers inositol phospholipid breakdown and eicosanoid synthesis.

Adenosine Triphosphate↗
PubMed1989

Differential effects of heparin on inositol 1,4,5-trisphosphate binding, metabolism, and calcium release activity in the bovine adrenal cortex.

In a wide variety of cells, inositol-1,4,5-triphosphate is a second messenger that interacts with specific intracellular receptors and triggers the release of sequestered Ca2+ from an intracellular store. We have looked at the influence of heparin on the action and metabolism of inositol-1,4,5-triphosphate in the bovine adrenal cortex. Heparin blocked inositol-1,4,5-trisphosphate binding with half-maximal efficiency around 10 micrograms/ml. Scatchard analyses revealed that heparin did not change the affinity but decreased the number of available binding sites. The Ca2+-releasing activity of inositol-1,4,5-trisphosphate was monitored with the fluorescent indicator, fura-2. Heparin blocked this activity with half-maximal effeciency around 10 micrograms/ml. The effect of heparin could be overcome by a supramaximal dose of inositol-1,4,5-trisphosphate (25 microM). The activity of inositol-1,4,5-trisphosphate-3-kinase from bovine adrenal cortex cytosol was also studied. Heparin inhibited the activity of the kinase with a half-maximal effeciency around 0.4 microgram/ml. Lineweaver-Burk plots revealed that this potent effect was noncompetitive. Finally, we observed that heparin is without effect on inositol-1,4,5-trisphosphate-5-phosphatase (at concentrations as high as 2 mg/ml). These results are consistent with the suggestion that the binding sites for inositol-1,4,5-trisphosphate are the intracellular receptors responsible for the Ca2+-mobilizing effects of inositol-1,4,5-trisphosphate. These results also show that the kinase, the phosphatase, and the receptor are three different molecular entities, which are affected in a different manner by heparin.

Adrenal Cortex↗
PubMed1989

Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane.

The depletion of an inositol 1, 4,5-trisphosphate-sensitive intracellular Ca2+ pool has been proposed to be the signal for Ca2+ entry in agonist-activated cells. Consistent with this idea, thapsigargin, which releases intracellular Ca2+ without inositol phosphate formation, has been reported to activate Ca2+ entry in certain cells. We now report the effects of thapsigargin on Ca2+ entry in parotid acinar cells. In fura-2-loaded parotid acinar cells, thapsigargin caused a sustained elevation of [Ca2+], but did not increase inositol phosphate formation. In the absence of extracellular Ca2+, the increase in [Ca2+], was transient, suggesting that thapsigargin activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from the extracellular space. In the absence of extracellular Ca2+, pretreatment with methacholine, an agonist believed to mobilize Ca2+ through the production of inositol 1,4,5-trisphosphate, inhibited but did not completely block the response to thapsigargin; likewise, pretreatment with thapsigargin inhibited the response to methacholine. In permeabilized cells, thapsigargin gradually released Ca2+, whereas inositol 1,4,5-trisphosphate caused a rapid and transient discharge of Ca2+. The simultaneous addition of thapsigargin with inositol 1,4,5-trisphosphate evoked a maximum Ca2+ release similar to that for inositol 1,4,5-trisphosphate alone, but the reuptake seen with inositol 1,4,5-trisphosphate alone was abolished. In intact cells, methacholine and thapsigargin together produced a greater initial release of Ca2+ than either alone, but they were not additive in the sustained phase of Ca2+ mobilization. These results demonstrate that the mechanisms for activation of Ca2+ entry by thapsigargin and methacholine are the same and are consistent with the idea that entry is initiated by the depletion of the intracellular inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. The results also indicate that, in contrast to previously proposed models, Ca2+ entry into agonist-activated cells occurs directly across the plasma membrane to the cytoplasm rather than through a cycle of uptake and release by the intracellular Ca2+ pool.

Animals↗
PubMed1987

The metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase and 3-kinase enzymes.

Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate to form both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,2-cyclic 4,5-trisphosphate (cInsP3). The further metabolism of these inositol trisphosphates is determined by two enzymes: a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate (InsP4), while the latter forms inositol 1,4-bisphosphate and inositol 1,2-cyclic 4-bisphosphate from Ins(1,4,5)P3 and cInsP3, respectively. The current studies show that the 3-kinase is unable to phosphorylate cInsP3. Also, the 5-phosphomonoesterase hydrolyzes InsP4 with an apparent Km of 0.5-1.0 microM to form inositol 1,3,4-trisphosphate at a maximal velocity approximately 1/30 that for Ins(1,4,5)P3. The apparent affinity of the enzyme for the three substrates is InsP4 greater than Ins(1,4,5)P3 greater than cInsP3; however, the rate at which the phosphatase hydrolyzes these substrates is Ins(1,4,5)P3 greater than cInsP3 greater than InsP4. The 5-phosphomonoesterase and 3-kinase enzymes may control the levels of inositol trisphosphates in stimulated cells. The 3-kinase has a low apparent Km for Ins(1,4,5)P3 as does the 5-phosphomonoesterase for InsP4, implying that the formation and breakdown of InsP4 may proceed when both it and its precursor are present at low levels. Ins(1,4,5)P3 is utilized by both the 3-kinase and 5-phosphomonoesterase, while cInsP3 is utilized relatively poorly only by the 5-phosphomonoesterase. These findings imply that inositol cyclic trisphosphate may be metabolized slowly after its formation in stimulated cells.

Animals↗
PubMed1987

Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium.

Inositol 1,4,5-trisphosphate is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. We have recently identified high levels of specific binding sites for inositol 1,4,5-trisphosphate in brain membranes (Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161) and have now further characterized these sites. In cerebellar membranes, inositol 1,4,5-trisphosphate binding sites are abundant (20 pmol/mg protein) and display high affinity and selectivity for inositol 1,4,5-trisphosphate (KD approximately equal to 40 nM), whereas other inositol phosphates such as inositol 1,3,4,5-tetrakisphosphate (Ki approximately equal to 10 microM) and inositol 1,4-bisphosphate (Ki approximately equal to 10 microM) exhibit much lower affinity for this site. Submicromolar concentrations of calcium strongly inhibit inositol 1,4,5-trisphosphate binding (IC50 approximately equal to 300 nM). A sharp increase in binding occurs at slightly alkaline pH. These results suggest that actions of inositol 1,4,5-trisphosphate are regulated by physiological alterations in intracellular pH and calcium concentrations.

Animals↗
PubMed2003

Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae.

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Journal Article↗
PubMed1988

The purification and properties of myo-inositol monophosphatase from bovine brain.

1. An inositol monophosphatase was purified to homogeneity from bovine brain. 2. The enzyme is a dimer of subunit Mr 29,000. 3. The enzyme hydrolyses both enantiomers of myo-inositol 1-phosphate and both enantiomers of myo-inositol 4-phosphate, but has no activity towards inositol bisphosphates, inositol trisphosphates or inositol 1,3,4,5-tetrakisphosphate. 4. Several non-inositol-containing monophosphates are also substrates. 5. The enzyme requires Mg2+ for activity, and Zn2+ supports activity to a small extent. 6. Other bivalent cations (including Zn2+) are inhibitors, competitive with Mg2+. 7. Phosphate, but not inositol, is an inhibitor competitive with substrate. 8. Li+ inhibits hydrolysis of inositol 1-phosphate and inositol 4-phosphate uncompetitively with different apparent Ki values (1.0 mM and 0.26 mM respectively).

Animals↗
PubMed1994

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.

Adenosine Triphosphate↗
PubMed1999

Myo-inositol-dependent sodium uptake in ice plant

In salt-stressed ice plants (Mesembryanthemum crystallinum), sodium accumulates to high concentrations in vacuoles, and polyols (myo-inositol, D-ononitol, and D-pinitol) accumulate in the cytosol. Polyol synthesis is regulated by NaCl and involves induction and repression of gene expression (D.E. Nelson, B. Shen, and H.J. Bohnert [1998] Plant Cell 10: 753-764). In the study reported here we found increased phloem transport of myo-inositol and reciprocal increased transport of sodium and inositol to leaves under stress. To determine the relationship between increased translocation and sodium uptake, we analyzed the effects of exogenous application of myo-inositol: The NaCl-inducible ice plant myo-inositol 1-phosphate synthase is repressed in roots, and sodium uptake from root to shoot increases without stimulating growth. Sodium uptake and transport through the xylem was coupled to a 10-fold increase of myo-inositol and ononitol in the xylem. Seedlings of the ice plant are not salt-tolerant, and yet the addition of exogenous myo-inositol conferred upon them patterns of gene expression and polyol accumulation observed in mature, salt-tolerant plants. Sodium uptake and transport through the xylem was enhanced in the presence of myo-inositol. The results indicate an interdependence of sodium uptake and alterations in the distribution of myo-inositol. We hypothesize that myo-inositol could serve not only as a substrate for the production of compatible solutes but also as a leaf-to-root signal that promotes sodium uptake.

Journal Article↗
PubMed1978

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates.

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis.

Journal Article↗
PubMed1982

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb.

A Mg(2+)-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and beta-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg(2+). At 4 millimolar, Co(2+), Fe(2+) or Mn(2+) are less effective. Substantial inhibition is obtained with 0.25 molar Li(+). With beta-glycerophosphate as substrate the K(m) is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, beta-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.In higher plants this myo-inositol-1-phosphatase links myo-inositol biosynthesis to the myo-inositol oxidation pathway to produce an alternative path from d-glucose 6-phosphate to UDP-d-glucuronate that bypasses UDP-d-glucose dehydrogenase. myo-Inositol-1-phosphatase also furnishes free myo-inositol for reactions that lead to other cyclitols and cyclitol-containing compounds of biosynthetic and/or regulatory significance in plant growth and development.

Journal Article↗
PubMed1981

Equilibrium aspects of the binding of myo-inositol hexakisphosphate to human hemoglobin as studied by 31P NMR and pH-stat techniques.

The interaction of myo-inositol hexakisphosphate (P6-inositol) with human hemoglobin has been studied as a function of pH using pH-stat techniques and 31P NMR. With the pH-stat method the following data were obtained: the association constants for the P6-inositol/deoxyhemoglobin and P6-inositol/carboxyhemoglobin complexes at alkaline and acid pH respectively and the proton absorption curves associated with the protein/phosphate interaction for both complexes from pH 5.5 to pH 9. From these data affinities of P6-inositol towards deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been calculated as a function of pH. The shape of the proton absorption curves was found to be strongly dependent on the ligation state of the hemoglobin molecule. The pH dependence of the 31P NMR spectra of P6-inositol bound to Hb or HbCO provides a monitor for the proton-binding behaviour of the phosphate groups of P6-inositol when present in the central cavity of the protein. It appears that this behaviour is only slightly dependent on the ligation state of the hemoglobin molecule. The NMR spectral data were interpreted in terms of a model which takes into account the electrostatic interaction between the phosphate groups within the P6-inositol molecule as well as the electrostatic interaction between the phosphate groups and positively charged groups on the protein. To account for the discrepancy between the pH-stat and 31P NMR results, i.e. a strong dependence of the proton-absorption curves and a weak dependence of the proton-binding behaviour of P6-inositol on the ligation state of the protein respectively, it is proposed that a conformational change takes place in HbCO upon P6-inositol binding.

Carboxyhemoglobin↗
PubMed1988

Noradrenaline stimulation of the phosphoinositide system: evidence for a novel hydrophobic inositol-containing compound in resistance arterioles.

1. Five inositol phosphates were extracted from adult rat resistance arterioles and separated by ion-exchange high performance liquid chromatography. 2. By use of this technique, inositol phosphates liberated were identified as inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Stimulation of phosphoinositide hydrolysis with noradrenaline produced increases in inositol phosphate production. 3. Three inositol-containing phospholipids extracted from resistance arterioles were measured as their glycerol esters following deacylation, thereby permitting an analysis of both membrane and cytosolic components of the phosphoinositide signalling system. 4. A substantial agonist-sensitive pool of a previously undescribed inositol but not glycerol-containing lipid extract component was also identified in this tissue. 5. These experiments for the first time allow a precise description of phosphoinositide metabolism in resting and agonist-stimulated resistance arterioles and provide data on a novel compound possibly similar to that recently described in other tissues.

Animals↗
PubMed1988

Beta-adrenoceptor stimulation inhibits histamine-stimulated inositol phospholipid hydrolysis in bovine tracheal smooth muscle.

1. Histamine and carbachol produced concentration-related increases in the accumulation of 3H-inositol phosphates in slices of bovine tracheal smooth muscle. 2. Noradrenaline alone produced a small stimulation of 3H-inositol phosphate accumulation which was inhibited by the alpha-adrenoceptor antagonist phentolamine. In contrast, when noradrenaline (0.1 mM) was added simultaneously with histamine it significantly reduced the inositol phosphate response to high (greater than or equal to 0.1 mM) concentrations of histamine. However, noradrenaline had no inhibitory effect on the carbachol-induced inositol phosphate response. 3. The non-selective beta-agonist isoprenaline (IC50 = 0.08 microM) and the beta 2-selective agonist salbutamol (IC50 = 0.29 microM) both produced a dose-related inhibition of the inositol phosphate response to 0.1 mM histamine. The inhibitory effect of salbutamol was antagonized by propranolol (KA = 2.4 x 10(9) M-1) and the beta 2-selective adrenoceptor antagonist ICI 118551 (KA = 1.7 x 10(9) M-1). 4. The accumulation of 3H-inositol phosphates induced by histamine increased steadily over a 40 min period after an initial lag period of 3-4 min. Following the simultaneous addition of histamine and salbutamol there was a further delay of 3-4 min before the appearance of the inhibitory effect of salbutamol. 5. The effect of histamine on inositol phosphate accumulation was accompanied by a stimulation of [3H]-inositol incorporation into membrane phospholipids which was reduced by the presence of salbutamol. However, when histamine was used to stimulate maximally [3H]-inositol incorporation during the prelabelling period, salbutamol produced a marked inhibition of histamine-stimulated 3H-inositol phosphate accumulation under conditions in which there was no change in the level of incorporation.

Adrenergic beta-Agonists↗
PubMed1987

Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+.

We recently identified an enzyme which we have designated inositol polyphosphate 1-phosphatase that hydrolyzes both inositol 1,3,4-trisphosphate (Ins-1,3,4-P3) and inositol 1,4-bisphosphate (Ins-1,4-P2), yielding inositol 3,4-bisphosphate and inositol 4-phosphate, respectively, as products (Inhorn, R. C., Bansal, V.S., and Majerus, P.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). We have now purified the inositol polyphosphate 1-phosphatase 3600-fold from calf brain supernatant. The purified enzyme has an apparent molecular mass of 44,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphatase activities. The enzyme hydrolyzes Ins-1,4-P2 with an apparent Km of approximately 4-5 microM, while it degrades Ins-1,3,4-P3 with an apparent Km of approximately 20 microM. The enzyme hydrolyzes these substrates at approximately the same maximal velocity. Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation. A Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively. Calcium and manganese inhibit enzyme activity, with 50% inhibition at approximately 6 microM. Lithium inhibits Ins-1,4-P2 hydrolysis uncompetitively with a Ki of approximately 6 mM. This mechanism of lithium inhibition is similar to that observed for the inositol monophosphate phosphatase (originally designated myo-inositol-1-phosphatase; Hallcher, L.M., and Sherman, W.R. (1980) J. Biol. Chem. 255, 10896-10901), suggesting that these two enzymes are related. Lithium also inhibits Ins-1,3,4-P3 hydrolysis with an estimated Ki of 0.5-1 mM.

Animals↗
PubMed2000

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.

Animals↗
PubMed1992

Heterologous regulation of inositol lipid hydrolysis in human breast cancer cells by oestradiol 17 beta, bombesin and fluoroaluminate.

Inositol lipid turnover has been implicated in the action of oestradiol 17 beta and bombesin-related peptides on the human breast cancer cell line MCF-7. In the present study, in addition to measuring inositol lipid turnover as indicated by inositol monophosphate (IP) accumulation, we have also monitored the effect of oestradiol on the incorporation of both 3H-inositol and 14C-glycerol into MCF-7 cell phospholipids. Pre-treatment of MCF-7 cells with oestradiol (10 nM) for 48 hr stimulated a 4.3-fold increase in IP production. This was similarly accompanied by a 3.4-fold increase in the incorporation of 3H-inositol into total phosphoinositides and a 40% increase in cell growth. The oestrogen antagonist LYI 17018 completely attenuated these effects. Oestradiol also stimulated 14C-glycerol incorporation into phosphatidyl inositol, -choline and -ethanolamine by 97%, 82% and 99%, respectively. IP production in response to bombesin was potentiated by oestradiol in a dose-dependent fashion. Fluoroaluminate (AlF4-) stimulated a dose-dependent increase in IP production and oestradiol pre-treatment increased the sensitivity of this IP response to AlF4-. Medroxyprogesterone acetate inhibited bombesin-stimulated IP production but had no effect on the response to AlF4-. Our data suggest that the oestrogenic action on basal IP production in MCF-7 cells may reflect an effect on inositol lipid synthesis rather than turnover. However, the potentiation by oestradiol of both bombesin- and AlF4(-)-stimulated inositol lipid hydrolysis suggests the operation of a post-receptor regulatory mechanism(s) which is independent of the inositol lipid pool size.

Aluminum↗
PubMed1992

The effect of ischaemia-reperfusion on [3H]inositol phosphates and ins(1,4,5)P3 levels in cardiac atria and ventricles--a comparative study.

In this study incorporation of [3H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P3 levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [3H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2-3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[3H]Ins(1,4,5)P3 assay system, the Ins(1,4,5)P3 levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles. Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P3 levels as well as incorporation of [3H]inositol into ventricular InsP2 and InsP3. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P3 levels and also [3H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P3 levels as well as the incorporation of [3H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.

Animals↗