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PubMed1996

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 5. Mechanism of the myo-inositol efflux pathway.

PURPOSE: Cultured bovine lens epithelial cells (BLECs) exposed to sodium hypertonicity respond with an accumulation of intracellular myo-inositol. Using BLECs initially maintained at hypertonicity and reacting to a decrease in medium osmolality, a mechanism for the tonicity-activated release of myo-inositol was recognized. Alternatively, BLECs acclimated to sodium hypertonicity and subsequently transferred to high sodium osmolality plus hypergalactosemia rapidly accumulate intracellular galactitol, an experimental manipulation that permitted characterization of the role of sugar alcohols in polyol-activated myo-inositol efflux. The authors identify a communal transport route for tonicity-activated and polyol- activated myo-inositol release from cell to medium and demonstrate an association for myo-inositol efflux with chloride movement. METHODS: Two distinct experimental approaches were designed to delineate the physiological circumstances that initiate myo-inositol efflux. For tonicity-induced inositol efflux, BLECs were maintained at confluence in sodium hypertonic medium (473+/-6 mOsm) for 48 hours; afterward, the medium was replaced with isotonic medium (285+/-4 mOsm) containing 40 mM galactose +/- Sorbinil. For polyol-induced inositol release, hypertonically adapted BLECs were transferred to fresh sodium hypertonic medium containing 40 mM galactose (513+/- 10 mOsm). RESULTS: On reduction in medium osmolality, intracellular myo-inositol was lost because of a rapid, transient efflux during the first 30 minutes, which was followed by a slow, sustained decrease in efflux during the next 12 hours. Inhibition of aldose reductase activity substantially diminished myo-inositol efflux from cell to galactose-containing, isotonic medium. Administration of phloretin significantly inhibited both tonicity-activated and polyol-activated myo-inositol release, as did the chloride channel blocker, niflumic acid. CONCLUSIONS: In cultured bovine lens epithelial cells, tonicity-activated movement of myo-inositol from cell to medium and myo-inositol efflux as induced by intracellular polyol accumulation appear to be interactively associated with chloride movement and moderated by a common anionic (chloride) channel, carrier-mediated transport protein, or both.

Animals↗
PubMed1989

Chemoattractant and guanosine 5'-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.

Animals↗
PubMed1988

Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation.

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.

Adenosine Triphosphate↗
PubMed1997

Identification of chiro-inositol and its formation by isomerization of myo-inositol during hydrolysis of glycosylphosphatidylinositol-anchored proteins.

myo-Inositol has been believed to be a sole inositol isomer existing in phosphatidylinositol (PI) and related derivatives. In this experiment, chiro-inositol, an inositol isomer other than myo-inositol, was identified in hydrolytic products from several GPI-anchored proteins. The chiro-inositol contents in several different GPI-anchored proteins including 5'-nucleotidase of bovine liver and alkaline phosphatase of mouse NS-1 varied with hydrolytic conditions of these GPI anchor. Isomerization of 20-60% of myo-inositol occurred on the hydrolysis in 6 N HCl solution. Under the hydrolytic conditions of a HCl gas stream in place of solution, however, isomerization was very low (less than 0.1%). Even in the hydrolysis under HCl gas stream, existence of CNBr accelerated the isomerization of inositol in GPI up to 70-95%. In the hydrolysis of phosphatidylinositol or myo-inositol 1-phosphate, however, a significant amount of chiro-inositol was not detected in 6 N HCl solution or in the existence of CNBr under the HCl stream. These facts indicated that isomerization occurred during the hydrolysis of the GPI anchor, when myo-inositol is substituted by glucosamine at 6-OH and is substituted by phosphate at 1-OH. It also suggested that the former identification of chiro-inositol in GPI structure in the various reports might be due to isomerization.

5'-Nucleotidase↗
PubMed1999

Epi-inositol and inositol depletion: two new treatment approaches in affective disorder.

Inositol is a simple polyol precursor in a second messenger system important in brain myo-insitol, the natural isomer, which has been found to be therapeutically effective in depression, panic disorder, and obsessive-compulsive disorder in double-blind controlled trials. Recently, epi-inositol, an unnatural stereoisomer of myo-inositol, was found to have effects similar to those of myo-inositol to reverse lithium-pilocarpine seizures. We measured the behavior of rats in an elevated plus maze model of anxiety after chronic treatment of 11 daily intraperitoneal injections of epi-inositol, myo-inositol, or control solution. Epi-inositol reduced anxiety levels of rats compared with controls, and its effect was stronger than that of myo- inositol. Lithium has been hypothesized to alleviate mania by reducing brain inositol levels. Inositol in brain derives from the second messenger cycle, from new synthesis, or from diet via transport across the blood brain barrier. Because the first two are inhibited by lithium, we propose that an inositol-free diet will augment lithium action in mania by enhancing restriction of inositol.

Animals↗
PubMed1990

Glucose inhibits myo-inositol uptake and reduces myo-inositol content in cultured rat glomerular mesangial cells.

Although activation of polyol pathway has been proposed as one of the etiologic factors of diabetic complications, precise mechanism of the effect of polyol accumulation is still unclear. In order to test the hypothesis that there is an association of polyol pathway with myo-inositol metabolism, we measured myo-inositol content in cultured rat glomerular mesangial cells. By exposing the cells to high concentrations of glucose, intracellular myo-inositol content was reduced from 12.39 +/- 0.64 nmol/mg protein at 0 mmol/L glucose to 6.54 +/- 0.38 nmol/mg protein at 27.5 mmol/L glucose and 4.88 +/- 0.43 nmol/mg protein at 55 mmol/L glucose. This decrease of myo-inositol content was partially prevented by co-incubation with aldose reductase inhibitor, sorbinil. To examine further the mechanism of myo-inositol depletion, myo-inositol uptake by mesangial cells was studied. Major myo-inositol uptake process was sodium-dependent, saturable, and ouabain sensitive with Vmax of 171 pmol/mg protein/20 min and Km of 33 mumol/L. Sodium-dependent myo-inositol uptake was significantly inhibited by glucose in a dose-dependent manner only when glucose was present during uptake experiment, and kinetic analysis revealed the inhibition was competitive. Aldose reductase inhibition failed to prevent inhibitory effect of glucose on myo-inositol uptake. These data suggest that myo-inositol content of glomerular mesangial cells, which is reduced by high concentrations of glucose, is maintained by two processes: a glucose-sensitive but sorbitol-insensitive process, sodium-dependent myo-inositol uptake; and a sorbitol (aldose reductase) sensitive process, myo-Inositol depletion under high glucose condition may induce dysfunction of mesangial cells seen in diabetes.

Aldehyde Reductase↗
PubMed2002

Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis.

Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg(2+) being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC(50) = 0.9 mM). As anticipated, inositol-1-phosphate was the preferred substrate (K(m) = 0.177 +/- 0.025 mM; k(cat) = 3.6 +/- 0.2 s(-)(1)); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2'-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-1-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC(50) = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.

Amino Acid Sequence↗
PubMed1976

myo-Inositol metabolism during lactation and development in the rat. The prevention of lactation-induced fatty liver by dietary myo-inositol.

Effects of dietary myo-inositol deprivation were examined during prenatal and postnatal development and during lactation in the rat. The deficient diet contained no detectable myo-inositol while the supplemented diet contained 0.5% (w/w) myo-inositol while the supplemented diet ct contained 0.5% (w/w) myo-inositol at the expense of sucrose. Both diets contained 25% casein, adequate amounts of all known vitamins, choline, and essential fatty acids as well as 0.5% (w/w) phthalylsulfathiazole to depress myo-inositol contribution to the diet by microorganisms. Pregnant rats of the Holtzman strain were fed the respective diets during gestation and lactation, and pups were fed the corresponding diet after weaning until 3 months of age. There were no significant differen-es in body weight between experimental groups. Supplementation of the diet with myo-inositol significanly increased the levels of myo-inositol in plasma, liver, kidney, and intestine of pups at all ages examined, and significantly increased the levels of myo-inositol in the milk and mammary tissue during lactation. During lactation, the myo-inositol deprived dams developed severe fatty livers (31% w/w) characterized by diminished phosphatidyl-inositol (50%) and total phospholipid phosphorus (57%) levels as compared with controls. After weaning, the liver lipid content of the myo-inositol deprived dams returned to normal (4.5%). The data suggest that a possible threshold level of free myo-inositol (approximately 0.15 mumoles/g lipid-free tissue) was required to prevent fatty liver in lactating dams under these dietary conditions. Effects of the deficient diet on fertility were also examined. Based on sperm count and production of offspring, there were no differnences between the experimental and control males. Females of both groups showed equal ability to produce offspring.

Amniotic Fluid↗
PubMed1993

Upregulation of myo-inositol transport compensates for competitive inhibition by glucose. An explanation for the inositol paradox?

High glucose concentrations inhibit the uptake of myo-inositol into cells. However, whether this leads to a depletion of intracellular myo-inositol levels has been debated, because unchanged, decreased, and increased cellular myo-inositol levels all have been reported for diabetic tissues. To evaluate whether cells are capable of counterregulating impaired uptake, we have investigated myo-inositol uptake in porcine aortic endothelial cells under short- and long-term hyperglycemic conditions. Although increasing glucose concentrations inhibited acute myo-inositol uptake competitively, the uptake was increased markedly, when cells were already preincubated in a high glucose medium for > 6 h. The stimulation was maximal at 20 mM of glucose with no further increase at 40 mM glucose. The same stimulation of uptake could be induced by 5 mM of glucose plus 35 mM of raffinose, whereas 35 mM of sorbitol or mannitol, which do not compete for myo-inositol uptake, were ineffective. Lineweaver-Burk analysis revealed an increased Vmax for the induced myo-inositol transport activity, whereas the Km for myo-inositol remained constant (18 microM). The upregulated inositol transporter was still Na+ and ATP dependent, indicating that the same carrier system was operating. Uptake returned to control values when cells were again exposed to normoglycemic medium conditions for an additional 24 h. When endothelial cells were incubated with D-[U-14C]glucose and 10 microM myo-[2-3H]inositol of equal specific radioactivity for 24 h, no 14C radioactivity was found in intracellular myo-inositol, indicating that conversion of glucose to myo-inositol was rather low.

Animals↗
PubMed1992

Modulation of myo-[3H]inositol uptake by glucose and sorbitol in cultured bovine lens epithelial cells. I. Restoration of myo-inositol uptake by aldose reductase inhibition.

The association between high-ambient glucose, the polyol pathway, and aldose reductase inhibition on in vitro myo-[3H]inositol uptake was examined in cultured bovine lens epithelial cells (BLECs). Myo-[3H]inositol accumulation in the presence of 5.5 mmol/l D-glucose was rapid and linear for 8 hr. When Na+ was replaced on an equal molar basis with N-methyl-D-glucamine chloride, myo-[3H]inositol uptake was reduced by more than 95%. The myo-inositol transport system appear to be distinct from glucose transport, based upon three criteria: (1) 2-deoxy-D-[3H]glucose uptake, unlike myo-[3H]inositol uptake, was largely sodium independent; (2) L-glucose was a competitive inhibitor of myo-[3H]inositol uptake but had no effect on 2-deoxy-D-[3H]glucose uptake; and (3) 2-deoxy-D-[3H]glucose uptake appeared independent of myo-inositol concentration. Sodium-dependent myo-[3H]inositol uptake was substantially inhibited after chronic (20 hr) exposure of cultured cells to 40 mmol/l glucose. Inhibition of aldose reductase activity partially prevented the inhibitory effect of glucose on myo-[3H]inositol accumulation. No significant difference in the rates of passive efflux of myo-[3H]inositol from preloaded high glucose-treated and control cultures was observed. Although the coadministration of sorbinil to the high-glucose medium partially protected against the attendant decrease in transport activity, the failure to normalize myo-[3H]inositol uptake suggested that glucose-sensitive and sorbitol-sensitive processes were involved in the uptake of myo-inositol.

Aldehyde Reductase↗
PubMed1987

Reduction of inositol triphosphate in retinal microvessels by glucose and restimulation by myo-inositol.

The formation of inositol phospholipids (IPLs) and inositol phosphate esters (IPEs) in response to glucose was studied in isolated retinal microvessels from porcine eyes. Retinal microvessels incubated from 60 hr with myo-[3H]inositol were sequentially extracted to obtain IPLs and IPEs. [3H]Inositol-labelled IPLs were deacylated to produce the corresponding glycero derivatives. Both deacylation products and water-soluble IPEs were monitored by anion-exchange chromatography. In the presence of high glucose (30 mM) the labelling in inositol triphosphate (IP3) was reduced to 77% and was restimulated by adding myo-inositol (final concentration 0.4 mM) to 158% of the control under physiological conditions of glucose (5 mM) and myo-inositol (0.04 mM). With a fixed glucose concentration (5 mM), IPE accumulation was observed with increasing concentrations of exogenous myo-inositol. Under physiological conditions (glucose 5 mM, myo-inositol 0.04 mM) the distribution (percentage) of radioactivity in phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) was 63:19:18. The myo-inositol concentration dependence of IPL formation was also demonstrated. A decrease in IP3 in response to high glucose without changing PIP2 but with a reduction in PI indicated that PI may act as a reservoir to replace a possible loss of PIP2. These findings suggest that availability of myo-inositol by retinal microvessels may be essential to maintain the normal signal transduction and cell proliferation associated with IPL turnover under high glucose concentration.

Animals↗
PubMed1999

Synthesis, calcium mobilizing, and physicochemical properties of D-chiro-inositol 1,3,4,6-tetrakisphosphate, a novel and potent ligand at the D-myo-inositol 1,4,5-trisphosphate receptor.

The synthesis of a novel and potent ligand at the D-myo-inositol 1,4, 5-trisphosphate receptor (InsP3R) is described. D-chiro-Inositol 1,3, 4,6-tetrakisphosphate (7) and L-chiro-inositol 1,3,4, 6-tetrakisphosphate (ent-7) have been synthesized from D-2, 5-di-O-benzyl-chiro-inositol and L-2,5-di-O-benzyl-chiro-inositol, respectively. The potency of binding and Ca2+ release of 7 and ent-7 were examined in L15 and Lvec cells. 7 was a full agonist at the InsP3R in both cells, and ent-7 was inactive. The results are compared to those from D-myo-inositol 1,4,5-trisphosphate (1), DL-scyllo-inositol 1,2,4-trisphosphate (2), DL-myo-inositol 1,2,4, 5-tetrakisphosphate (3), scyllo-inositol 1,2,4,5-tetrakisphosphate (4), D-myo-inositol 2,4,5-trisphosphate (5), and D-chiro-inositol 1, 3,4-trisphosphate (6). The protonation processes of 7 have also been investigated by 31P NMR titration experiments.

Animals↗
PubMed2006

Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants.

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.

2,3-Diphosphoglycerate↗
PubMed1982

Redistribution of Tritium during Germination of Grain Harvested from myo-[2-H]Inositol- and scyllo-[R-H]Inositol-Labeled Wheat.

Wheat kernels from myo-[2-(3)H]inositol- or scyllo-[R-(3)H]inositol-labeled plants (Sasaki and Loewus 1980 Plant Physiol 66: 740-745) were used to study redistribution of (3)H into growing regions during germination. Most of the labeled 1-alpha-galactinol (or the analogous scyllo-inositol galactoside) was hydrolyzed within 1 day. Water-soluble phytate was dephosphorylated within 3 days. A large reserve of bound phytate continued to release myo-inositol over several days. Translocation of free myo-inositol to growing regions provided substrate for the myo-inositol oxidation pathway and incorporation of (3)H into new cell wall polysaccharides.Cell wall polysaccharides in the kernel were degraded during germination. The labeled residues were translocated to growing regions and reutilized for new cell wall formation. Pentosyl residues accounted for most of this label.Free scyllo-inositol followed a path of translocation from kernel to seedling similar to that of myo-inositol. Unlike myo-inositol, it did not furnish substrate for the myo-inositol oxidation pathway but accumulated as free scyllo-inositol in the seedling.The fate of phytate-derived myo-inositol during germination of wheat is discussed in relation to a recent scheme of phytate metabolism proposed by De and Biswas (1979 J Biol Chem 254: 8717-8719) for germinating mung bean seedlings.

Journal Article↗
PubMed1989

Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O.

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.

Animals↗
PubMed1985

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.

Angiotensin II↗
PubMed1986

Inositol 1,3,4,5-tetrakisphosphate and not phosphatidylinositol 3,4-bisphosphate is the probable precursor of inositol 1,3,4-trisphosphate in agonist-stimulated parotid gland.

When [3H]inositol-prelabelled rat parotid-gland slices were stimulated with carbachol, noradrenaline or Substance P, the major inositol trisphosphate produced with prolonged exposure to agonists was, in each case, inositol 1,3,4-trisphosphate. Much lower amounts of radioactivity were present in the inositol 1,4,5-trisphosphate fraction separated by anion-exchange h.p.l.c. Analysis of the inositol trisphosphate head group of phosphatidylinositol bisphosphate in [32P]Pi-labelled parotid glands showed the presence of phosphatidylinositol 4,5-bisphosphate, but no detectable phosphatidylinositol 3,4-bisphosphate. Carbachol-stimulated [3H]inositol-labelled parotid glands contained an inositol polyphosphate with the chromatographic properties and electrophoretic mobility of an inositol tetrakisphosphate, the probable structure of which was determined to be inositol 1,3,4,5-tetrakisphosphate. Since an enzyme in erythrocyte membranes is capable of degrading this tetrakisphosphate to inositol 1,3,4-trisphosphate, it is suggested to be the precursor of inositol 1,3,4-trisphosphate in parotid glands.

Animals↗
PubMed1986

Perturbation of the human T-cell antigen receptor-T3 complex leads to the production of inositol tetrakisphosphate: evidence for conversion from inositol trisphosphate.

Antibodies directed against the T-cell antigen receptor-T3 complex mimic antigen and lead to cellular changes consistent with activation. When cells of the human T-cell line Jurkat were stimulated with a monoclonal antibody directed against T3, inositol phosphates were produced. In addition to inositol trisphosphate, which is the product of phosphatidylinositol bisphosphate cleavage, a second inositol polyphosphate was formed. This compound was more polar than inositol trisphosphate but less polar than inositol pentakisphosphate. It cochromatographed with inositol tetrakisphosphate from ostrich erythrocytes. In permeabilized Jurkat cells, this compound was shown to be formed from inositol 1,4,5-trisphosphate, but only in the presence of ATP, and 32P was incorporated into it from [gamma-32P]ATP. There also was coincident formation of inositol 1,3,4-trisphosphate. We conclude that the more polar compound is inositol tetrakisphosphate, which is formed by phosphorylation of inositol 1,4,5-trisphosphate and may be the precursor of inositol 1,3,4-trisphosphate.

Cell Line↗