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PubMed1985

5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown.

Phosphoinositide breakdown has been linked to the receptor mechanism involved in the elevation of cytosolic Ca2+. In a cell-free system prepared from [3H] inositol-labeled blowfly salivary glands, 5-hydroxytryptamine stimulated the rapid production of inositol phosphates. Within 30 s of hormone addition, there was a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositol monophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol or glycerol phosphoinositol formation. Half-maximal activation of inositol phosphate production was obtained with 0.33 microM 5-hydroxytryptamine. Ethylene glycol bis(beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid, (EGTA) (0.3 mM) inhibited the basal formation of inositol phosphates and decreased the net accumulation of inositol bisphosphate and inositol trisphosphate due to hormone as compared to homogenates incubated in the absence of added Ca2+. EGTA, however, had little effect on the per cent stimulation of inositol phosphate production due to hormone. In homogenates, ATP, GTP or guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was required for a hormone effect. Gpp(NH)p, unlike ATP or GTP, increased the basal formation of inositol phosphates. In membranes, GTP, Gpp(NH)p, or guanosine 5'-(3-O-thio)trisphosphate (GTP gamma S) sustained a hormone effect whereas ATP was ineffective. GTP did not affect production while Gpp(NH)p and GTP gamma S increased inositol phosphate production. Half-maximal effects of Gpp(NH)p and GTP gamma S on hormone-stimulated inositol phosphate formation occurred at 10 microM and 100 nM, respectively. In the presence of 1 microM GTP gamma S, 5-methyltryptamine stimulated inositol phosphate formation within 2 s in membranes. These results indicate that in a cell-free system, GTP is involved in mediating the effects of Ca2+-mobilizing hormones on phosphoinositide breakdown.

Animals↗
PubMed1999

Inositol synthesis and catabolism in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that synthesizes and catabolizes inositol. This study demonstrates inositol synthesis from glucose-6-phosphate via inositol-1-phosphate synthase and catabolism to glucuronic acid via inositol oxygenase in this organism. These inositol synthetic and catabolic pathways are regulated in opposition; repressing conditions for one are inducing conditions for the other. An inositol-requiring strain was generated by UV mutagenesis. Without inositol, this mutant strain undergoes 'inositol-less' death, during which time the phosphatidylinositol composition of the membranes decreases without alteration of the proportion of other phospholipids. The mutation on this strain results in no detectable inositol synthetic activity but normal (wild-type) inositol catabolic activity. This inositol-requiring mutant strain reverted at a high frequency. Classical genetic experiments revealed that the majority of the reverting mutations are at second sites. Interestingly, the revertants exhibited unusual morphological phenotypes when deprived of inositol, while provision of inositol restored wild-type morphology. Inositol metabolism is clearly important for growth and development of C. neoformans and may be involved in this organism's mechanism for survival as both a saprophyte in soil and a parasite in humans.

Base Sequence↗
PubMed1987

Intracellular receptors for inositol 1,4,5-trisphosphate in angiotensin II target tissues.

Many cells (including angiotensin II target cells) respond to external stimuli with accelerated hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, a rapidly diffusible and potent Ca2+-mobilizing factor. Following its production at the plasma membrane level, inositol 1,4,5-trisphosphate is believed to interact with specific sites in the endoplasmic reticulum and triggers the release of stored Ca2+. Specific receptor sites for inositol 1,4,5-trisphosphate were recently identified in the bovine adrenal cortex (Baukal, A. J., Guillemette, G., Rubin, R., Spät, A., and Catt, K. J. (1985) Biochem. Biophys. Res. Commun. 133, 532-538) and have been further characterized in the adrenal cortex and other target tissues. The inositol 1,4,5-trisphosphate-binding sites are saturable and present in low concentration (104 +/- 48 fmol/mg protein) and exhibit high affinity for inositol 1,4,5-trisphosphate (Kd 1.7 +/- 0.6 nM). Their ligand specificity is illustrated by their low affinity for inositol 1,4-bisphosphate (Kd approximately 10(-7) M), inositol 1-phosphate and phytic acid (Kd approximately 10(-4) M), fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (Kd approximately 10(-3) M), with no detectable affinity for inositol 1-phosphate and myo-inositol. These binding sites are distinct from the degradative enzyme, inositol trisphosphate phosphatase, which has a much lower affinity for inositol trisphosphate (Km = 17 microM). Furthermore, submicromolar concentrations of inositol 1,4,5-trisphosphate evoked a rapid release of Ca2+ from nonmitochondrial ATP-dependent storage sites in the adrenal cortex. Specific and saturable binding sites for inositol 1,4,5-trisphosphate were also observed in the anterior pituitary (Kd = 0.87 +/- 0.31 nM, Bmax = 14.8 +/- 9.0 fmol/mg protein) and in the liver (Kd = 1.66 +/- 0.7 nM, Bmax = 147 +/- 24 fmol/mg protein). These data suggest that the binding sites described in this study are specific receptors through which inositol 1,4,5-trisphosphate mobilizes Ca2+ in target tissues for angiotensin II and other calcium-dependent hormones.

2,3-Diphosphoglycerate↗
PubMed1987

Novel aspects of gonadotropin-releasing hormone action on inositol polyphosphate metabolism in cultured pituitary gonadotrophs.

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone secretion via receptor-mediated activation of phosphoinositide hydrolysis to yield inositol phosphates and diacylglycerol. Application of anion-exchange high-performance liquid chromatography together with absorbance and radiochemical flow detection has enabled both the characterization and quantitative estimation of pituitary cell inositol phosphates and phosphoinositides. In cultured pituitary cells, GnRH caused a rapid and progressive rise in the formation of inositol 1,4,5-trisphosphate and of higher polyphosphoinositols corresponding to inositol tetrakisphosphate, pentakisphosphate, and hexakisphosphate. The inositol 1,4,5-trisphosphate formed during GnRH action was dephosphorylated predominantly via inositol 4-monophosphate rather than the expected metabolite, inositol 1-monophosphate. The catabolism of inositol 4-monophosphate, like that of inositol 1-monophosphate, was inhibited by lithium. For these reasons and because it was the major metabolite of [3H] inositol 1,4,5-trisphosphate in permeabilized gonadotrophs, inositol 4-monophosphate appears to represent a specific marker for ligand-stimulated inositol polyphosphate formation and metabolism. The marked and sustained elevations of inositol 4-monophosphate and inositol 1,4-bisphosphate in GnRH-stimulated gonadotrophs indicate that polyphosphoinositides rather than phosphatidylinositol are the preferred substrates of phospholipase C during GnRH action.

Animals↗
PubMed1993

Chemical and kinetic mechanism of the inositol monophosphatase reaction and its inhibition by Li+.

Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21-DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li+], Li+ inhibited the hydrolysis of racemic myo-inositol 1-phosphate, myo-inositol 4-phosphate and adenosine 2'-phosphate in a linear uncompetitive manner with apparent Ki values of 1.1, 0.11 and 1.52 mM, respectively. At Li+ concentrations higher than 4 mM, Li+ acted as a non-linear noncompetitive inhibitor for myo-inositol 1-phosphate, Ki greater than 1.5 mM. The enzyme was unable to catalyze the transesterification of [14C]inositol in the presence of inositol 1-phosphate or adenosine 2'-phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li+, the enzyme was able to release inositol from inositol 1-phosphate, in a burst, faster than the rate of steady-state substrate turnover suggesting that Li+ binds after P-O bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of 18O from [18O] water into phosphate was shown to be completely dependent upon inositol. The Km for inositol for 18O exchange was 190 mM and in the presence of saturating phosphate, VEx was at least 60% of Vmax for the hydrolysis reaction. Thus, the enzyme operates via a ternary-complex mechanism, and Li+ exerts its action by binding to enzyme/product complexes. At low concentration, Li+ inhibition with respect to the cofactor, Mg2+ was non-competitive. Mg2+ acted as a non-competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium-ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg2+ with inositol 1-phosphate and 2'AMP as the substrate but not with inositol 4-phosphate. The combined results indicate that Mg2+ and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non-competitive inhibitor for inositol 1-phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent (Ki = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li+ and phosphate, increasing [Li+] caused the Ki for phosphate to decrease by a factor of (1 + [Li+]/KLi). The Ki for the first product off (inositol) was, however, unaltered by Li+. The results indicate that Li+ can bind to the species E.Ins.Pi and E.Pi, but not to enzyme/substrate complexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Aging-related regulation of myo-inositol 1,4,5-trisphosphate signal transduction pathway in the rat striatum.

To determine the effects of the aging process on the regulation of phosphoinositide signal transduction pathway, inositol 1,4,5-trisphosphate and inositol 1,4,5-trisphosphate receptor-associated parameters were examined in the striatum of brains removed from young (3 months), adult (12 months) and senescent (25 months) male Fischer 344 rats. Inositol 1,4,5-trisphosphate content was significantly increased (P < or = 0.01) at 25 months of age compared to 3 and 12 months. No age-related differences in phosphatidylinositol 4,5-bisphosphate hydrolysis were found in striatal slices after stimulation with trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate, a metabotropic glutamatergic receptor agonist. Phosphatidylinositol 4,5-bisphosphate hydrolysis following stimulation with (R,S)-alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid, a glutamatergic/quisqualate agonist, showed a significantly increased accumulation of net [3H]inositol 1,4,5-trisphosphate in senescent striatum whereas the muscarinic cholinergic agonist carbachol induced highest response in the young striatum. In each case, agonist-stimulated response was significantly reduced in the presence of the receptor-associated antagonist. The density of inositol 1,4,5-trisphosphate receptor in the particulate membranes derived from 12- and 25-month-old rats was decreased (P < 0.01) compared to that from young rats. Binding affinity of inositol 1,4,5-trisphosphate receptor for [3H]inositol 1,4,5-trisphosphate was increased (P = 0.05) only at 25 months of age when compared with 3 months of age. Incubation of partially purified inositol 1,4,5-trisphosphate receptor with striatal cytosol in the presence of Ca2+ showed an age-dependent susceptibility to proteolytic degradation of this receptor that was completely inhibited by calpain I inhibitor peptide. Paradoxically, the quantity of inositol 1,4,5-trisphosphate receptor mRNA-encoding transcripts was increased (P < or = 0.01) at 25 months of age, suggesting an age-dependent change in either transcriptional rate, stability or processing of inositol 1,4,5-trisphosphate receptor mRNAs in the striatum. The activity of inositol 1,4,5-trisphosphate3-kinase decreased (P < or = 0.01) with age whereas the activity of soluble inositol 1,4,5-trisphosphate 5-phosphatase was highest at 3 months but significantly decreased at 12 months of age. However, the activity of inositol 1,4,5-trisphosphate 5-phosphatase remained unchanged between 12 and 25 months of age, suggesting possible developmental modulation of the activity of the enzyme. Taken together with the established 'cross-talk' between signal transduction systems, the present data suggest that molecular/cellular changes in striatal inositol 1,4,5-trisphosphate/Ca2+ signal transduction pathway along with neuronal cell loss may contribute to aging-related decrease in striatal functioning.

Aging↗
PubMed2000

Characterization of the myo-inositol transport system in Trypanosoma cruzi.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.

Adenosine Triphosphatases↗
PubMed1999

Restoration of inositol prototrophy in the fission yeast Schizosaccharomyces pombe.

The biosynthesis of inositol requires only two enzymes, inositol-1-phosphate synthase (encoded by INO1) and an inositol monophosphatase, but the regulation of inositol biosynthesis is under multiple controls and is exquisitely regulated. In the budding yeast Saccharomyces cerevisiae, mutations in any of 26 different genes lead to inositol auxotrophy. The fission yeast Schizosaccharomyces pombe, however, is a natural inositol auxotroph. An investigation has been initiated to examine the possible reasons that might have led to inositol auxotrophy in Sch. pombe. Complementation with a genomic library of an inositol prototrophic yeast indicated that a Pichia pastoris INO1 gene alone could confer inositol prototrophy to Sch. pombe and that the gene was absent in Sch. pombe. To investigate possible reasons for the loss of INO1 gene in Sch. pombe, an attempt was made to disrupt inositol homeostasis in Sch. pombe by overproduction of intracellular inositol, but this did not lead to any discernible adverse effects. The sources of inositol in the natural environment of Sch. pombe were also examined. As the natural environment of Sch. pombe contains significant amounts of phytic acid (inositol hexaphosphate), an investigation was carried out and it was discovered that Sch. pombe can utilize phytic acid as a source of inositol under very specific conditions.

Cloning, Molecular↗
PubMed1992

Phosphoinositide metabolism in the developing conceptus. Effects of hyperglycemia and scyllo-inositol in rat embryo culture.

Culture of the postimplantation rat conceptus in hyperglycemic medium causes developmental abnormalities and is associated with a diminished water-soluble myo-inositol content. We investigated the effect myo-inositol depletion has on lipid-soluble phosphoinositides, precursors, and water-soluble inositol phosphates. Rat conceptuses were cultured from gestational day 9.5 (presomite, early head fold) to day 10.5 (7-15 somites) in 6.7-73.3 mM D-glucose. Significant decreases in the phosphoinositides of the embryo were observed with increased culture D-glucose concentrations. PI was reduced 15-34%, PIP 18-46%, and PIP2 26-46%. Yolk sac phosphoinositides also were reduced but to a lesser degree. Culture in hyperglycemic media also mediated significant reductions of conceptus inositol phosphates. To investigate whether effects similar to those induced by D-glucose could be mediated by another agent capable of decreasing myo-inositol content, we used scyllo-inositol, a transported but nonmetabolized isomer of myo-inositol. Conceptuses cultured in medium containing scyllo-inositol (0.06-16.7 mM) had dose-dependent decreases of myo-[3H]inositol in water-soluble and lipid-soluble fractions. Incorporation of myo-[3H]inositol into phosphoinositides and inositol phosphates was decreased concomitantly. Developmental effects of D-glucose and scyllo-inositol were assessed in rat conceptuses cultured from day 9.5 (presomite, early head fold) to day 11.5 (22-28 somites). Culture in 40.0-73.3 mM glucose and 0.06-33.3 mM scyllo-inositol impaired growth while increasing dysmorphogenesis in a dose-dependent manner. The results suggest that decreases in conceptus myo-inositol and associated diminution of phosphoinositides, which are the inositol/lipid cycle precursors, are dysmorphogenic and may contribute to the etiology of diabetic embryopathy.

Analysis of Variance↗
PubMed1987

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.

Animals↗
PubMed1998

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes.

The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.

Animals↗
PubMed1995

Modulation by inositol of cholinergic- and serotonergic-induced seizures in lithium-treated rats.

Hippocampal and cortical EEG recordings in rats were used to monitor the in vivo modulation by lithium of responses to agonists for 5HT2/5HT1c serotonergic (DOI) and cholinergic (pilocarpine) receptors and the influence of inositol administration. Administration of DOI (8 mg/kg) or pilocarpine (30 mg/kg) to rats pretreated with lithium acutely (3 mmol/kg) or chronically (dietary, 4 weeks) resulted in seizures, whereas these doses did not cause seizures without lithium pretreatment. This indicated that lithium most likely affects a signal transduction process common to both systems, which is the phosphoinositide second messenger system. To examine the potential influence of altered inositol levels on these responses, we tested the effects of infusions (10 mg, i.c.v.) of myo-inositol, a precursor of phosphoinositide synthesis, and of epi-inositol, an isomer not used for phosphoinositide synthesis. Administration of myo-inositol (10 mg) slightly reduced the incidence of seizures induced by acute lithium plus DOI but almost completely blocked seizures induced by acute lithium plus pilocarpine. This was surprising since seizures induced by acute lithium plus DOI were less severe than those after acute lithium plus pilocarpine, but myo-inositol was more effective in blocking the latter. Epi-inositol also blocked seizures under both conditions but it was less effective than myo-inositol after treatment with acute lithium plus pilocarpine. The latencies to seizures and/or severity of seizures were potentiated more by chronic than acute lithium pretreatment with both DOI and pilocarpine, but attenuation by myo-inositol was less with each agonist after chronic lithium compared with acute lithium treatment. Peripheral administration of a high dose of myo-inositol blocked seizures induced by acute lithium plus pilocarpine, but the inositol treatment itself was toxic and caused seizures prior to pilocarpine administration, so the mechanism of action cannot simply be attributed to increased brain inositol levels. These results demonstrate that lithium modulates the in vivo responses to DOI and pilocarpine, most probably through an effect on the phosphoinositide signal transduction system. They also show that centrally administered myo-inositol modifies responses to these agents, but the effectiveness of epi-inositol and other results leave unclear the mechanistic basis of its actions.

Amphetamines↗
PubMed1993

Kinetic evidence for compartmentalization of myo-inositol in hepatocytes.

myo-Inositol uptake and conversion to phosphatidylinositol (PI) was studied in isolated rat hepatocytes. Uptake of myo-[2-3H]-inositol into the trichloroacetic acid (TCA)-soluble fraction showed no evidence of saturation, while incorporation into lipid had an apparent Km of 0.28 mmol/L for external myo-inositol. With 50 mumol/L myo-[2-3H]-inositol, approximately half of the radiolabel was found in lipid at 30 minutes. Glucose and galactose were weak inhibitors, while phlorizin at 1 mmol/L reduced uptake by 50%. Metabolic inhibitors reduced incorporation of myo-[2-3H]-inositol into lipid, but had no effect on uptake. Hepatocytes maintained myo-inositol levels of 0.4 mmol/L for 60 minutes when incubated with 50 mumol/L myo-inositol, but levels increased when incubated with 1 mmol/L myo-inositol. Efflux of label was studied in hepatocytes prelabeled for 20 minutes with myo-[2-3H]-inositol. Loss of label was initially rapid, but had slowed by 20 minutes, with much of the label remaining in the cells. Phlorizin inhibited the loss of myo-[2-3H]-inositol, while increasing myo-inositol concentration in the medium enhanced efflux. The effects of these agents on the rate of efflux was found in lipid rather than in the TCA-soluble myo-inositol fraction. These findings suggest that myo-inositol is compartmentalized within hepatocytes, with a bulk metabolically inert pool and a smaller active pool that equilibrates with extracellular myo-inositol via an energy-independent carrier-mediated mechanism, and is preferentially available for efflux or for synthesis of phosphoinositides.

Animals↗
PubMed1991

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils.

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.

Biological Transport, Active↗
PubMedDate not supplied

Dietary myo-inositol supplementation does not prevent retinal and glomerular vascular structural changes in chronically diabetic rats.

To assess effects of dietary myo-inositol supplementation on diabetes-induced vascular structural lesions, diabetes was induced in Sprague-Dawley rats with streptozotocin; one-third of these rats was fed a 2% myo-inositol diet for 9 months, one-third was left untreated for 5 months then treated with myo-inositol for the last 4 months, and one-third was untreated for the entire 9 months. Controls included untreated and myo-inositol-treated groups. Weight gain was impaired and plasma glucose, glycosylated hemoglobin, food consumption, urine volume, and albuminuria were increased significantly in diabetic versus age-matched control rats. Plasma myo-inositol levels were increased approximately fivefold in controls and approximately six- to eightfold in diabetic rats treated with myo-inositol. In general, myo-inositol did not affect any of the above parameters in control or diabetic rats. Retinal capillary basement membrane width (CBMW) was increased significantly (approximately 50% versus controls) after 9 months of diabetes. In the control group myo-inositol increased CBMW to the level of untreated diabetic rats; myo-inositol had no effect on CBMW in each diabetic group. The number of retinal capillaries containing pericyte nuclei and pericyte capillary coverage were increased in untreated as well as myo-inositol-treated diabetic rats and in the myo-inositol-treated control group. Glomerular CBMW was increased after 5 and 9 months of diabetes versus age-matched controls, and was increased even more by myo-inositol. Mesangial fractional volume of the glomerulus was increased 36% by diabetes and was decreased slightly but significantly by myo-inositol. These results indicate that diets supplemented with 2% myo-inositol (1) cause capillary basement membrane (CBM) thickening and pericyte changes in retinal capillaries of normal rats, (2) are ineffective in preventing or reversing diabetes-induced retinal CBM thickening, and (3) cause further thickening of glomerular CBM in diabetic rats.

Albuminuria↗
PubMed1988

L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.

Homogenates of primary-cultured murine bone macrophages contain an enzyme capable of synthesizing myo-[3H]inositol pentakisphosphate from myo-[3H]inositol tetrakisphosphate fractions derived from myo-[3H]inositol-labelled mouse macrophages and chick erythrocytes. D-myo-inositol 1,3,4,5-tetrakis[32P]-phosphate present in the same incubations was not phosphorylated. Since the myo-[3H]inositol-labelled tetrakisphosphate fractions used as substrates consist of a mixture of L-myo-inositol 1,4,5,6-tetrakisphosphate (60-85%) and a periodate-resistant tetrakisphosphate(s) whose characteristics are consistent with those of D-myo-inositol 1,3,4,5-tetrakisphosphate (the preceding paper [Stephens, Hawkins, Carter, Chahwala, Morris, Whetton & Downes (1988) Biochem. J. 249, 271-282] ), these data suggest the existence of a kinase that phosphorylates L-myo-inositol 1,4,5,6-tetrakisphosphate to give a myo-inositol pentakisphosphate. A similar activity was identified in homogenates of rat cerebrum, liver, heart and parotid gland. D-myo-Inositol 1,3,4,5-tetrakis[32P]phosphate in the same incubations was not a substrate. The activity was almost entirely soluble in all the tissues investigated and was found at its greatest specific activity in brain cytosol. The activity was purified 120-fold from a rat brain homogenate by (NH4)2SO4 fractionation and anion-exchange chromatography. The activity was clearly distinct from D-myo-inositol 1,4,5-trisphosphate (3-hydroxy)kinase. Incubation of this partially purified preparation with L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate from chick erythrocytes and [gamma-32P]ATP resulted in the formation of L-myo-[3H]-inositol [1-32P]1,3,4,5,6-pentakisphosphate. The enzyme is therefore identified as an L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.

Animals↗
PubMed2000

neo-inositol polyphosphates in the amoeba Entamoeba histolytica.

We have reexamined the structure of inositol phosphates present in trophozoites of the parasitic amoeba Entamoeba histolytica and show here that, rather than being myo-inositol derivatives (Martin, J.-B., Bakker-Grunwald, T., and Klein, G. (1993) Eur. J. Biochem. 214, 711-718), these compounds belong to a new class of inositol phosphates in which the cyclitol isomer is neo-inositol. The structures of neo-inositol hexakisphosphate, 2-diphospho-neo-inositol pentakisphosphate, and 2, 5-bisdiphospho-neo-inositol tetrakisphosphate, which are present in E. histolytica at concentrations of 0.08-0.36 mM, were solved by two-dimensional (31)P-(1)H NMR spectroscopy. No evidence for the co-existence of their myo-inositol counterparts has been found. These neo-inositol compounds were not substrates of 6-diphospho-inositol pentakisphosphate 5-kinase, an enzyme purified from Dictyostelium discoideum that phosphorylates 6-diphospho-myo-inositol pentakisphosphate and more slowly also myo-inositol hexakisphosphate, specifically on position 5. Because preliminary data indicate that large amounts of the same neo-inositol phosphate and diphosphate esters are also present in another primitive amoeba, Phreatamoeba balamuthi, the occurrence of high concentrations of neo-inositol polyphosphates may be much more general than previously thought.

Animals↗
PubMed1979

myo-Inositol deficiency: studies on the mechanism of lactation-dependent fatty liver formation in the rat.

Intravenous administration of [1-14C]palmitate to 8-day lactating rats fed a myo-inositol supplemented or deficient diet resulted in rapid labeling of liver triglycerides and phospholipids. Compared with myo-inositol deficient rats, those supplemented with myo-inositol showed a greater loss of isotope from liver triglycerides with a more rapid appearance of isotope in serum triglyceride. Loss of 14C from liver phospholipids was similar for both groups, whereas the appearance of labeled phospholipids in serum was slightly greater for myo-inositol supplemented controls compared with myo-inositol deficient rats. The labeling pattern of liver microsomal triglycerides and phospholipids of the two dietary groups was similar; however, liver microsomal protein was significantly reduced in myo-inositol deficient rats relative to the control group. Concurrent administration of Triton WR-1339 with [1-14C]palmitate resulted in significantly less label accumulation in serum triglycerides of myo-inositol deficient rats compared with myo-inositol supplemented rats. Labeled triglycerides in whole liver and in liver microsomes of myo-inositol supplemented rats turned over more rapidly than those of myo-inositol deficient rats while no significant difference was noted for the [14C]palmitate labeled phospholipid of either source. The incorporation of [guanido-14C]arginine into total liver and serum protein 1 hour after injection of the precursor was similar whether the 14-day lactating dams were myo-inositol supplemented or deficient, but total serum protein specific radioactivity of myo-inositol deficient rats was 66% that of myo-inositol supplemented rats. Thus, the significantly reduced release of hepatic triglycerides appears to be the cause of the fatty liver observed during lactation-dependent myo-inositol deficiency.

Animals↗