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At least 19 recordsLinked to original sources
PubMed2002

Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls.

Previous data from our and other labs demonstrated a decreased chiro-inositol content in urine and tissues of human subjects and animals with type 2 diabetes. In urine this decrease in chiro-inositol was accompanied by an increase in myo-inositol content. Decreased urine levels of chiro-inositol in monkeys were next correlated with the severity of underlying insulin resistance determined by five separate assays. To investigate the decreased chiro-inositol and the accompanying increased myo-inositol excretions in urine in humans and monkeys, we postulated a defect in the epimerization of myo-inositol to chiro-inositol. [(3)H]Myo-inositol was then shown to be converted to [(3)H]chiro-inositol in rats in vivo and in fibroblasts in vitro in a process stimulated by insulin. We next demonstrated that the conversion of [(3)H]myo-inositol to [(3)H]chiro-inositol in vivo was markedly decreased in GK type 2 diabetic rats compared to Wistar controls in liver, muscle, and fat, insulin sensitive tissues. Decreases of 20-25% conversion to baseline levels of under 5% conversion were observed. In the present work, we initially compared the total contents of myo-inositol and chiro-inositol in GK type 2 diabetic rat kidney, liver, and muscle compared to Wistar controls. We demonstrated a consistent decreased total chiro-inositol to myo-inositol ratio in kidney, liver, and muscle compared to controls. We next established the presence of a myo-inositol to chiro-inositol epimerase activity in rat liver cytosol. Enzyme activity was shown to be time and enzyme concentration dependent with a broad pH optimum. It required NADH and NADPH for full activity, which is compatible with its action via an oxido-reductive mechanism. Lastly, we demonstrated that the epimerase enzyme bioactivity was significantly decreased in muscle, liver, and fat cytosolic extracts of GK type 2 diabetic rats versus Wistar controls. Decreased myo-inositol to chiro-inositol epimerase activity may therefore play a role in explaining the decreased chiro-inositol to myo-inositol urine and tissue ratios observed here and in previous animal and human studies. Further it may also possibly play a role in the underlying insulin resistance.

Adipose Tissue↗
PubMed1998

In vivo chiro-inositol metabolism in the rat: a defect in chiro-inositol synthesis from myo-inositol and an increased incorporation of chiro-[3H]inositol into phospholipid in the Goto-Kakizaki (G.K) rat.

We report our comparative studies of myo- and chiro-[3H]inositol distribution in control nondiabetic Wistar and spontaneous nonobese insulin-resistant Type II diabetic G.K. rat tissues following 78-h labeling period. From various tissue extracts, free inositols, inositol phosphates, and inositol phospholipids were isolated and analyzed. Our findings demonstrate (1) a significant difference in the metabolism of myo- and chiro-inositol in normal Wistar and G.K. rats, (2) a severe defect in conversion of myo-[3H]inositol to chiro-[3H]inositol at the inositol phospholipid level of insulin-sensitive tissues of the G.K. rat, (3) an increased incorporation of myo-[3H]inositol into purified inositol phospholipids of the G.K. rat consistent with a decreased conversion to chiro-[3H]inositol, (4) the presumed presence of a pathway which incorporates chiro-[3H]inositol into inositol phospholipids, and which is overactive in the G.K. rat compared to the Wistar rat, and (5) no or minimal conversion of chiro-[3H]inositol to myo-[3H]inositol.

Animals↗
PubMed1990

Product-precursor relationships amongst inositol polyphosphates. Incorporation of [32P]Pi into myo-inositol 1,3,4,6-tetrakisphosphate, myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 3,4,5,6-tetrakisphosphate and myo-inositol 1,3,4,5,6-pentakisphosphate in intact avian erythrocytes.

Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.

Adenosine Triphosphate↗
PubMed1989

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase.

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.

Animals↗
PubMed1991

Multiple isomers of inositol pentakisphosphate in Epstein-Barr-virus- transformed (T5-1) B-lymphocytes. Identification of inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate.

Substantial amounts of three [3H]InsP5 isomers were detected in [3H]inositol-labelled human lymphoblastoid (T5-1) cells. Their structures were determined by h.p.l.c. [Phillippy & Bland (1988) Anal. Biochem. 175, 162-166], and by utilizing a stereospecific D-inositol 1,2,4,5,6-pentakisphosphate 3-kinase from Dictyostelium discoideum [Stephens & Irvine (1990) Nature (London) 346, 580-583]. The structures were: inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate. The relative proportions of these isomers (approx. 73:14:14 respectively) were unaffected by cross-linking anti-IgD receptors. The T5-1 cells also contained InsP6 and three Ins P4s, which were identified as the 1,3,4,5, 1,3,4,6 and 3,4,5,6 isomers. In incubations with permeabilized T5-1 cells, both 1,3,4,6 and 3,4,5,6 isomers of InsP4 were phosphorylated solely to Ins(1,3,4,5,6)P5. Permeabilized cells also dephosphorylated InsP6, even in the presence of a large excess of glucose 6-phosphate to saturate non-specific phosphatases. In the latter experiments the following isomers of InsP5 accumulated: D- and/or L-Ins(1,2,3,4,5)P5, plus D- and/or L-Ins(1,2,4,5,6)P5. This demonstration that multiple isomers of InsP5 may be formed in vivo and in vitro by a transformed lymphocyte cell line adds a new level of complexity to the study of inositol polyphosphate metabolism and function.

Animals↗
PubMed1987

myo-Inositol phosphorothioates, phosphatase-resistant analogues of myo-inositol phosphates. Synthesis of DL-myo-inositol 1,4-bisphosphate and DL-myo-inositol 1,4-bisphosphorothioate.

Syntheses of a metabolite of the second messenger myo-inositol 1,4,5-trisphosphate, myo-inositol 1,4-bisphosphate, and an analogue, the 1,4-bisphosphorothioate, are reported, by using phosphite chemistry on (+/-)-1,2:4,5-di-isopropylidene-myo-inositol. The synthesis of (+/-)-1,2:4,5-di-isopropylidene 3,6-bis[di-(2-cyanoethyl)]phosphite provides an intermediate that can be oxidized to either the corresponding bisphosphate or bisphosphorothioate. myo-Inositol phosphorothioates are proposed as novel analogues of myo-inositol phosphates; their resistance to phosphatase-catalysed breakdown is reported.

Alkaline Phosphatase↗
PubMed1991

Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate are inhibitors of the soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase and the inositol 1,4,5-trisphosphate/1,3,4,5-tetrakisphosphate 5-phosphatase from pig brain.

The influence of highly phosphorylated inositol phosphates on the Ins(1,3,4,5)P4 3-phosphatase enriched from the soluble fraction of pig brain was tested, using [5-32P]Ins(1,3,4,5)P4 as substrate. Both Ins(1,3,4,5,6)P5 and InsP6 were very potent inhibitors of the Ins(1,3,4,5)P4 3-phosphatase. The Ki values were approximately 60 nM and approximately 3 nM for Ins(1,3,4,5,6)P5 and InsP6 respectively. Ins(1,3,4,5,6)P5 and InsP6 also inhibited the Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. Using Ins(1,3,4,5)P4 as substrate, the Ki values were about 35 microM and 15 microM for Ins(1,3,4,5,6)P5 and InsP6 respectively. The concentrations which led to a 50% inhibition of Ins(1,4,5)P3 (0.5 microM) degradation by the 5-phosphatase were about 20 and 10 microM for the pentakis- and hexakis-phosphate respectively. As the intracellular concentrations of Ins(1,3,4,5,6)P5 and InsP6 are high (up to 60 microM) compared with those of the inositol trisphosphates and tetrakisphosphates, it is possible that the highly phosphorylated inositol phosphates act as regulators in the metabolism of Ca(2+)-mobilizing inositol phosphates.

Animals↗
PubMed1993

Myo-inositol 1,3,4,5-tetrakisphosphate can independently mobilise intracellular calcium, via the inositol 1,4,5-trisphosphate receptor: studies with myo-inositol 1,4,5-trisphosphate-3-phosphorothioate and myo-inositol hexakisphosphate.

Myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] acts as a full agonist for Ca2+ release in saponin-permeabilised SH-SY5Y neuroblastoma cells. Studies were conducted in the presence of myo-inositol hexakisphosphate (InsP6, 10 microM), to inhibit the Ins(1,3,4,5)P(4)-3-phosphatase catalysed back conversion of Ins(1,3,4,5)P4 to Ins(1,4,5)P3. HPLC analysis confirmed that Ins(1,3,4,5)P4 releases the entire content of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores, independent of 3-phosphatase activity. Further we utilised racemic myo-inositol 1,4,5-trisphosphate-3-phosphorothioate [DL-Ins(1,3,4,5)P(4)-3S], a novel intrinsically Ins(1,3,4,5)P(4)-3-phosphatase resistant Ins(1,3,4,5)P4 analogue. DL-Ins(1,3,4,5)P(4)-3S specifically displaced [3H]Ins(1,4,5)P3 from bovine adrenal cortex Ins(1,4,5)P3 binding sites (IC50 = 889 nM, compared to Ins(1,4,5)P3, IC50 = 4.4 nM and Ins(1,3,4,5)P4, IC50 = 152 nM). DL-Ins(1,3,4,5)P(4)-3S was a full agonist for Ca2+ release (EC50 = 4.7 microM), being 90- and 2-fold less potent than Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (with InsP6), respectively. DL-Ins(1,3,4,5)P(4)-3S will be an important tool for identification of potentially exclusive Ins(1,3,4,5)P4 second messenger functions, since its resistance to 3-phosphatase action precludes the inconvenient artefact of steady state Ins(1,4,5)P3 generation.

Animals↗
PubMed1987

Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate.

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)P3.

Blood Platelets↗
PubMed1990

Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate.

In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.

Animals↗
PubMed2000

Inositol for respiratory distress syndrome in preterm infants.

BACKGROUND: Inositol is an essential nutrient required by human cells in culture for growth and survival. Inositol promotes maturation of several components of surfactant and may play a critical role in fetal and early neonatal life. OBJECTIVES: To assess the effectiveness/safety of supplementary inositol in preterm infants with RDS in reducing adverse neonatal outcomes. SEARCH STRATEGY: Medline, Embase, and Reference Update Databases were searched in August 1997 using key words: inositol and infant-newborn and random allocation or controlled trial or randomized trial (RCT). The reference lists of identified RCTs, personal files and Science Citation Index were searched. Unpublished additional information was obtained from the authors of one RCT published in abstract form. SELECTION CRITERIA: All randomized controlled trials of inositol supplementation to preterm infants with a control group that received a placebo or no intervention were included. Outcomes of interest were bronchopulmonary dysplasia (BPD), death, BPD or death, retinopathy of prematurity (ROP), intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), and sepsis. DATA COLLECTION AND ANALYSIS: Data on neonatal outcomes were abstracted independently by the two researchers and any discrepancy was resolved through consensus. Revman was used for analysis of the data. MAIN RESULTS: Four reports of three RCTs were identified. One report was a duplicate publication. The outcome of death or bronchopulmonary dysplasia was reported in two trials, and was found to be significantly reduced (RR 0.56, 95% CI 0.42, 0.77; RD -0.215, 95% CI -0.323, -0.107). The outcome of death was reported in two trials and was found to be significantly reduced (RR 0.48, 95% CI 0.28, 0.80; RD -0.131, 95% CI -0.218, -0.043). Retinopathy of prematurity, stage 4 or needing therapy, was reported in two trials, and was found to be significantly reduced (RR 0.09, 95% CI 0.01, 0.67; RD -0.078, 95% CI -0.128, -0.027). Intraventricular hemorrhage, grade III-IV, was significantly decreased (RR 0.55, 95% CI 0.32, 0.95; RD -0.090, 95% CI -0.170, -0.010). Neither sepsis nor necrotizing enterocolitis outcomes were increased. When a secondary analysis was done excluding a study published in abstract form, the results differed only in that there was a significant reduction in retinopathy of prematurity, any stage (RR 0.53, 95% CI 0.29, 0.97; RD -0.082, 95% CI -0.159,-0.005). REVIEWER'S CONCLUSIONS: Inositol supplementation results in statistically significant and clinically important reductions in important short-term adverse neonatal outcomes. A multi-center RCT of appropriate size is justified to confirm these findings.

Dietary Supplements↗
PubMed1986

Rapid formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands may both result indirectly from receptor-stimulated release of inositol 1,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate.

Addition of 1 mM-carbachol to [3H]inositol-labelled rat parotid slices stimulated rapid formation of [3H]inositol 1,3,4,5-tetrakisphosphate, the accumulation of which reached a peak 20 s after stimulation, and then declined rapidly towards a new steady state. The initial rate of formation of inositol 1,3,4,5-tetrakisphosphate was slower than that for inositol 1,4,5-trisphosphate. The radioactivity in [3H]inositol 1,3,4,5-tetrakisphosphate fell quickly in carbachol-stimulated and then atropine-blocked parotid slices, suggesting that it is rapidly metabolized during stimulation. Parotid homogenates rapidly dephosphorylated inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and, less rapidly, inositol 1,3,4-trisphosphate. Inositol 1,3,4,5-tetrakisphosphate was specifically hydrolysed to a compound with the chromatographic properties of inositol 1,3,4-trisphosphate. The only 3H-labelled phospholipids that we could detect in parotid slices labelled with [3H]inositol for 90 min were phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Parotid homogenates synthesized inositol tetrakisphosphate from inositol 1,4,5-trisphosphate. This activity was dependent on the presence of ATP. We suggest that, during carbachol stimulation of parotid slices, the key event in inositol lipid metabolism is the activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. The inositol 1,4,5-trisphosphate thus liberated is metabolized in two distinct ways; by direct hydrolysis of the 5-phosphate to form inositol 1,4-bisphosphate and by phosphorylation to form inositol 1,3,4,5-tetrakisphosphate and hence, by hydrolysis of this tetrakisphosphate, to form inositol 1,3,4-trisphosphate.

Animals↗
PubMed1993

Effects of inositol starvation on the levels of inositol phosphates and inositol lipids in Neurospora crassa.

An inositol-requiring strain of Neurospora crassa was labelled during growth in liquid medium with [3H]inositol, and the levels of inositol phosphates and phosphoinositides were determined under inositol-sufficient and inositol-starved conditions. Because the mutant has an absolute requirement for inositol, the total mass of inositol-containing compounds could be determined. Inositol-containing lipids were identified by deacylation and co-migration with standards on h.p.l.c.; PtdIns3P, PtdIns4P, and PtdIns(4,5)P2 were found in approximately equal amounts, in addition to large amounts of PtdIns. Inositol starvation decreased the level of PtdIns to 10% of the sufficient level, and decreased the levels of the other phosphoinositides to about 25%. A number of inositol phosphates were found, including several InsP3s, InsP4s and InsP5s and phytic acid. Ins(1,4,5)P3 was identified by co-migration with standards on h.p.l.c. and by digestion with inositol phosphomonoesterase. High concentrations of all inositol phosphates were found in the extracellular medium in inositol-starved cultures. Inositol starvation on both liquid and solid agar media decreased the intracellular levels of some inositol phosphates, but increased the levels of phytic acid and several other inositol phosphates which may be its precursors and/or breakdown products. These results may indicate that inositol starvation induces phytic acid synthesis as a protection against the free-radical production and lipid peroxidation characteristic of inositol-less death.

Chromatography, High Pressure Liquid↗
PubMed1988

Formation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate from inositol 1,3,4,5-tetrakisphosphate and their pathways of degradation in RBL-2H3 cells.

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.

Animals↗
PubMed1989

The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover.

Some, though not all, previous studies have suggested that the inositol lipid which is hydrolysed during transmembrane signalling in response to receptor activation might be drawn from a metabolically discrete and relatively small hormone-sensitive lipid pool that turns over more rapidly than the bulk of membrane inositol lipid. In order to seek evidence for the existence of this putative hormone-sensitive lipid pool, we have double-labelled cells by growing them for 3 days in a medium containing [14C]inositol and then supplying them with [3H]inositol for the final 2 h before stimulation. We anticipated that stimulation of these doubly labelled cells might provoke the formation, from the postulated hormone-sensitive pool, of small quantities of relatively 3H-enriched inositol phosphates, and that these could be harvested from cells (provided that the cytosolic inositol monophosphatase and inositol 1,4-bisphosphate/inositol 1,3,4-trisphosphate 1-phosphatase activities are first inhibited by Li+). Experiments of this type, using both vasopressin-stimulated WRK1 rat mammary tumour cells and 3T3 mouse fibroblasts stimulated by prostaglandin F2 alpha, have largely failed to demonstrate the formation of relatively 3H-enriched inositol phosphates. There was a tendency for phosphatidyl-inositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to have slightly higher 3H: 14C ratios than phosphatidylinositol, but the 3H: 14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the 3H: 14C ratios of the inositol lipids. We therefore conclude, at least for the two cell lines that we studied, that hormone-stimulated inositol lipid hydrolysis can call, either directly or indirectly, upon the majority of the inositol lipid complement of the stimulated cell.

Animals↗
PubMed1989

Microinjection of inositol 1,2-(cyclic)-4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium.

Inositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives into Xenopus laevis oocytes. These cells have calcium-regulated chloride channels but do not have a calcium-induced calcium release system. Microinjection of inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,2-(cyclic)-4,5-trisphosphate (cIP3), inositol 1,4,5-trisphosphate (IP3), or inositol 4,5-bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1-phosphate (IP1), inositol 1,3,4,5,6-pentakisphosphate (IP5), inositol 1,4-bisphosphate, or inositol 3,4-bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3 and cIP3 induce similar half-maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4 induces a mono- or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3 and cIP3, (4,5)IP2 induces a long-term (greater than 40 min) depolarization. The efficacy (cIP3 = IP3 = (4,5)IP2 much greater than IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meiotic cell division are discussed.

Animals↗
PubMed1996

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates.

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate, (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Binding Sites↗