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PubMed1991

Evidence for attenuation of myo-inositol uptake, phosphoinositide turnover and inositol phosphate production in aortic vasculature of rats during pregnancy.

We postulated that vascular phosphoinositide metabolism is attenuated during pregnancy, and thereby could contribute to maternal vasodilation and reduced vascular reactivity. The basal rate of incorporation of [3H]myo-inositol and [3H]glycerol into phosphoinositides of aortae from pregnant rats in vitro was significantly reduced, when compared with vessels from virgin animals. After injection of [3H]myo-inositol intravenously into chronically instrumented conscious pregnant and virgin rats, the incorporation of the label by phosphatidylinositol was 66 +/- 4% less in aortae of gravid versus virgin animals (P less than 0.001), despite comparable plasma concentrations of radioactivity. Fold stimulation of total [3H]inositol phosphates by arginine vasopressin, norepinephrine, and angiotensin II over a 15-min period was not different between aortic segments from virgin and gravid rats, although both absolute basal and stimulated levels were significantly less in vessels from pregnant animals. After 45 s of incubation with 10(-7) M arginine vasopressin, however, the fold-stimulation of [3H]inositol trisplus tetrakisphosphate was reduced in aortae from gravid rats, when compared with vessels from virgin animals (P less than 0.005). By HPLC, greater than 90% of the radioactivity in the [3H]inositol trisplus tetrakisphosphate column fraction after 30 and 60 s of agonist stimulation was [3H]inositol-1,4,5-trisphosphate. We further observed that the rate of uptake of [3H]myo-inositol by aortic vasculature obtained from gravid rats was significantly (24%) less than uptake by vessels from virgin animals. Plasma myo-inositol concentrations were not significantly different, but presumably as a consequence of reduced uptake, aortic segments freshly isolated from pregnant rats contained 22 +/- 6% less myo-inositol than vessels from virgin controls as measured by gas chromatography-mass spectrometry (P less than 0.03). We conclude that myo-inositol uptake and content, phosphoinositide turnover, and inositol phosphate production are reduced in aortic vasculature of gravid rats.

Animals↗
PubMed1991

Synthesis and Ca(2+)-release activity of D- and L-myo-inositol 2,4,5-trisphosphate and D- and L-chiro-inositol 1,3,4-trisphosphate.

Partial benzoylation of the 3,4-dibenzyl ethers of D- and L-chiro-inositol provided the 1,2,5-tri-O-benzoyl-3,4-di-O-benzyl-chiro-inositols. Inversion of the free axial hydroxyl group gave a mixture of chiral 1,3,4- and 1,2,4-tri-O-benzoyl-5,6-di-O-benzyl-myo-inositols [W. Tegge and C. E. Ballou, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 94-98]. Catalytic hydrogenolysis cleaved the benzyl ether groups of the 1,3,4-tri-O-benzoyl-5,6-di-O-benzyl-myo-inositols (D- and L-) to yield the 1,3,4-tri-O-benzoyl-myo-inositols, which were phosphorylated by a dibenzyl phosphoramidite method. Removal of all blocking groups gave the pure enantiomeric myo-inositol 2,4,5-trisphosphates. Syntheses of the chiro-inositol 1,3,4-trisphosphates, which are analogs of the myo-inositol 1,4,5-trisphosphates having an axial phosphate group at position 1, or analogs of the myo-inositol 2,4,5-triphosphates having an axial hydroxyl at position 1, were also devised starting with the 1,2,5-tri-O-benzoyl-3,4-di-O-benzyl-chiro-inositols. In a calcium-release assay with saponin-permeabilized rat basophilic leukemia cells, the D isomers of both of these analogs had EC50 values of 4 microM, compared with a value of 0.17 microM for D-myo-inositol 1,4,5-trisphosphate, whereas the L isomers had EC50 values of about 100 microM.

Animals↗
PubMed1997

A convenient synthesis of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and L-myo-inositol 1,4,5-trisphosphate (Ins(3,5,6)P3).

An efficient synthesis of an optically active inositol derivative that is a precursor to D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, (-)) is described. Crystallization of the diastereomers of (+/-)-1-O-[(+)-menthoxycarbonyl]-6-O-benzyl-2,3:4,5-di-O-isopropyl idene-myo- inositol diastereomers from methanol gives only one diastereomer. Alkaline hydrolysis gives the useful inositol derivative (-)-6-O-benzyl-2,3:4,5-di-O-isopropylidene-myo-inositol. Likewise, crystallization of the diastereomers of (+/-)-3-O-[(-)-menthoxycarbonyl]-4-O-benzyl-1,2:5,6-di-O-isopropyl idene-myo- inositol from methanol gave a pure compound which could be hydrolyzed to give (+)-4-O-benzyl-1,2:5,6-di-O-isopropylidene-myo-inositol, a precursor to D-myo-inositol 3,5,6-trisphosphate (Ins(3,5,6)P3,(+)). The ease with which these enantiomerically pure inositol derivatives were isolated may facilitate the synthesis of more complex inositol phosphate derivatives such as D-myo-inositol 1,3,4,5-tetrakisphosphate.

Inositol 1,4,5-Trisphosphate↗
PubMed1986

Metabolism and function of myo-inositol and inositol phospholipids.

Alterations in the level of dietary inositol can significantly influence the concentration of free inositol and inositol-containing phospholipid in the circulation and in selected mammalian tissues and cells. The 1-stearoyl 2-arachidonyl molecular species that commonly predominates in cellular phosphoinositides may be of considerable importance for the functioning of these phospholipids in biological membranes. Retailoring reactions subsequent to the de novo biosynthesis of PI involving the acylation of lyso(1-acyl) PI allow for the preferential enrichment of this phospholipid in arachidonic acid. The impaired release of plasma lipoprotein, increased fatty acid mobilization from adipose tissue, and enhanced fatty acid synthesis in liver have all been implicated as causative factors in the hepatic triacylglycerol accumulation occurring with experimental inositol deficiency. The severe intestinal lipodystrophy that develops in female gerbils consuming inositol-deficient diets is likely mediated by a reduced synthesis of PI and the associated impairment of chylomicron assembly and secretion. Membrane PI can potentially regulate enzyme activities and transport processes as well as providing a source of free arachidonic acid for production of the eicosanoids. There has been mounting evidence recently to indicate that an accelerated turnover of the phosphoinositides may play a key role in mediating cellular responses to external stimuli. The transient rise of phosphoinositide-derived 1,2-diacylglycerol in stimulated cells may serve as a signal for the transmembrane control of protein phosphorylation by activating protein kinase C. Receptor occupancy also elicits the phosphodiesterase-catalyzed release of the second messenger inositol 1,4,5-trisphosphate, which appears to provide for the mobilization of calcium from internal stores. Subnormal levels of free inositol and inositol phospholipid, as found in the nerves of animals with experimental diabetes and in sciatic nerves removed postmortem from diabetic patients, have been implicated in the impaired nerve conduction of human diabetics. Patients with renal failure exhibit a dramatic hyperinositolemia that may have clinical significance. Nutritional intervention may offer an approach for counteracting abnormalities in inositol and inositol phospholipid profiles and associated physiological responses in certain disease states.

Absorption↗
PubMed1988

Prolactin induces the formation of inositol bisphosphate and inositol trisphosphate in cultured mouse mammary gland explants.

The effect of prolactin on [3H]inositol metabolism in cultured mouse mammary gland explants derived from 12-14-day pregnant mice was determined. In mammary gland explants that were prelabeled by culturing the tissues with 3 microCi/ml myo-[3H]inositol for 48 h, the levels of 3H in inositol derivatives were determined. The temporal effect of prolactin on the quantity of 3H present in phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and various inositol phosphate containing fractions were examined. Prolactin significantly stimulated the accumulation of 3H label in inositol monophosphate (IP1), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) 1-3 h after addition of prolactin. An effect of prolactin on the accumulation of inositol derivatives was not apparent at prolactin-exposure periods of less than 60 min; nor was an effect of prolactin apparent when exposure periods of 4 h or longer were employed. Prolactin did not significantly decrease the 3H label in PI, PIP or PIP2 except at 1 and 2 h. These data when considered with other apropos studies are compatible with the conclusion that the turnover of inositol lipid derivatives may be involved in the mechanism by which prolactin regulates metabolic processes in the mammary gland. The primary action of prolactin on mammary cells, however, would not appear to involve its action on the metabolism of the inositol derivatives in view of the extended time required (1 h) before effects of prolactin on perturbations of inositide metabolism are manifested.

Animals↗
PubMed2000

Antihyperglycemic effects of 3-O-methyl-D-chiro-inositol and D-chiro-inositol associated with manganese in streptozotocin diabetic rats.

The acute effects after administration of 3-O-Methyl-D-Chiro-inositol, D-Chiro-inositol and manganese, components of the pH 2.0 putative mediator of insulin action, on plasma glucose were examined in low dose streptozotocin-treated rats. 3-O-Methyl-D-Chiro-inositol at a bolus dose of 5 mg/kg decreased plasma glucose 6% at 120 min (p < 0.05). A higher bolus dose of 3-O-Methyl-D-Chiro-inositol (15 mg/kg) promoted a more persistent hypoglycemic effect of 22% at 120 min (p < 0.05). Infusion of 8.3 microg/min of manganese chloride lowered plasma glucose by 23% (p < 0.05). 3-O-Methyl-D-Chiro-inositol (15 mg/kg) together with manganese chloride (8.3 microg/min) promoted a reduction of 49% in 120 min (p < 0.05). D-Chiro-inositol at a bolus dose of 5 mg/kg had no effect. A single dose of 15 mg/kg produced a reduction of 21% (p < 0.05) in 120 min. D-Chiro-inositol (15 mg/kg) associated with manganese chloride (8.3 microg/min) decreased elevated plasma glucose 47% (p < 0.05) in 120 min. D-Chiro-inositol coadministered with manganese reduced glucose concentrations during the final 60 min (p < 0.05). 3-O-Methyl-D-Chiro-inositol and D-chiro-inositol are components of the mediator structure. Manganese is also a presumed component of the mediator, having an important role in glucose uptake, insulin release and mediator generation. These compounds have also been identified in the literature as hypoglycemic agents.

Animals↗
PubMed1991

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol.

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.

Animals↗
PubMed1986

Phosphatidylinositol:myo-inositol exchange activity in intact nerve endings: substrate and cofactor dependence, nucleotide specificity, and effect on synaptosomal handling of myo-inositol.

Micromolar concentrations of CMP produced a large increase in Mn2+-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 microM, and that for myo-inositol was 38 microM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP. Manganese did not affect the uptake of myo-inositol into the synaptosomal cytosolic fraction or myo-inositol levels. Determinations of myo-inositol specific activity showed that the Mn2+-enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo-inositol. This lack of an effect on intrasynaptosomal myo-inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+-dependent exchange activity, it is the myo-inositol in the incubation medium that is being directly incorporated into membrane-bound phosphatidyl-inositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP-diacylglycerol:inositol phosphatidyl transferase, this enzyme is likely to be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+-dependent exchange in the absence of added nucleotide.

Animals↗
PubMed1987

Effects of cerebral ischemia on [3H]inositol lipids and [3H]inositol phosphates of gerbil brain and subcellular fractions.

Intracerebral injection of [3H]inositol into gerbil brain resulted in labeling of phosphoinositides and inositol-phosphates in various subcellular membrane fractions. Phosphatidylinositol (PI) comprised greater than 90% of the radioactivity of inositol lipids. However, the level of labeled poly-PI (with respect to PI) was higher in synaptosomes than in other membrane fractions. Ischemia induced in gerbils by ligation of the common carotid arteries resulted in a 30% decrease in labeled poly-PI in brain homogenates and this decrease was largely attributed to the poly-PI in synaptosomes (50% decrease). Among the inositol phosphates, the ischemia induction resulted in a decrease in labeling of inositol triphosphate (63%) and inositol biphosphate (38%), but labeling of inositol phosphate (IP) was increased by 59%. The results suggested a rapid turnover of the inositol phosphates in the gerbil brain. In general, changes in inositol lipids and inositol phosphates due to ischemia were attenuated after pretreatment with lithium (3 meq/kg) injected intraperitoneally 5 h prior to ligation. Surprisingly, lithium treatment alone did not cause an increase in IP labeling in the gerbil brain.

Animals↗
PubMed1989

Metabolism of inositol pentakisphosphate to inositol hexakisphosphate in Xenopus laevis oocytes.

The formation and metabolism of inositol pentakis-and hexakisphosphates (InsP5 and InsP6) were investigated in Xenopus laevis oocytes. After [3H]inositol injection, [3H]InsP5 and subsequently [3H]Insp6 increased progressively over 72 h. In intact oocytes, [3H]InsP5 was progressively converted to [3H]InsP6 from 6 to 72 h of incubation and was not metabolized to lower inositol phosphates. In contrast, [3H]InsP6 remained unmetabolized for up to 72 h. These data are consistent with the kinetics of the increases in [3H]InsP5 and [3H]InsP6 in [3H]inositol-labeled oocytes. The highly phosphorylated inositols showed significant changes during oogenesis and maturation. In oocytes incubated for 48 h after [3H]inositol injection, the radioactive incorporation into polyphosphoinositols increased progressively from stage 3 to stage 6, with 5- and 6-fold rises (cpm/mg protein) for [3H]InsP5 and [3H]InsP6, respectively. These developmental changes were associated with 5-fold increases in [3H]inositol tetrakisphosphate between stages 3 and 6 of oogenesis. Induction of oocyte maturation by progesterone (1 microM) during the last 12 of a 36-h incubation with [3H]inositol doubled the levels of [3H]InsP6 relative to [3H]InsP5, suggesting that the activity of inositol pentakisphosphate kinase increases during maturation. These results provide direct evidence for metabolic conversion of InsP5 to InsP6 in animal cells and show that the higher inositol polyphosphates, unlike the lower phosphoinositols, are extraordinarily stable. These species increase markedly during ovum development and may play a regulatory role in oogenesis and maturation.

Animals↗
PubMed2006

Appel-Lee synthesis of glycosyl inositols, substrates for inositol dehydrogenase from Bacillus subtilis.

We recently reported that inositol dehydrogenase (EC 1.1.1.18) from Bacillus subtilis can catalyze the highly stereoselective oxidation of 1l-4-O-substituted myo-inositol derivatives, as well as disaccharides melibiose and isomaltose, but not gentiobiose or maltose, consistent with the requirement of an alpha-(1-->6) linkage. We believed that the enzyme might therefore catalyze efficient stereoselective oxidation of the appropriate alpha-linked glycosyl inositols. We have synthesized alpha-D-glucopyranosyl-(1-->4)-(DL)-myo-inositol and alpha-d-galactopyranosyl-(1-->4)-(DL)-myo-inositol using the Appel-Lee protocol to couple benzyl-protected glycosyl donors to protected inositols. This method failed in our hands using glycosyl donors derived from D-mannose and 2-azido-2-deoxy-D-glucose. When myo-inositol 1,3,5-monoorthoformate is used as the acceptor, the reaction is regiospecific for the 4/6-position. We report here the mildest conditions known for the removal of the orthoformate group. 2-Azido-2-deoxy-alpha-D-glucopyranosyl-(1-->4)-(DL)-myo-inositol was synthesized using the trichloroacetimidate derivative as the donor, and all three pseudo-disaccharides were substrates for inositol dehydrogenase. The glucopyranosyl and galactopyranosyl derivatives displayed apparent second-order rate constants comparable to that of myo-inositol.

Bacillus subtilis↗
PubMed2002

Selective epimerization of L-chiro-inositol to L-muco- and D-chiro-inositol derivatives.

In a one step procedure, L-1-O-benzyl-2-O-methyl-chiro-inositol (1) was acetalized to the L-muco-inositol derivatives 2, 3 and D-2-O-benzyl-3-O-cyclohexylcarbamoyl-4-deoxy-4-(N,N'-dicyclohexylureido)-1-O-methyl-5,6-O-trichloroethylidene-chiro-inositol (4). Complete conversion of L-1-O-benzyl-6-O-cyclohexylcarbamoyl-3-O-formyl-2-O-methyl-4,5-O-trichloroethylidene-muco-inositol (3) into L-1-O-benzyl-6-O-cyclohexylcarbamoyl-2-O-methyl-4,5-O-trichloroethylidene-muco-inositol (2) is feasible by deformylation in boiling methanolic triethylamine. Furthermore, stepwise deprotection of 2 and 4 is described. Thus, compounds 5, 10, and 7 were obtained by decarbamoylation of 2, 4, and 6, respectively, with boiling methanolic sodium methoxide. The trichloroethylidene group of L-1-O-benzyl-2-O-methyl-4,5-O-trichloroethylidene-muco-inositol (5) was removed in a two step procedure (hydrodechlorination-deacetalization) via the ethylidene acetal 7 to give L-1-O-benzyl-2-O-methyl-muco-inositol (9). On refluxing D-chiro-inositol derivative 4 with 99% acetic acid, the ureido moiety was cleaved generating D-2-O-benzyl-4-cyclohexylamino-3-O-cyclohexylcarbamoyl-4-deoxy-1-O-methyl-5,6-O-trichloroethylidene-chiro-inositol (11). By contrast, cleavage of the ureido moiety of 10 was relatively difficult. The corresponding D-2-O-benzyl-4-cyclohexylamino-4-deoxy-1-O-methyl-5,6-O-trichloroethylidene-chiro-inositol (12) was only formed in small amounts. The structures of 1, 3 and 10 were confirmed by X-ray analysis.

Acetylation↗
PubMed1990

Partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (indoleacetic acid-inositol synthase).

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-beta-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-beta-d-glucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.

Acyltransferases↗
PubMed2006

Identification of a functional 2-keto-myo-inositol dehydratase gene of Sinorhizobium fredii USDA191 required for myo-inositol utilization.

Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.

The metabolism of inositol 1,3,4-trisphosphate is a pivotal branch point of inositol phosphate turnover; its dephosphorylation replenishes cellular inositol pools, its phosphorylation at the 6-position supports the synthesis of inositol pentakisphosphate, and its phosphorylation at the 5-position produces inositol 1,3,4,5-tetrakisphosphate (Shears, S.B. (1989) J. Biol. Chem. 264, 19879-19886). In order to increase understanding of the control of inositol-1,3,4-trisphosphate kinase activity, the enzyme was highly purified from rat liver by precipitation with polyethylene glycol, MonoQ ion-exchange chromatography, heparin-agarose affinity chromatography, and a novel affinity chromatography procedure that utilized Affi-Gel resin to which InsP6 was coupled (Marecek, J.F., and Prestwich, G.D. (1991) Tetrahedron Lett. 32, 1863-1866). The final purification was about 26,000-fold, with a 6% yield. This final preparation performed both 5- and 6-kinase activities in the ratio of approximately 1:5. The affinity of the enzyme for inositol 1,3,4-trisphosphate was 0.04 microM, the highest yet determined for an inositol phosphate kinase. Both inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate were competitive inhibitors of the kinase (Ki values of 2-4 microM). The enzyme was determined to have a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity was unaffected by Ca2+/calmodulin, protein kinase A, or protein kinase C.

Animals↗
PubMed1986

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent.

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.

Adenosine Triphosphate↗
PubMed2003

Synthesis of D- and L-myo-inositol 1,2,4,6-tetrakisphosphate, regioisomers of myo-inositol 1,3,4,5 tetrakisphosphate: activity against Ins(1,4,5)P3 binding proteins.

We report here the synthesis of D- and L-myo-inositol 1,2,4,6-tetrakisphosphate 3a and 3b and the racemic modification 3ab. Racemic myo-inositol 1,2,4,6-tetrakisphosphate 3ab was synthesised from DL-1,2,4,6-tetra-O-allyl-myo-inositol 9ab. Benzylation and de-allylation provided the tetraol 11ab, which was phosphitylated in the presence of bis(benzyloxy)diisopropylaminophosphine and 1H-tetrazole, then oxidised to give the fully protected 1,2,4,6-tetrakisphosphate 13ab. Hydrogenolysis of 13ab and purification of product by ion exchange chromatography gave racemic myo-inositol 1,2,4,6-tetrakisphosphate 3ab, which showed no demonstrable agonism or antagonism for Ca2+ release at 200 microM in permeabilised hepatocytes. The chiral derivatives, D-3a and L-myo-inositol 1,2,4,6-tetrakisphosphate 3b were synthesised from 5-O-benzyl-1,4,6-tri-O-p-methoxybenzyl-myo-inositol 19ab, which was resolved using R-(-)-O-acetylmandelic acid providing two diastereoisomers 21 and 22 which were separated and deacylated to give the corresponding enantiomers. Further transformations gave the corresponding chiral 1,2,4,6-tetraols which were phosphitylated, oxidised, deprotected and purified as for the racemic mixture. The enantiomeric tetrakisphosphates 3a and 3b were evaluated for inhibition of the metabolic enzymes inositol 1,4,5-trisphosphate 5-phosphatase and 3-kinase in comparison with the enantiomers of another synthetic regioisomer D- and L-myo-inositol 1,2,4,5-tetrakisphosphate. Both D- and L-myo-inositol 1,2,4,6-tetrakisphosphate inhibit 5-phosphatase with an IC50 value of 3.8 microM and 14 microM, repectively. However, both enantiomers were poorly recognised by the 3-kinase enzyme, with IC50 values greater than 100 microM. The enantiomers of the 1,2,4,5-tetrakisphosphate showed the same relative pattern of activity towards the two enzymes but were more potent against 5-phosphatase (0.47 microM and 3 microM respectively).

Animals↗
PubMed1993

Beryllium competitively inhibits brain myo-inositol monophosphatase, but unlike lithium does not enhance agonist-induced inositol phosphate accumulation.

Despite limiting side-effects, lithium is the drug of choice for the treatment of bipolar depression. Its action may be due, in part, to its ability to dampen phosphatidylinositol turnover by inhibiting myo-inositol monophosphatase. Beryllium has been identified as a potent inhibitor of partially purified myo-inositol monophosphatase isolated from rat brain (Ki = 150 nM), bovine brain (Ki = 35 nM), and from the human neuroblastoma cell line SK-N-SH (Ki = 85 nM). It is over three orders of magnitude more potent than LiCl (Ki = 0.5-1.2 mM). Kinetic analysis reveals that beryllium is a competitive inhibitor of myo-inositol monophosphatase, in contrast with lithium which is an uncompetitive inhibitor. Inhibition of exogenous [3H]inositol phosphate hydrolysis by beryllium (IC50 = 250-300 nM) was observed to the same maximal extent as that seen with lithium in permeabilized SK-N-SH cells, reflecting inhibition of cellular myo-inositol monophosphatase. However, in contrast with that observed with lithium, agonist-induced accumulation of inositol phosphate was not observed with beryllium in permeabilized and non-permeabilized SK-N-SH cells and in rat brain slices. Similar results were obtained in permeabilized SK-N-SH cells when GTP-gamma-S was used as an alternative stimulator of inositol phosphate accumulation. The disparity in the actions of beryllium and lithium suggest that either (1) selective inhibition of myo-inositol monophosphatase does not completely explain the action of lithium on the phosphatidylinositol cycle, or (2) that uncompetitive inhibition of myo-inositol monophosphatase is a necessary requirement to observe functional lithium mimetic activity.

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