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PubMed1984

Thyrotropin-releasing hormone-stimulated [3H]inositol metabolism in GH3 pituitary tumor cells. Studies with lithium.

GH3 pituitary tumor cells were labeled to isotopic equilibrium with [3H]inositol. Thyrotropin-releasing hormone (TRH), which has been shown to stimulate inositol phospholipid metabolism in these cells, enhanced the accumulation of [3H]inositol-derived radioactivity in the cell's acid-soluble fraction. Separation of the [3H]inositol metabolites by ion-exchange chromatography revealed that TRH induced a rapid rise in the cellular content of [3H]inositol mono-, bis-, and trisphosphate. The latter two metabolites accumulated in a multiphasic manner with an initial peak 5-10 sec after TRH addition. This was followed by a short-lived decline and a secondary rise which left the metabolite levels elevated for at least 50 min. The GH3 cell [3H]inositol monophosphate and [3H] inositol content also rose in response to TRH, but the latter accumulated with a considerably slower time course than the phosphorylated derivatives. None of these responses could be mimicked by the calcium ionophore A23187. Incubation of GH3 cells with TRH in the presence of lithium led to an enhanced accumulation of [3H]inositol monophosphate and, to a lesser extent, of [3H]inositol bis- and trisphosphate. This accumulation rose in a linear fashion with time for at least 20 min, by which point 50% of the [3H]inositol-containing phospholipids had been depleted. When lithium was added 30 min after TRH, [3H]inositol monophosphate accumulated at the same rate as was found when TRH and lithium were added together, indicating that the TRH-induced phospholipid response in GH3 cells does not desensitize. Under normal conditions, approximately equal amounts of the three [3H]inositol phosphates were formed within 5 sec of TRH addition. However, when TRH was added to cells grown chronically in lithium-containing medium, or to cells incubating at a subphysiological temperature (25 degrees), greater than 90% of the metabolites formed were the bis- or trisphosphates. This indicates that the primary event stimulated by TRH is the breakdown by phospholipase C of phosphatidylinositol 4,5-bisphosphate and, perhaps also, of phosphatidylinositol 4-phosphate.

Animals↗
PubMed1986

Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH.

GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2-3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone-stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH-treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release.

Animals↗
PubMed1994

Effects of L-690,488, a prodrug of the bisphosphonate inositol monophosphatase inhibitor L-690,330, on phosphatidylinositol cycle markers.

In order to enhance the entry into cells of L-690,330, a bisphosphonate inhibitor of inositol monophosphatase (IMPase; a key, enzyme in the phosphatidylinositol (Pl) cell signaling pathway), the tetrapivaloyloxymethyl ester prodrug, L-690,488 [tetrapivaloyloxymethyl 1-(4-hydroxyphenoxy)ethane-1,1-bisphosphonate], was synthesized. The effects of L-690,488 were studied in cholinergically (carbachol)-stimulated rat cortical slices and Chinese hamster ovary cells stably transfected with the human muscarinic m1 receptor (m1 CHO cells). The accumulation of [3H]inositol monophosphates or [3H]cytidine monophosphorylphosphatidate ([3H]CMP-PA) after [3H]inositol or [3H]cytidine prelabeling, respectively, and inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate mass were measured. In rat cortical slices and m1 CHO cells, the maximum response and time course of accumulation of [3H]inositol monophosphates for L-690,488 and lithium were similar. However, the concentrations of L-690,488 required to produce these effects (EC50 values of 3.7 +/- 0.9 and 1.0 +/- 0.2 microM in cortical slices and m1 CHO cells, respectively) were much lower than with lithium (0.3-1.5 mM). Likewise, the time course and maximum accumulation of [3H] CMP-PA in L-690,488-treated m1 CHO cells was similar to lithium but L-690,488 was again much more potent (EC50 values = 3.5 +/- 0.3 microM and 0.52 +/- 0.03 mM for L-690,488 and lithium, respectively). In addition, L-690,488 attenuated the carbachol-induced elevation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in m1 CHO cells, an effect reported previously with lithium. These results are all consistent with L-690,488 and lithium both depleting intracellular inositol as a consequence of inhibition of IMPase. That these effects of L-690,488 on the PI cycle are indeed due to inositol depletion is shown by the observation that the effects of L-690,488 on CMP-PA accumulation could be overcome by addition of exogenous myo-inositol (EC50 = 1.7 +/- 0.5 mM). These data show that inhibition of IMPase produces effects on the PI cycle comparable to lithium. As a corollary, the effects of lithium on the PI cycle are therefore consistent with its major mechanism of action being inhibition of IMPase.

Animals↗
PubMed1996

Dissociation of cyclic inositol phosphohydrolase activity from annexin III.

Cyclic inositol phosphohydrolase is a phosphodiesterase that cleaves the cyclic bond of cyclic inositol monophosphate. In 1990, Ross et al. (Ross, T. S., Tait, J. F., and Majerus, P. W. (1990) Science 248, 605-607) purified this enzyme from human placenta and reported that cyclic inositol phosphohydrolase is identical to annexin III. Independent confirmation of this finding has not been provided. The relative distribution of annexin III and cyclic inositol phosphohydrolase activity in rat kidney and spleen indicated that annexin III can be dissociated from cyclic inositol phosphohydrolase activity. Rat spleen contains large quantities of annexin III, but has very little cyclic inositol phosphohydrolase activity. In contrast, rat kidney, one of the richest sources of cyclic inositol phosphohydrolase activity, possesses very little (immunohistochemistry) or no (Western blot) annexin III. Similar to cytosol of human placenta, cytosol of guinea pig kidney contains both annexin III and cyclic inositol phosphohydrolase. On SDS-gel electrophoresis, guinea pig kidney annexin III has a slightly different mobility than the human placental annexin III. Human placental annexin III co-migrates with cyclic inositol phosphohydrolase on ion exchange chromatography, while guinea pig kidney annexin III is clearly dissociated from cyclic inositol phosphohydrolase on ion exchange chromatography. Both guinea pig kidney annexin III and human placental annexin III pellet with the addition of calcium and centrifugation, while cyclic inositol phosphohydrolase activity in both of these tissues remains in the supernatant. Our studies clearly show that cyclic inositol phosphohydrolase and annexin III are two different proteins.

Animals↗
PubMed1992

The inositol phosphate pathway as a mediator in the proliferative response of rat calvarial bone cells to cyclical biaxial mechanical strain.

Isolated newborn rat calvarial bone cells grown in monolayer on polyurethane membranes in specially constructed culture chambers and subjected to a cyclical biaxial mechanical strain of 0.17% at a frequency of 1 Hz for 30 min demonstrated a 16% increase in DNA synthesis during the subsequent 24 h. The metabolites of the inositol phosphate pathway, shown to be an important second messenger in many cell types, were shown to be elevated using high-performance liquid chromatography to separate and quantitate the various inositol polyphosphates. Inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1,3,4,5-tetrakisphosphate reached peak accumulations after 20 s of mechanical strain. Inositol 1,3,4-trisphosphate reached a peak accumulation after 2 min, and inositol 1,2,3,4,5,6 phosphate reached a peak accumulation after 60 min of mechanical strain. Neomycin, an inhibitor of phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate to start the inositol phosphate cascade, completely inhibited accumulation of the above inositol phosphates during mechanical straining of the bone cells. Neomycin also completely abolished the increase in DNA synthesis that was seen after a mechanical strain of 0.17%. It is concluded from this study that the inositol phosphate pathway is activated by mechanical strain in bone cells and that this pathway is an important and primary mediator in the transduction of mechanical strain into cellular proliferation in these cells.

Analysis of Variance↗
PubMed1995

Inositol 1,4,5-trisphosphate receptor in skeletal muscle: differential expression in myofibres.

The role of inositol 1,4,5-trisphosphate as a second messenger in signal transduction has been well established in many cell types. However, conflicting reports have led to a controversy regarding the role, if any, of inositol 1,4,5-trisphosphate signalling in skeletal muscle. Indeed, expression of the inositol 1,4,5-trisphosphate receptor has not previously been demonstrated in skeletal muscle. In the present study we used in situ hybridization, immunohistochemistry, and [3H]-inositol 1,4,5-trisphosphate binding to demonstrate that rat skeletal muscle fibres contain inositol 1,4,5-trisphosphate receptors. RNAse protection and partial sequencing suggested that the inositol 1,4,5-trisphosphate receptors expressed in skeletal muscle was most similar to the non-neuronal form of the type 1 inositol 1,4,5-trisphosphate receptor. While in situ hybridization showed inositol 1,4,5-trisphosphate receptor mRNA in all types of skeletal myofibres, immunodetectable inositol 1,4,5-trisphosphate receptor protein and specific [3H]-inositol 1,4,5-trisphosphate binding sites were preferentially expressed in slow oxidative (type I) and fast oxidative-glycolytic (type IIA) fibres, but not in fast glycolytic (type IIB) fibres. These findings indicate that an inositol 1,4,5-trisphosphate receptor is preferentially expressed in oxidative fibres of skeletal muscle.

Animals↗
PubMed1992

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

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

Animals↗
PubMed1994

Alteration of inositol 1,4,5-trisphosphate receptor after six-hour hemispheric ischemia in the gerbil brain.

In order to evaluate the influence of cerebral ischemia on the inositol 1,4,5-trisphosphate receptor, the alterations of inositol 1,4,5-trisphosphate receptor binding sites and local cerebral blood flow were examined 6 h after occlusion of the right common carotid artery in the gerbil brain. The autoradiographic method developed in our laboratory enabled us to measure both parameters within the same brain. Animals attaining ischemic scores of more than 5, as assessed 1 h after occlusion, were utilized. The local cerebral blood flow was measured 6 h after occlusion by the [14C]iodoantipyrine method. The inositol 1,4,5-trisphosphate binding sites were evaluated in vitro using [3H]inositol 1,4,5-trisphosphate as a specific ligand. The local cerebral blood flow fell below 15 ml/100 g per min in most of the cerebral regions on the occluded side. In contrast, a significant reduction in inositol 1,4,5-trisphosphate binding sites was noted only in the hippocampus CA1 on the occluded side. Inositol 1,4,5-trisphosphate binding tended to decrease when the values of local cerebral blood flow were below 20 ml/100 g per min in this region. On the other hand, the inositol 1,4,5-trisphosphate receptor immunoreactivity in the brain examined with a monoclonal antibody against inositol 1,4,5-trisphosphate receptor protein did not reveal any differences between the ischemia and sham groups on both sides, suggesting that the inositol 1,4,5-trisphosphate receptors may not undergo significant morphological degradation. These findings indicate that the suppression of inositol 1,4,5-trisphosphate binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of the inositol 1,4,5-trisphosphate metabolism in this region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Valproate decreases inositol biosynthesis.

BACKGROUND: Lithium and valproate (VPA) are used for treating bipolar disorder. The mechanism of mood stabilization has not been elucidated, but the role of inositol has gained substantial support. Lithium inhibition of inositol monophosphatase, an enzyme required for inositol recycling and de novo synthesis, suggested the hypothesis that lithium depletes brain inositol and attenuates phosphoinositide signaling. Valproate also depletes inositol in yeast, Dictyostelium, and rat neurons. This raised the possibility that the effect is the result of myo-inositol-1-phosphate (MIP) synthase inhibition. METHODS: Inositol was measured by gas chromatography. Human prefrontal cortex MIP synthase activity was assayed in crude homogenate. INO1 was assessed by Northern blotting. Growth cones morphology was evaluated in cultured rat neurons. RESULTS: We found a 20% in vivo reduction of inositol in mouse frontal cortex after acute VPA administration. As hypothesized, inositol reduction resulted from decreased MIP synthase activity: .21-.28 mmol/LVPA reduced the activity by 50%. Among psychotropic drugs, the effect is specific to VPA. Accordingly, only VPA upregulates the yeast INO1 gene coding for MIP synthase. The VPA derivative N-methyl-2,2,3,3,-tetramethyl-cyclopropane carboxamide reduces MIP synthase activity and has an affect similar to that of VPA on rat neurons, whereas another VPA derivative, valpromide, poorly affects the activity and has no affect on neurons. CONCLUSIONS: The rate-limiting step of inositol biosynthesis, catalyzed by MIP synthase, is inhibited by VPA; inositol depletion is a first event shown to be common to lithium and VPA.

Amides↗
PubMed1992

The preparation and phosphorylation of 2,5- and 1D-2,6-di-O-benzyl-myo-inositol.

1,3,4,6-Tetra-O-allyl-myo-inositol was converted into the 2,5-di-O-benzyl- and 2,5-di-O-p-methoxybenzyl ethers, and the products were deallylated to give the 2,5-di-O-benzyl (and p-methoxybenzyl) ethers of myo-inositol, which were converted into the mono-O-isopropylidene derivatives. Both the 2,5-di-O-benzyl ether and its mono-O-isopropylidene derivative were converted into the crystalline octa(2-cyanoethyl) ester of 2,5-di-O-benzyl-myo-inositol 1,3,4,6-tetrakisphosphate. (+-)-1,3,4,5-Tetra-O-allyl-myo-inositol was converted into (+-)-2,4-di-O-benzyl-myo-inositol which gave a separable mixture of the 1,6- and 5,6-O-isopropylidene derivatives. The 1,6-O-isopropylidene derivative was resolved via (-)- and (+)-omega-camphanates and was also converted into (+-)-2,6-di-O-benzyl-1,5-di-O-p-methoxybenzyl-myo-inositol, which was resolved via the (-)-omega-camphanates. The 5,6-O-isopropylidene derivative and 1,3-di-O-allyl-myo-inositol were converted into (+-)-1,3-di-O-allyl-2,6-di-O-benzyl-myo-inositol, which was resolved as the (-)-omega-camphanates. 1D-1,3,4,5-Tetra-O-allyl-myo-inositol and the above described, relevant diaste reoisomers were converted into 1D-2,6-di-O-benzyl-myo-inositol which gave the syrupy octabenzyl ester of 1D-2,6-di-O-benzyl-myo-inositol 1,3,4,5-tetrakisphosphate.

Benzyl Compounds↗
PubMed2003

Valproate disrupts regulation of inositol responsive genes and alters regulation of phospholipid biosynthesis.

Valproate (VPA) is one of the two drugs approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorder. The therapeutic mechanism of VPA has not been established. We have shown previously that growth of the yeast Saccharomyces cerevisiae in the presence of VPA causes a decrease in intracellular inositol and inositol-1-P, and a dramatic increase in expression of INO1, which encodes the rate limiting enzyme for de novo inositol biosynthesis. To understand the underlying mechanism of action of VPA, INO1, CHO1 and INO2 expression, intracellular inositol and phospholipid biosynthesis were studied as a function of acute and chronic exposure of growing cells to the drug. A decrease in intracellular inositol was apparent immediately after addition of VPA. Surprisingly, expression of genes that are usually derepressed during inositol depletion, including INO1, CHO1 and INO2 (that contain inositol-responsive UASINO sequences) decreased several fold during the first hour, after which expression began to increase. Incorporation of 32Pi into total phospholipids was significantly decreased. Pulse labelling of CDP-DG and PG, shown previously to increase during inositol depletion, increased within 30 min. However, pulse labelling of PS, which normally increases during inositol depletion, was decreased within 30 min. PS synthase activity in cell extracts decreased with time, although VPA did not directly inhibit PS synthase enzyme activity. Thus, in contrast to the effect of chronic VPA treatment, short-term exposure to VPA abrogated the normal response to inositol depletion of inositol responsive genes and led to aberrant synthesis of phospholipids.

5' Untranslated Regions↗
PubMed1999

Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol.

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.

Antineoplastic Agents↗
PubMed1993

Insulin stimulates the biosynthesis of chiro-inositol-containing phospholipids in a rat fibroblast line expressing the human insulin receptor.

HIRc-B cells (rat fibroblasts expressing the human insulin receptor) were incubated with myo-[3H]inositol for 48 hr, and the biosynthesis of chiro-[3H]inositol was investigated in the absence and presence of insulin following a time course up to 60 min. After phase separation, treatment with insulin for 15 min caused a 2.2-fold increase in the specific radioactivity of chiro-[3H]inositol-containing phospholipids in contrast to a 1.2-fold increase in the specific radioactivity of myo-[3H]inositol-containing phospholipids. No insulin-mediated change in the specific radioactivity was observed in the inositol phosphates or free inositols. Further detailed analysis of individual [3H]inositol-containing phospholipids demonstrated marked increases in specific activity of the chiro-[3H]inositol phospholipids after 15 min of incubation with insulin: phosphatidylinositol 4-phosphate and 4,5-bisphosphate, 4.2-fold; lysophosphatidylinositol, 1.5-fold; phosphatidylinositol, 3.2-fold. In contrast, myo-[3H]inositol-containing phospholipids demonstrated relatively small increases (1.1- to 1.4-fold) after 5 min of incubation with insulin. These findings indicate that insulin stimulates de novo synthesis of chiro-inositol-containing phospholipids at the inositol phospholipid level.

Animals↗
PubMed1992

Levels of inositol metabolites within normal myeloid blast cells and changes during their differentiation towards monocytes.

A homogeneous population of undifferentiated myeloid blast cells was purified from human fetal liver by rosette sedimentation of erythroblasts and macrophages, after coating these cells with monoclonal antibodies, followed by a cell elutriation step. The undifferentiated blast cells were maintained in culture, in a serum-free medium containing 1 mg l-1 inositol, by the presence of a high concentration of interleukin-3 (100 U ml-1). This allowed equilibrium labelling of cells with [2-3H]myo-inositol and analysis of the concentrations of inositol metabolites. The myeloid blast cells contained high concentrations of an unidentified inositol metabolite, possibly sn-glycero-3-phospho-1-inositol (GroPIns, 22 microM), inositol monophosphate (InsP, 16 microM), an unidentified inositol bisphosphate (InsP2, 9.4 microM), inositol pentakisphosphate (InsP5, 37 microM) and inositol hexakisphosphate (InsP6, 31 microM). These high concentrations are similar to those reported in the promyeloid cell line, HL60. Treatment of the blast cells with 10 nM phorbol myristate acetate (PMA) resulted in rapid differentiation of 48% of the cells towards monocytes. Notable changes in the levels of inositol metabolites included an increase in the putative GroPIns peak (to 73 microM) and decreases in the concentrations of InsP4 (from 4 microM to 1 microM) and InsP5 (to 21 microM). These changes in response to PMA, with the exception of the rise in the putative GroPIns, are similar to those reported in HL60 cells undergoing monocyte differentiation. These observations suggest that the abundant inositol polyphosphates may have an as yet unknown role in myeloid differentiation.

Abortion, Spontaneous↗
PubMed1986

Isolation of D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase from human placenta.

We have isolated D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase (EC 3.1.4.36) from human placenta. This enzyme catalyzes the conversion of inositol 1:2-cyclic phosphate to inositol 1-phosphate. The enzyme was purified 1300-fold to apparent homogeneity from the soluble fraction of human placenta. The enzyme requires Mn2+ or Mg2+ ions for activity, has an apparent Km for inositol 1:2-cyclic phosphate of 0.15 mM and forms 2.2 mumol of inositol 1-phosphate/min/mg protein. The enzyme does not utilize the cyclic esters of inositol polyphosphates as substrates. The molecular weight determined by gel filtration chromatography is approximately 55,000. Upon electrophoresis in polyacrylamide gels in sodium dodecyl sulfate, the molecular weight was found to be 29,000 both in the presence and absence of beta-mercaptoethanol. The enzyme was inhibited by inositol 2-phosphate (IC50 = 4 microM) and to a lesser degree by inositol 1-phosphate (IC50 = 2 mM) and inositol (IC50 = 4 mM). Zn2+ is a potent inhibitor of enzyme activity (IC50 = 10 microM). Neither Li+ nor Ca2+ had any effect on enzyme activity. This enzyme may serve to generate inositol from inositol cyclic phosphate metabolites produced by the phosphoinositide signaling pathway in cells.

Annexin A3↗
PubMed1980

Alcohol acutely depresses myo-inositol 1-phosphate levels in the male rat cerebral cortex.

The effects of acute alcohol administration on myo-inositol and myo-inositol 1-phosphate levels in the male rat cerebral cortex were examined. At a dose of 3 g/kg, alcohol reduced myo-inositol 1-phosphate levels in the cortex by 60% within 30 min after its injection. A significant depression in myo-inositol 1-phosphate was found 5 min after the injection of alcohol, and maximal levels of depression occurred at 30 min. Levels of myo-inositol 1-phosphate remained significantly depressed for 6 hr and then gradually recovered, reaching normal values of 24 hr after the initial injection. There was an excellent inverse correlation (rxy = 0.96) between myo-inositol 1-phosphate levels and blood alcohol levels at all time intervals examined. Dose-response analyses indicated that the half-maximal (ED50) decrease in myo-inositol 1-phosphate levels occurred at a dose of 1.6 g/kg of alcohol. In contrast to these effects, myo-inositol levels were not significantly altered by any dose of alcohol at any time interval examined. Because of the rapidity of changes in myo-inositol 1-phosphate after acute alcohol administration and the excellent negative correlation between blood alcohol levels and changes in myo-inositol 1-phosphate, alcohol-induced alterations in myo-inositol 1-phosphate appear to be the direct effect of alcohol per se and may participate in the acute pharmacological effects of the drug.

Animals↗
PubMed1985

Use of chemical ionization in gas chromatographic/mass spectrometric screening of human urine for disaccharides containing inositol.

Gas chromatography/mass spectrometry (GC/MS) in the positive chemical ionization (CI) mode was used to screen normal urine for inositol-containing disaccharides in the form of permethylated derivatives, after borodeuteride reduction of the reducing saccharides. Ammonia was the reactant gas. The results revealed the existence of deoxyhexosyl-inositol and hexosyl-inositol disaccharides, and of a new compound, N-acetylhexosaminyl-inositol disaccharide. Up to four isomers of deoxyhexosyl-inositol could be present in the same sample even though only one of them has so far been fully characterized in man. As regards the hexosyl-inositols, one to three isomers were present in the same sample and probably corresponded to the three isomers of galactosyl-inositol recently described in man. N-Acetylhexosaminyl-inositol (identified elsewhere by us as N-acetylgalactosaminyl-alpha (1-1)-myo-inositol) was seen in only a few samples. No relationship can be found between the excretion of all these inositol-containing disaccharides on the one hand, ABO(H) blood group and 'Secretor' status (Se or sese) of the donors on the other. The gas chromatographic CI mass spectrometric technique used here with various ammonia pressures can be applied to the screening of other biological fluids or tissues for inositol glycosides.

Disaccharides↗
PubMed1987

Receptor coupled events in bradykinin action: rapid production of inositol phosphates and regulation of cytosolic free Ca2+ in a neural cell line.

The addition of bradykinin to NG115-401L cells grown on coverslips results in the generation of rapid transient increases in intracellular [Ca2+] and inositol phosphates. Changes in intracellular Ca2+, measured using the fluorescent indicator dye Fura-2, show two components; an initial rapid peak in [Ca2+]i which is essentially independent of extracellular Ca2+, and a sustained plateau dependent on the presence of extracellular Ca2+. Analysis of bradykinin stimulated production of [3H]inositol phosphates, by h.p.l.c., shows a rapid biphasic production of inositol 1,4,5-trisphosphate, inositol tetrakisphosphate and inositol bisphosphates, followed by a sustained rise in inositol 1,3,4-trisphosphate production. Quantitative measurements have indicated the presence of other, more polar, [3H]inositol-labelled metabolites which do not show major changes on bradykinin stimulation. The initial phase of inositol phosphate production parallels the rapid transient increase in intracellular [Ca2+], however, the second phase of inositol phosphate production occurs when intracellular [Ca2+] is declining and implies a complex series of regulatory events following receptor stimulation. Similar time courses of inositol 1,4,5-trisphosphate and Ca2+ signals provides supporting evidence that inositol 1,4,5-trisphosphate is the second messenger coupling bradykinin receptor stimulation to release of Ca2+ from intracellular stores.

Animals↗