Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “INOSITOL”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33Linked to original sources
PubMed1983

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

Animals↗
PubMed1989

Inhibition of inositol phospholipid synthesis and norepinephrine-stimulated hydrolysis in rat brain slices by excitatory amino acids.

Inhibition by excitatory amino acid agonists of norepinephrine (NE)-stimulated phosphoinositide hydrolysis was studied in rat brain slices. Inhibition was not observed in cortical slices prelabeled with [3H]inositol but was observed when slices were incubated simultaneously with [3H]inositol, glutamate, and NE. Therefore, we hypothesized that glutamate inhibits the synthesis of inositol phospholipids available to the alpha 1-adrenergic receptor, thereby reducing NE-stimulated phosphoinositide hydrolysis. To test this hypothesis, the distribution of [3H]inositol in cortical slices was measured after 5, 10, 20, 40 and 60 min of incubation, with some slices being exposed to 200 microM NE, 1 mM glutamate, 1 mM N-methyl-D-aspartate (NMDA), 1 mM kainate, 1 mM quisqualate, or to NE in the presence of each of the excitatory amino acid agonists. Glutamate had little effect on the slice content of free [3H]inositol, but it severely reduced the synthesis of [3H]inositol phospholipids, in the presence or absence of NE. Glutamate also abolished NE-induced production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Quisqualate mimicked the effects of glutamate, whereas NMDA and kainate caused less inhibition of the synthesis of [3H]inositol phospholipids and did not inhibit the response to NE. Glutamate produced similar inhibitory effects in slices from hippocampus and striatum. To test if the inhibitory effect of glutamate was the result of irreversible cell damage, cortical slices were incubated with 1 mM glutamate for 60 min prior to exposure to [3H]inositol and NE. Preincubation with glutamate did not reduce the synthesis of [3H]inositol phospholipids or inhibit NE-stimulated [3H]inositol monophosphate production. These results indicate that glutamate impairs the synthesis of inositol phospholipids. Each of the excitatory amino acid agonists, quisqualate, NMDA and kainate, inhibited [3H]inositol phospholipid synthesis, but only quisqualate affected [3H]inositol phospholipids available to the alpha 1-adrenergic receptor.

Animals↗
PubMed1983

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.

2,4-Dinitrophenol↗
PubMed1988

Inositol trisphosphate releases intracellularly stored calcium and modulates ion channels in molluscan neurons.

Stimulation of the bag cell neurons of Aplysia triggers a long-lasting afterdischarge in these cells. In vivo, such a discharge causes the onset of a sequence of reproductive behaviors. We have found that treatments that trigger discharges in vitro stimulate the hydrolysis of phosphoinositides in the bag cell neurons, as measured by increased incorporation of 3H-inositol into fractions containing membrane lipids and water-soluble inositol phosphates. The electrophysiological effects of inositol trisphosphate, one of the products of phosphoinositide turnover that has been shown to mobilize intracellular calcium in non-neuronal cells, were investigated using isolated bag cell neurons in cell culture. Microinjection of inositol trisphosphate into cultured bag cell neurons caused a transient hyperpolarization of the membrane (approximately 35 sec), together with an increase in conductance. This effect of inositol trisphosphate was abolished by 50 mM tetraethylammonium ions. Inositol trisphosphate also reduced the amplitude of action potentials. Injection of calcium ions directly into bag cell neurons mimicked these responses seen after inositol trisphosphate injection. Using the cell-attached patch-clamp technique in conjunction with inositol trisphosphate microinjection, we observed that inositol trisphosphate evoked increases in the activity of a channel carrying outward current at the resting potential and more positive potentials. The estimated slope conductance of the channel modulated by inositol trisphosphate was approximately 40 pS, and its reversal potential was close to that predicted for potassium ions. The increased opening of this channel in response to inositol trisphosphate injection appeared to result from a transient shift of its voltage-dependence to more negative potentials. In a few cases, inositol trisphosphate injection also elicited an increase in the activity of a channel passing inward current at rest. Direct measurements of changes in intracellular calcium in response to inositol trisphosphate were made using digital imaging of isolated neurons loaded with the fluorescent calcium indicator fura-2. These revealed that injection of inositol trisphosphate significantly elevated intracellular calcium levels, and that this inositol trisphosphate-induced rise in cytosolic calcium was not affected by removal of extracellular calcium. In contrast to the effects of trains of action potentials in calcium-containing media, which produced increases in calcium primarily in neurites, the inositol trisphosphate-induced elevation of calcium appeared more localized to the somata of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Inositol phosphates in rat atria and the importance of the extraction procedure.

The nature of the inositol phosphates present in adult rat left atria have been examined. Trichloroacetic acid extracts of [3H] inositol-labelled left atria contained the 1 (or 3)-, 2- and 4-isomers of inositol monophosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate. Addition of noradrenaline increased all of these isomers. However, even in the presence of noradrenaline, there was no evidence for the presence of inositol-1,3,4,5-tetrakisphosphate or its dephosphorylation products including inositol-1,3,4-trisphosphate and the 1,3- and 3,4-isomers of inositol bisphosphate. Total accumulation of inositol phosphates in the presence of LiCl was 98% accounted for by dephosphorylation of inositol-1,4,5-trisphosphate and its cyclic isomer. These data indicate that, in intact left atria, metabolism of inositol-1,4,5-trisphosphate is by dephosphorylation and that significant activity of the phosphorylation pathway is not present. When extractions were performed using either acidified chloroform-methanol or perchloric acid, other [3H] inositol-labelled compounds were detected in atrial extracts. One of these chromatographed with ATP and might be mistaken for inositol-1,3,4-trisphosphate if co-chromatography with ATP was used to identify that compound. Another compound had a similar, but not identical chromatographic profile to inositol-1,3,4,5-tetrakisphosphate and is thus likely to be mistaken for that compound. In addition, perchloric acid extraction caused phosphate group migration generating extra isomers of the inositol phosphates and other unidentified [3H]-labelled compounds. Such extraction artifacts are likely especially problematic in studies of intact heart tissue because of the time required to effectively homogenize the tissue. These findings demonstrate the need for caution in interpreting data relating to estimations of inositol phosphates in intact heart tissue.

Animals↗
PubMed1992

Determination of inositol polyphosphates from human T-lymphocyte cell lines by anion-exchange high-performance liquid chromatography and post-column derivatization.

The intracellular amounts of several inositol tris-, tetrakis- and pentakisphosphates and inositol hexakisphosphate were determined in resting and stimulated cells from human T-lymphocyte lines. The inositol polyphosphates were separated by anion-exchange high-performance liquid chromatography and were detected on-line by a recently developed post-column dye system. In the human T-lymphocyte cell line Jurkat, basal intracellular concentrations ranged between 25 +/- 10 pmol per 10(9) cells for inositol 1,4,5-trisphosphate to 6380 +/- 355 pmol per 10(9) cells for inositol hexakisphosphate. Similar basal concentrations were observed in the human T-lymphocyte cell line HPB.ALL, with the exception that inositol hexakisphosphate was approximately 665 +/- 10 pmol per 10(9) cells. Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor by a monoclonal antibody directed against the T-cell receptor-CD3 complex induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate isomers, including inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate, an as yet unidentified inositol tetrakisphosphate isomer, inositol 1,3,4,5,6-pentakisphosphate, inositol 1,2,3,4,6-pentakisphosphate and DL-inositol 1,2,4,5,6-pentakisphosphate. Inositol 1,4,5-trisphosphate increased only transiently after 5 min, whereas DL-inositol 1,4,5,6-tetrakisphosphate (determined as the enantiomeric mixture) increased after 20 min.

Anions↗
PubMed1989

Accumulation of inositol polyphosphate isomers in agonist-stimulated cerebral-cortex slices. Comparison with metabolic profiles in cell-free preparations.

1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.

Animals↗
PubMed1990

Low urinary chiro-inositol excretion in non-insulin-dependent diabetes mellitus.

BACKGROUND AND METHODS: Inositol is a major component of the intracellular mediators of insulin action. To investigate the possible role of altered inositol metabolism in non-insulin-dependent diabetes mellitus (NIDDM), we used gas chromatography and mass spectrometry to measure the myo-inositol and chiro-inositol content of urine specimens from normal subjects and patients with NIDDM: The study subjects were whites, blacks, and Pima Indians. The type of inositol and its concentration in insulin-mediator preparations from muscle-biopsy specimens from normal subjects and diabetic patients were also determined. RESULTS: The urinary excretion of chiro-inositol was much lower in the patients with NIDDM (mean [+/- SE], 1.8 +/- 0.8 mumol per day) than in the normal subjects (mean, 84.9 +/- 26.9 mumol per day; P less than 0.01). In contrast, the mean urinary myo-inositol excretion was higher in the diabetic patients than in the normal subjects (444 +/- 135 vs. 176 +/- 46 mumol per day; P less than 0.05). There was no correlation between chiro-inositol excretion and the age, sex, or weight of the diabetic patients, nor was there any correlation between urinary chiro-inositol and myo-inositol excretion in either group. The results were similar in a primate model of NIDDM, and chiro-inositol excretion was decreased to a lesser extent in animals with prediabetic insulin resistance. chiro-Inositol was undetectable in insulin-mediator preparations from muscle-biopsy samples obtained from patients with NIDDM: Similar preparations from normal subjects contained substantial amounts of chiro-inositol. Furthermore, the chiro-inositol content of such preparations increased after the administration of insulin during euglycemic-hyperinsulinemic-clamp studies in normal subjects but not in patients with NIDDM: CONCLUSIONS: NIDDM is associated with decreased chiro-inositol excretion and decreased chiro-inositol content in muscle. These abnormalities seem to reflect the presence of insulin resistance in NIDDM:

Age Factors↗
PubMed1990

Modulation of hemodynamic and vascular filtration changes in diabetic rats by dietary myo-inositol.

To assess the potential of myo-inositol-supplemented diets to prevent diabetes-induced vascular functional changes, we examined the effects of diets supplemented with 0.5, 1, or 2% myo-inositol on blood flow and vascular filtration function in nondiabetic control rats and rats with streptozocin-induced diabetes (STZ-D). After 1 mo of diabetes and dietary myo-inositol supplementation, 1) 131I-labeled bovine serum albumin (BSA) permeation of vessels was assessed in multiple tissues, 2) glomerular filtration rate (GFR) was estimated as renal plasma clearance of 57Co-labeled EDTA, 3) regional blood flows were measured with 15-microns 85Sr-labeled microspheres, and 4) endogenous albumin and IgG urinary excretion rates were quantified by radial immunodiffusion assay. In STZ-D rats, 131I-BSA tissue clearance increased significantly (2- to 4-fold) in the anterior uvea, choroid-sclera, retina, sciatic nerve, aorta, new granulation tissue, diaphragm, and kidney but was unchanged in skin, forelimb muscle, and heart. myo-Inositol-supplemented diets reduced diabetes-induced increases in 131I-BSA clearance (in a dose-dependent manner) in all tissues; however, only in new granulation tissue and diaphragm did the 2% myo-inositol diet completely normalize vascular albumin permeation. Diabetes-induced increases in GFR and in urinary albumin and IgG excretion were also substantially reduced or normalized by dietary myo-inositol supplements. Increased blood flow in anterior uvea, choroid-sclera, kidney, new granulation tissue, and skeletal muscle in STZ-D rats also was substantially reduced or normalized by the 2% myo-inositol diet. myo-Inositol had minimal if any effects on the above parameters in control rats. These observations indicate that diabetes-induced increases in regional blood flow, 131I-BSA permeation, GFR, and urinary protein excretion can be markedly reduced or normalized by consumption of myo-inositol-supplemented diets that raise plasma myo-inositol levels approximately fivefold. The failure of the 2% myo-inositol diet to normalize GFR and blood flow and albumin permeation in several tissues despite markedly elevated plasma myo-inositol levels and normal or elevated tissue myo-inositol levels indicates that if vascular functional changes in these tissues are linked to altered myo-inositol levels, they are resistant to normalization by elevation of plasma myo-inositol levels. These results suggest that other factors independent of changes in relative or absolute tissue myo-inositol levels may play an important role in the pathogenesis of diabetes-induced vascular functional changes in these tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Attenuation of agonist-induced desensitization of the rat substance P receptor by microinjection of inositol pentakis-and hexakisphosphates in Xenopus laevis oocytes.

Recently, inositol hexakisphosphate (phytic acid) was shown to bind to photoreceptor arrestin and block its interaction with rhodopsin. Such an interaction might predict that inositol polyphosphates could alter G protein-coupled receptor desensitization. To investigate the possible roles of higher inositol polyphosphates on receptor desensitization, we have expressed the rat substance P receptor in Xenopus laevis oocytes. The functional expression of substance P receptor was monitored by voltage-clamp recording of substance P-induced Ca(2+)-dependent Cl- currents. When control oocytes were stimulated with substance P (30 nM), after 10 min of washing the second responses to substance P were approximately 15% of the first responses. Cytosolic injection of inositol pentakisphosphate (100 microM) or inositol hexakisphosphate (100 microM) inhibited the reduction of the second substance P-induced current responses, maintaining the second responses to 57-58% of the initial responses. The protective effects of inositol pentakisphosphate and inositol hexakisphosphate against agonist-induced desensitization were concentration and time dependent and structurally specific, in that inositol hexasulfate and inositol tris- and tetrakisphosphate isomers were inactive. Microinjection of inositol hexakisphosphate did not (a) change the potency of substance P or the sensitivity of the expressed substance P receptor to substance P, (b) inhibit 12-O-tetradecanoylphorbol-13-acetate-induced loss of substance P-induced current responses, or (c) alter the currents elicited by microinjection of inositol-1,4,5-trisphosphate. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate have specific inhibitory effects on the agonist-induced loss of responsiveness of the rat substance P receptor. Moreover, these protective effects of inositol hexakisphosphate against desensitization were also observed with the endogenous lysophosphatidic acid/phosphatidic acid receptor, indicating that this mechanism is not specific to ectopic receptors. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate may be novel pharmacological tools for the study of agonist-induced desensitization.

Animals↗
PubMed1981

Phosphatidylinositol-inositol exchange in a rabbit lung.

A microsomal fraction prepared from rabbit lung tissue was found to catalyze CDPdiacylglycerol-independent incorporation of [3H]inositol into phosphatidylinositol. This incorporation resulted from CMP-dependent phosphatidylinositol-inositol exchange and did not constitute a net synthesis of phosphatidylinositol. The phosphatidylinositol-inositol exchange activity was distinct from the phospholipid-base exchange enzymes and was specific for inositol. Optimal in vitro phosphatidylinositol-inositol exchange activity was observed at pH 8.5--8.8 and either Mn2+ or Mg2+ was essential for activity. Mercaptoethanol stimulated phosphatidylinositol-inositol exchange and Hg2+ inhibited this activity. In the absence of CMP, no phosphatidylinositol-inositol exchange was observed. CDP (and to a smaller extent CTP) also supported phosphatidylinositol-inositol exchange and this appeared to occur via the generation of CMP during incubations. The apparent Km values of the phosphatidylinositol-inositol exchange enzyme for CMP and inositol were 0.4 mM and 11 microM, respectively. When CDPdiacylglycerol was present at a concentration optimal for CDPdiacylglycerol : inositol transferase activity, CMP-dependent phosphatidylinositol-inositol exchange activity was still observed. However, in the presence of Hg2+ CDPdiacylglycerol inhibited phosphatidylinositol-inositol exchange activity. Several properties of the phosphatidylinositol-inositol exchange enzyme resemble those of CDPdiacylglycerol : inositol transferase, but the two enzymes appear distinct on the basis of different degrees of inhibition by either Ca2+, Hg/+ or heat, and on the basis of different changes in activity during lung development.

Animals↗
PubMed1976

Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells.

Inositol biosynthesis was studied in soluble, cell extracts of a wild-type (Ino) strain of Saccharomyces cerevisiae. Two reactions were detected: (i) conversion of D-glucose-6-phosphate to a phosphorylated form of inositol, presumably inositol-1-phosphate (IP synthethase, EC5.5.1.4), and (ii) conversion of phosphorylated inositol to inositol (IP phosphatase, EC3.1.3.25). The in vitro rate of conversion of glucose-6-phosphate to inositol was proportional to incubaion time and enzyme concentration. The pH optimum was 7.0. The synthesis of inositol required oxidized nicotinamide adenine dinucleotide (NAD) and was stimulated byNH4C1 and MgC12. NADP substituted poorly for NAD, and NADH inhibitedthe reaction. Phosphorylated inositol accumulated in the absence of MgC12, suggesting that inositol-phosphate is an intermediate in the pathway and that Mg ions stimulate the dephosphorylation of inositol-phosphate. IP synthetase was inhibited approximately 20% in the presence of inositol in the reaction mixture at concentrations exceeding 1 mM. The enzyme was repressed approximately 50-fold when inositol was present in the growth medium at concentrations exceeding 50 muM. IP synthetase reached the fully repressed level approximately 10 h after the addition of inositol to logarithmic cultures grown in the absence of inositol. The specific activity of the enzyme increased with time in logarithmically growing cultures lacking inositol andapproached the fully depressed level as the cells entered stationary phase.

Ammonium Chloride↗
PubMed1997

Application of stable isotope tracer combined with mass spectrometric detection for studying myo-inositol uptake by cultured neurons from fetal mouse: effect of trisomy 16.

A gas chromatographic (GC)/mass spectrometric method for studying myo-inositol uptake by neurons in vitro is described. Cultured cortical neurons from fetuses of diploid and trisomy 16 mouse (animal model for Down syndrome) were incubated with a physiological concentration of hexadeuterated myo-inositol for 2-40 min. Washed cells were lysed and scyllo-inositol (internal standard) was added to the intracellular material which contained labeled myo-inositol taken up by the cells as well as the endogenous, unlabeled myo-inositol. The samples were evaporated to dryness and the analytes were converted into acetate derivatives. The components were separated by capillary GC, and the m/z 379 ion for labeled myo-inositol and the m/z 373 ion for myo-inositol and scyllo-inositol generated by chemical ionization in an ion trap mass spectrometer were monitored. Quantitation of the deuterium-labeled myo-inositol taken up by the neuron along with endogenous myo-inositol was achieved for 2-40 min of incubation. The labeled myo-inositol uptake was linear for up to 20 min and was Na+ dependent in these neurons. This non-radioisotope method was used to demonstrate a significant (40%) increase in the rate of myo-inositol uptake by cortical neurons from the trisomy 16 mouse relative to control neurons. An increased myo-inositol uptake is consistent with evidence that the myo-inositol transporter gene is on both human chromosome 21 and mouse chromosome 16, and that myo-inositol concentrations are elevated in cerebrospinal fluid from adult Down syndrome individuals and brains from the fetal trisomy 16 mouse.

Adult↗
PubMed1985

Separation of inositol phosphates and glycerophosphoinositol phosphates by high-performance liquid chromatography.

We developed a HPLC method which separates the following nine inositol-containing compounds of biological interest: inositol, inositol 1-monophosphate, inositol 2- or 4-monophosphate, inositol 1,2-cyclic phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, glycerophosphoinositol, glycerophosphoinositol 4-monophosphate, and glycerophosphoinositol 4,5-bisphosphate. The method shows good resolution and sufficient recovery (70-80%) for each compound. By applying this method to human platelets prelabeled with [3H]inositol and stimulated with thrombin, we found an early increase of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of glycerophosphoinositol, inositol 1-monophosphate, and an inositol monophosphate which cochromatographs with inositol 2- and inositol 4-monophosphate occurs later. The method is simple, and--after removal of salts from the incubation buffer--can be directly applied to the measurement of aqueous soluble [3H]inositol-labeled compounds in biological samples.

Chromatography, High Pressure Liquid↗
PubMed1993

Ziskind-Somerfeld Research Award 1993. Biochemical, behavioral, and clinical studies of the role of inositol in lithium treatment and depression.

Lithium (Li) reduces brain inositol levels by inhibiting the enzyme inositol monophosphatase. The enzyme inositol-1-phosphatase was measured in human red blood cells of controls, Li-free bipolar patients, and Li-treated bipolar patients and was found to be reduced by 80% in Li-treated bipolars, thus supporting the concept that chronic Li at therapeutic concentrations inhibits this enzyme. Two behaviors in rats caused by Li, reduction of rearing, and Li-pilocarpine seizures, are reversed by intracerebroventricular replenishment of inositol. The reversal is stereospecific to the naturally occurring myo-inositol; whereas the stereoisomer L-chiro-inositol is ineffective. The reversal is dose-dependent, requiring a dose consistent with known quantities of brain inositol depletion; and is time-dependent, as inositol must be given 1-8 h before stimulation. High-dose peripheral inositol also reverses the limbic seizures induced by Li-pilocarpine, and using gas chromatography was shown to increase brain inositol levels that had been reduced by Li treatment. Low-dose inositol could be shown to reverse a peripheral Li-induced side effect, polyuria/polydipsia, in rats and in patients treated with Li. A higher dose of inositol markedly reduced Hamilton Depression Ratings in 9 of 11 unipolar major depressive disorder patients previously unresponsive to tricyclics, in an open design, but had no effect on chronic schizophrenics in a controlled double-blind randomized crossover trial. A new inositol monophosphatase inhibitor, a fungal product originally discovered as a complement inhibitor, was found to act like Li and lower the seizure threshold for subconvulsant doses of pilocarpine. These data suggest that inositol monophosphatase inhibition is a key mechanism of Li's therapeutic action and that design of new inositol monophosphatase inhibitors may be a practical strategy to create new compounds with Li-like therapeutic effects.

Adult↗
PubMed1997

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Amino Acid Sequence↗
PubMed1980

Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle.

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca(2+) was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50mum) increased the breakdown of phosphatidylinositol bisphosphate in [(3)H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of (3)H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the (3)H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20mum), but not tubocurarine (100mum); and it was abolished by depletion of extracellular Ca(2+) with EGTA, but restored on addition of low concentrations of Ca(2+) (20mum). 5. Calcium-antagonistic agents, such as verapamil (20mum), dibenamine (20mum) or La(3+) (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (;microsomes') was stimulated by 43% in the presence of 50mum-Ca(2+). 7. The results indicate that increased Ca(2+) influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca(2+) activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the (3)H radioactivity of this phospholipid.

Acetylcholine↗
PubMed1986

Mammalian aldolases are isomer-selective high-affinity inositol polyphosphate binders.

A search for target proteins of inositol polyphosphates in mammalian tissues revealed that fructose 1,6-bisphosphate aldolases are potent isomer-selective binders of inositol polyphosphates. Binding was measured by tryptophan fluorescence quenching, by difference spectroscopy, and, in aldolase A, by equilibrium dialysis. Among a series of inositol phosphates containing between one and six phosphates and varying in their positions, inositol 1,4,5-trisphosphate was found to be bound strongest both by aldolase A [( L]0.5 = 0.58 microM) and aldolase B [( L]0.5 = 0.83 microM). Aldolase A showed also a strong binding of inositol tetrakisphosphate [( L]0.5 = 0.83 microM), of inositol 2,4,5-trisphosphate [( L]0.5 = 1.4 microM) and of inositol 1,3,4,5,6-pentakisphosphate [( L]0.5 = 2.0 microM); in aldolase B but not in aldolase A inositol 4,5-bisphosphate was bound as strongly as inositol 1,4,5-trisphosphate [( L]0.5 = 0.95 microM) and also inositol 2,4,5-trisphosphate was tightly bound [( L]0.5 = 1.2 microM). Both in aldolase A and B, 4 mol inositol 1,4,5-trisphosphate were bound/mol tetramer, in aldolase A a total binding of 8 mol inositol 1,4-bisphosphate/mol tetramer was evaluated. Difference spectra revealed that the binding of inositol phosphates to both isoenzymes may be associated with conformational changes. The binding of all inositol phosphates led to an inhibition of the enzyme activity. In aldolase A the inhibition was purely competitive, in aldolase B a complex cooperative type of inhibition was evident with fructose 1,6-bisphosphate as a substrate whereas with fructose 1-phosphate the inhibition also was purely competitive. Model calculations based on the in vitro data indicated a significant potential of aldolase to bind preferentially inositol 1,4,5-trisphosphate also in the presence of excess fructose 1,6-bisphosphate.

Animals↗