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At least 19 recordsLinked to original sources
PubMed1991

Binding of sugar phosphates, inositol phosphates and phosphorylated amino acids to actin.

Binding of biological phosphate compounds to actin was investigated by the effect of these compounds on the critical concentration of the pointed ends of gelsolin-capped actin filaments. According to this assay millimolar concentrations of glucose 6-phosphate and the bisphosphorylated sugars fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and 2,3-bisphosphoglycerate were found to associate with actin. Glycerophosphoinositol phosphates bound to actin if they were present in millimolar concentrations, and if carbon atom 4 of the inositol ring was phosphorylated and carbon atom 5 was free of phosphate. Also phosphoserine and phosphotyrosine were found to interact with actin. Most of the actin-binding compounds stabilized actin filaments by decreasing the critical concentration suggesting that these compounds had a higher affinity for the subunits along actin filaments than for actin monomers. However, 2,3-bisphosphoglycerate and fructose 2,6-bisphosphate increased the critical concentration probably because these sugar phosphates bound to actin monomers thereby inhibiting actin polymerization.

Actins↗
PubMed1992

Rapid and sensitive anion-exchange high-performance liquid chromatographic determination of radiolabeled inositol phosphates and inositol trisphosphate isomers in cellular systems.

A rapid and sensitive high-performance liquid chromatographic method for the determination of multiple inositol phosphates and inositol trisphosphate isomers was developed. The separation of inositol phosphates was optimized by controlling the ionic strength with stepped gradient programs and the pH of mobile phase. Six inositol phosphates were determined within 22 min or the six compounds plus an inositol trisphosphate isomer within 24 min using a single anion-exchange column containing the quaternary ammonium functional group. This technique was successfully applied to the determination of inositol phosphatide turnover by AlF4-stimulation in a small amount (5.10(5)-1.10(6) cells) of cultured retinal capillary pericytes. Because of its efficiency, accuracy and applicability to the separation of inositol phosphates from biological samples, this method may be useful in signal transduction studies in cellular systems.

Animals↗
PubMed1977

Quantitative determination of myoinositol, inositol 1-phosphate, inositol cyclic 1 : 2-phosphate and glycerylphosphoinositol in normal and Rous-sarcoma-virus-transformed quail fibroblasts under different growth conditions.

Myoinositol and its phosphorylated derivatives have been quantitatively determined in normal and Rous-sarcoma-virus-transformed quail cells under various growth conditions using [2-(3)H]myoinositol at isotope equilibrium conditions. The following amounts were determined (nmol/mumol phospholipid, as a unit of cell mass): exponentially growing normal and tumor cells contained 25--40 nmol free inositol, 0.40--0.45 nmol myoinositol 1-phosphate, 0.30--0.50 nmol glycerylphosphoinositol, and 0.03--0.04 nmol myoinositol cyclic 1 : 2-phosphate. At high cell populations in the absence of serum, conditions which result in cessation of growth by normal but not by tumor cells, changed levels were found for glycerylphosphoinositol and free inositol. In tumor cells the levels of these two compounds increased to 0.64 nmol and 64 nmol, respectively. In normal cells glycerylphosphoinositol increased to 0.95 nmol and free inositol showed highly elevated levels of 144 nmol. At short pulses the specific activities of inositol 1-phosphate and inositol cyclic 1 : 2-phosphate were found to be higher than that of phosphatidylinositol. This was not the case for glycerylphosphoinositol.

Animals↗
PubMed1992

Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.

3T3 Cells↗
PubMed1991

Effect of desipramine on inositol phosphate formation and inositol phospholipids in rat brain and human platelets.

To examine the mechanism of action of antidepressant drugs, we studied the effect of desipramine (DMI) in vitro on agonist-stimulated inositol phosphate formation and inositol phospholipids in rat brain and human platelets. We observed that DMI inhibited thrombin-stimulated 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) but not 3H-inositol monophosphate (IP1) formation in human platelets. DMI also inhibited norepinephrine (NE) and serotonin (5-HT) stimulated 3H-IP1 formation in rat cerebral cortex. DMI increased levels of all three 3H-inositol phospholipids, 3H-phosphatidyl inositol (PI), 3H-PI-4-phosphate (PIP), and 3H-PI 4,5-bisphosphate (PIP2), in both platelets and rat cortex. The decreased formation of inositol phosphates and increased levels of [3H]-PI, [3H]-PIP, and [3H]-PIP2 by DMI appears to be due to the inhibition of the enzyme phospholipase C rather than its effects on receptors. It is thus possible that interaction of tricyclic antidepressant drugs with the PI-signaling system may be related to their mechanism of action.

Animals↗
PubMed1992

Calcium homeostasis in Trypanosoma cruzi amastigotes: presence of inositol phosphates and lack of an inositol 1,4,5-trisphosphate-sensitive calcium pool.

The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5 microM Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1 microM FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.

Animals↗
PubMed1992

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.

Animals↗
PubMed1990

Stimulation of a dopamine D1 receptor enhances inositol phosphates formation in rat brain.

Accumulation of inositol phosphates was determined in rat brain slices prelabeled with 2-[3H]inositol and incubated with various drugs. In the striatum, micromolar concentrations of dopamine, apomorphine and SKF38393 induced significant accumulations of inositol phosphates in a dose-dependent manner, whereas quinpirole lacked effect. The EC50 values for the accumulation of inositol monophosphate induced by dopamine, apomorphine and SKF38393 were, respectively, 148, 159 and 129 microM. SKF 38393 effect was time-dependent on the accumulation of all three inositol phosphates, with peak effects occurring 64-128 min after drug addition. The action of the dopamine D1 receptor agonist, SKF38393, was blocked by SCH23390 (D1-selective antagonist), but not by sulpiride (D2-selective antagonist), atropine (muscarinic antagonist), prazosin (alpha-1 adrenoceptor antagonist) or methiotepin and methysergide (serotonergic antagonists), indicating that the observed effects of dopaminergic agonists were selectively mediated through the D1 dopamine receptor. On examining the effect of SKF38393 in several brain regions, the highest dopaminergic stimulation of inositol phosphates formation was obtained in the amygdala, followed by the hippocampus and then the striatum and frontal cortex. The finding of an SKF38393-stimulated PI hydrolysis in amygdala, a brain region that is enriched in SCH23390 and SKF38393 binding sites but devoid of dopamine-stimulated adenylate cyclase, suggests that the D1 receptor that is linked to PI metabolism is independent of the D1 receptor which stimulates cyclic AMP formation.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Quantitative analysis of inositol lipids and inositol phosphates in synaptosomes and microvessels by column chromatography: comparison of the mass analysis and the radiolabelling methods.

Chromatographic methods that measure both the mass and the radiolabelling of various inositol lipids and inositol phosphates in tissues have been developed. The mass of phosphatidylinositol (PtdIns), phosphatidylinositol-4-monophosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] was quantitated by measuring the inorganic phosphate, whereas inositol monophosphate (IP), inositol bisphosphate (IP2), inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) were quantitated by using an enzymic method. The radiolabelling of various inositol lipids and inositol phosphates was determined by incubating the tissue samples with [3H]myo-inositol, separating individual inositol lipids and inositol phosphates, and measuring the radioactivity in each compound. Although the mass analysis method was sensitive enough to measure low levels of inositol lipids or inositol phosphates, the method was laborious and time-consuming. Compared with the enzymic method, the radiolabelling method was simple and fast, but it gave variable results. This study demonstrated differences in inositol lipid and inositol phosphate levels by radiolabelling and mass measurements, and agonist-stimulated phosphatidylinositol turnover of synaptosomes versus the blood-brain barrier as represented by microvessels. Although the mass of PtdIns, PtdIns(4)P and PtdIns(4,5)P2 was comparable in synaptosomes and microvessels, the incorporation of [3H]myo-inositol into phosphorylated PtdIns in microvessels was less than that in synaptosomes.

Animals↗
PubMed1976

Control of inositol biosynthesis in Saccharomyces cerevisiae; inositol-phosphate synthetase mutants.

Inositol-requiring mutants of Saacharomyces cerevisiae were tested in cell extracts for the ability to convert glucose-6-phosphate to inositol-phosphate (IP synthetase) and inositol (IP phosphatase). Mutants representing any one of 10 unlinked loci conferring the inositol requirement were unable to synthesize either compound in an assay with glucose-6-phosphate as the substrate. These results indicate that the mutants lack IP synthetase activity and that at least 10 genes control the conversion of glucose-6-phosphate to inositol-phosphate. In addition, a mutation known to be unlinked with the ino1 locus interacts with a leaky ino1 allele and may play a role in the regulation of IP synthetase. This mutation causes a 47% reduction in wild-type IP synthetase activity and, when combined in a haploid strain with the leaky ino1 allele, it reduced IP synthetase activity to a level below that which is growth supporting. Wild-type and IP synthetase-deficient strains were tested for reduced nicotinamide adenine dinucleotide (NADH) accumulation, since NAD+ is required in the conversion of glucose-6-phosphate to inositol. No detectable accumulation of NADH was observed in the wild-type strain, presumably because the NADH generated is rapidly oxidized during subsequent partial reactions of IP synthetase. Mutants representing three different loci accumulate NADH and may, therefore, lack the NADH-mediated reductase activity of IP synthetase. Other mutants tested fail to accumulate NADH and may, therefore, lack the NAD+-mediated oxidase activity of IP synthetase. Phospholipid synthesis was studied by 32P pulse labeling in one mutant under conditions of inositol supplementation and starvation. Starved cells incorporate 32P into phospholipids normally for 2 h, followed by a period in which the rate of phosphatidylinositol synthesis decreases and the rate of phosphatidylcholine synthesis increases. After 5 to 6 h starvation, all cellular phospholipid synthesis ceases.

Carbohydrate Epimerases↗
PubMed1992

The role of alpha 1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion.

The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [3H]inositol (1.5 microCi/ml). LiCl (10 mM) and prazosin (0.3 microM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [3H]inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP3 levels (Amersham InsP3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [3H]inositol incorporation into inositol phosphates and InsP3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [3H]inositol into inositol phosphates as well as InsP3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 microM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.

Animals↗
PubMed1992

Agonist-induced inositol phosphate responses in bovine airway submucosal glands.

Agonist-induced inositol phosphate responses were investigated in bovine airway submucosal glands prelabeled with [3H]inositol. The agents used included muscarinic, adrenergic, and histaminergic agonists and NaF as a direct stimulant of phosphoinositidase C. Carbachol (1 microM-1 mM) produced concentration-related [3H]-inositol phosphate formation [maximal response 7.3-fold, half-maximal effective concentration (EC50) 47 microM]. This response was inhibited by atropine [affinity constant (Ka) 1.5 x 10(9) M-1], suggesting the involvement of a muscarinic receptor. Histamine (EC50 66 microM, 2.4-fold stimulation) and norepinephrine (EC50 28 microM, 2.7-fold stimulation) also produced concentration-dependent [3H]inositol phosphate responses. The response to histamine was inhibited by mepyramine (Ka 1.9 x 10(8) M-1), and the response to norepinephrine was inhibited by phentolamine (Ka 1.3 x 10(8) M-1) and prazosin (Ka 2.3 x 10(8) M-1), indicating the involvement of H1 and alpha 1-receptors, respectively, in these responses. NaF also produced concentration-dependent [3H]inositol phosphate formation compatible with the involvement of a G protein in this response. These results demonstrate that a range of secretagogues induce inositol phosphate responses in bovine airway submucosal glands. This response may underlie stimulus response coupling in this tissue.

Animals↗
PubMed1991

Increased inositol phosphate accumulation in platelets from patients with NIDDM.

We evaluated thrombin-induced inositol phosphate accumulation in [3H]inositol-labeled platelets prepared from patients with non-insulin-dependent diabetes mellitus. There were no significant differences in [3H]inositol incorporation into and contents of phosphoinositides between the diabetic patients and their age-matched control subjects. Thrombin induced a dose- and time-dependent accumulation of inositol phosphate. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate by thrombin stimulation were significantly enhanced in platelets from the diabetic patients, although the accumulation of [3H]inositol monophosphate did not differ between the diabetic patients and the control subjects. In addition, the platelet aggregation rate induced by thrombin was also significantly enhanced in the diabetic patients in correlation with the enhanced inositol phosphate accumulation. These results suggest that increased inositol phosphate accumulation may cause accelerated platelet functions in diabetes mellitus.

Blood Platelets↗
PubMed1991

[Malignant hyperthermia and inositol phosphate metabolism in the heart and skeletal musculature].

There are recent reports that inositol phosphate metabolism is involved in the development of malignant hyperthermia (MH). Consequently, we investigated the basal concentration of inositol phosphate products in skeletal and heart muscles of malignant hyperthermia-susceptible (MHS) and healthy control (MHN) swine. Different inositol phosphates were measured by high pressure liquid chromatography, including inositol trisphosphate, tetrakisphosphate, pentakisphosphate and hexakisphosphate. All inositol phosphate products measured had a higher concentration in MHS than MHN in skeletal (304-1330%) as well as heart muscles (134-440%). An activation of the inositol phosphate metabolism has been shown to mobilise intracellular calcium from the sarcoplasmic reticulum. It is therefore concluded that, firstly, besides involvement of the skeletal muscles a primary myocardial abnormality in MHS is possible; and secondly, the idea that the inositol phosphate metabolism could be involved in the development of MH is additionally supported.

Animals↗
PubMed1992

Recent advances in methodology for analysis of phytate and inositol phosphates in foods.

This review summarises the methods available for the analysis of phytate and structurally related molecules, i.e., inositol polyphosphates. Phytate has been determined by colorimetry, low pressure ion exchange column chromatography, phosphorus-31 fourier transform nuclear magnetic resonance spectroscopy (31P FT NMR), near-infrared reflectance spectroscopy and high performance liquid chromatography (HPLC). Among these techniques anion exchange column chromatography and HPLC were shown to be best suited for separation of inositol phosphates. Since inositol phosphates do not have a characteristic absorption spectrum, their detection in HPLC analysis is limited to methods employing monitoring of refractive index, post column reaction products, conductivity or indirect detection although other detection methods may be feasible. As refractive index detection under isocratic eluent conditions is relatively easy to manipulate, anion-exchange HPLC methods using a low capacity column or ion-pair HPLC methods are recommended for the analysis of inositol phosphates in nutritional studies.

Chromatography↗
PubMed1991

Differences in inositol phosphate production in blood vessels of normotensive and spontaneously hypertensive rats.

1. Total inositol phosphate formation was measured in labelled femoral and iliac arteries and veins of 14 week-old spontaneously hypertensive rats (SHR) and age-matched Wistar Kyoto (WKY) controls, either unstimulated or in the presence of noradrenaline. 2. Basal levels of [3H]-inositol phosphates and [3H]-phosphatidylinositol were significantly enhanced in SHR femoral artery, but not in the other 3 vessels, compared with WKY. 3. Noradrenaline stimulated phosphoinositide hydrolysis in all four vessels of SHR and WKY. Pretreatment with prazosin (10(-7)-10(-6) M) but not with yohimbine (10(-7) M), inhibited the noradrenaline-induced inositol phosphate formation indicating an alpha 1-adrenoceptor-mediated response. 4. In the femoral artery of SHR compared to WKY, [3H]-inositol phosphate accumulation induced by noradrenaline (10(-7)-10(-5) M) was significantly reduced when expressed relative to basal values although the response to higher concentrations (10(-4)-10(-3) M) was not altered. In contrast, a significant reduction of inositol phosphates was seen only with 10(-7) M noradrenaline when absolute values were compared. In the other three vessels, no difference in noradrenaline-induced [3H]-inositol phosphate formation was observed between strains. 5. These data suggest that phosphoinositide hydrolysis-mediated by alpha 1-adrenoceptors may be reduced in some but not all blood vessels of adult SHR.

Animals↗
PubMed1990

Inositol phosphate formation in arterial smooth muscle from rabbits with perinephritis hypertension.

Total inositol phosphate formation was measured in the aorta and femoral artery from rabbits at 1, 2 and 6 weeks after kidney wrapping, at which times the mean arterial pressures were 88 +/- 4, 96 +/- 3 and 126 +/- 7 mmHg against a control pressure of 74 +/- 3 mmHg. Noradrenaline-stimulated (10(-7) to 10(-4) mol/l) inositol phosphate formation was increased in the aorta and femoral artery from hypertensive rabbits at 2 weeks (aorta noradrenaline 10(-6) mol/l sham, 105 +/- 14%; hypertensive, 164 +/- 20% of control). Noradrenaline-stimulated inositol phosphate formation was unchanged at 1 and 6 weeks in the aorta. Endothelin-stimulated inositol phosphate formation was unchanged at 2 weeks. Basal inositol phosphate formation was not significantly different in normotensive and hypertensive animals. In perinephritis hypertension there is an alteration in phosphatidylinositol metabolism in arterial smooth muscle. This occurs at the time when the blood pressure is rising rapidly. This alteration may affect a specific phosphatidylinositol pool that is linked to the alpha-adrenoceptor but not to the endothelin receptor.

Animals↗
PubMed1991

A method to measure simultaneously cyclic AMP and inositol phosphate accumulation in rat brain slices.

The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion-exchange chromatography and their concentrations were determined by means of a double-labeling technique, the substrates adenine and inositol being labeled with 14C and 3H, respectively. The recoveries were 70-80% for inositol phosphates and 40-50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with alpha- and beta-adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of 3H-inositol phosphates and [14C]cyclic AMP in a concentration-dependent process. EC50 values were similar for the two, 8.5 +/- 3.9 microM for 3H-inositol phosphates and 20.2 +/- 6.3 microM for [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of 3H-inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two receptor-coupled effectors.

1-Methyl-3-isobutylxanthine↗