Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Angiotensin II-induced increase in inositol 1,4,5-trisphosphate in cultured rat mesangial cells: evidence by refined high performance liquid chromatography

Angiotensin II-induced change in inositol phosphates were studied in cultured rat mesangial cells prelabeled with [3H]myo-inositol. By using anion-exchange high performance liquid chromatography, we could analyzed the change in inositol mono-, bis-, and tris-phosphate more rapidly and easily with higher resolution than the previously reported methods. Angiotensin II rapidly increased inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate within 15 sec, followed by an increase in inositol 1-monophosphate at 30 sec. Angiotensin II-induced increases in inositol phosphates were dose-dependent and completely blocked by saralasin. These results indicate that angiotensin II induces the production of inositol phosphates including inositol 1,4,5-trisphosphate, an intracellular Ca2+-releasing factor, in cultured rat mesangial cells.     

In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo-[3H]Inositol or [1-14C]arachidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H-labeled inositol phosphates were analyzed by either ion-exchange chromatography or HPLC. The aqueous humor was analyzed for 14C-labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo-[3H]inositol into phosphoinositides and accumulation of 3H-labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo-inositol 1-phosphate, 36% in myo-inositol bisphosphate, 0.95% in myo-inositol 1,3,4-trisphosphate (1,3,4-IP3), and 2.6% in 1,4,5-IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for alpha 1-adrenergic receptors in this tissue. (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.     

The separation of myo-inositol phosphates by ion-pair chromatography

The separation of myo-inositol phosphates by ion pair, reverse-phase high performance liquid chromatography has been investigated. The retention of the inositol phosphates is dependent on both the polarity of the hetaeron utilized and on the pH of the solvent. A method is presented which permits the isocratic separation of multiple forms of inositol phosphates including isomers of myo-inositol trisphosphate. This method appears to be superior to the anion exchange based systems currently employed because of smaller retention volumes, the low ionic strength of the solvent employed, the absence of a requirement for reequilibration, and the ability to perform separations isocratically.     

High-performance liquid chromatographic analysis of radiolabeled inositol phosphates

Separation of inositol phosphates by low-pressure anion-exchange chromatography yields unsatisfactory results, while previously described anion-exchange HPLC methods require such extensive processing times that they preclude efficient sample analysis. Using a low-capacity Vydac nucleotide anion-exchange column, we have developed a method which allows complete separation of myo-inositol, inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in approximately 10 min followed by a 5-min column regeneration time. This method provided exceptional reproducibility and quantitative recovery of each inositol phosphate. One column was used for over 300 separations with no loss in performance or alteration in elution pattern. A modified procedure with a 14-min gradient was developed to separate the 1,3,4- and 1,4,5-isomers of inositol trisphosphate. These separation procedures were used to characterize the kinetics of degradation of inositol phosphates by lysates of erythrocytes and neutrophils. We conclude that these procedures are applicable for rapid and quantitative analysis of radiolabeled inositol phosphates in cellular extracts.     

Nuclear magnetic resonance spectroscopic analysis of myo-inositol phosphates including inositol 1,3,4,5-tetrakisphosphate

1H and 31P NMR spectra of a variety of phosphorylated myo-inositols have been analyzed using a Bruker WH-360 spectrometer. Proton and phosphorus chemical shifts and coupling constants are reported for myo-inositol 1-phosphate, myo-inositol 2-phosphate, myo-inositol 5-phosphate, myo-inositol 1,2-cyclic phosphate, myo-inositol 1,4-bisphosphate, myo-inositol 1,4,5-trisphosphate, and myo-inositol 1,3,4,5-tetrakisphosphate. These data provide the basis for the chemical identification and characterization of biologically relevant inositol phosphates.     

Gradient ion chromatography of inositol phosphates

Inositol phosphates including phytic acid were separated in 30 min by gradient ion chromatography with postcolumn derivatization. All four pentakisphosphates were resolved, while four tetrakisphosphate peaks were detected. The limits of detection for all polyphosphates, including tris- and bisphosphates, were between 1 and 2 nmol. The method was used to compare nonenzymatic dephosphorylation of inositol hexakisphosphate at pH 4.0 versus pH 10.8. The only pentakisphosphate detected in calf brains was identified as myo-inositol 1,3,4,5,6-pentakisphosphate. The major pentakisphosphate in raw soybean seeds was myo-inositol 1,2,4,5,6-pentakisphosphate of unknown enantiomeric composition, while lesser amounts of myo-inositol 1,2,3,4,5-pentakisphosphate of unknown enantiomeric composition, myo-inositol 1,2,3,4,6-pentakisphosphate, and myo-inositol 1,3,4,5,6-pentakisphosphate were also present.     

Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.

1. The effect of Li+ on the agonist-dependent metabolism of [3H]inositol has been studied in rat brain, rat parotid and the insect salivary gland. 2. When brain or parotid slices were incubated in the presence of [3H]inositol, Li+ was found to amplify the ability of agonists such as carbachol, phenylephrine, histamine, 5-hydroxytryptamine and Substance P to elevate the amount of label appearing in the inositol phosphates. 3. A different approach was used with the insect salivary gland, which was prelabelled with [3H]inositol. After washing out the label, the subsequent release of [3H]inositol induced by 5-hydroxytryptamine was greatly decreased by Li+. During Li+ treatment there was a large accumulation of [3H]inositol 1-phosphate. 4. This ability of Li+ to greatly amplify the agonist-dependent accumulation of myo-inositol 1-phosphate offers a novel technique for identifying those receptors that function by hydrolysing phosphatidylinositol. 5. The therapeutic action of Li+ may be explained by this inhibition of myo-inositol 1-phosphatase, which lowers the level of myo-inositol and could lead to a decrease in the concentration of phosphatidylinositol, especially in those neurons that are being stimulated excessively. This alteration in phosphatidylinositol metabolism may serve to reset the sensitivity of those multifunctional receptors that generate second messengers such as Ca2+, cyclic GMP and the prostaglandins.     

D-myo-inositol (1,4)-bisphosphate 1-phosphate. Partial purification from rat liver and characterization

A specific 1-phosphatase acting on myo-inositol (1,4)-biphosphate with a high affinity (Km = 0.9 microM) has been purified 49-fold from soluble proteins of rat liver by anion exchange chromatography followed by gel filtration. This enzyme has a molecular weight of 58,000 as estimated by gel filtration, a pH optimum of 7.5, and requires Mg++ for activity. The only product formed from myo-inositol (1,4)-bisphosphate is the 4-monophosphate. Of 7 other inositol phosphates examined only myo-inositol (1,3,4)-triphosphate was a substrate.     

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.     

Characterization of glycosyl phosphatidylinositol lipids of parasitic protozoans: Leishmania mexicana mexicana promastigotes, Trypanosoma cruzi Peru epimastigotes and Tritrichomonas foetus

Glycosyl phosphatidylinositol lipids of cultured L.mex, mexicana LV732 promastigotes, T. cruzi Peru epimastigotes and Tritrichomonas foetus have been isolated and characterized using metabolic labelling and chromatographic and mass spectrometric (MS) techniques. TLC of the unsaponifiable lipid fractions of L. mex. mexicana and T. cruzi obtained from DEAE Sephadex A-25 followed by Iatrobead column chromatography showed three inositol phosphate-containing lipid components. [3H]myo-inositol, [3H]palmitic acid or H3 32PO4 lipid precursors were incorporated into these three lipid components. Fraction 2 (LM2 and TCP-2) comprises inositol phosphate ceramides. The other two fractions appear to contain mono-O-alkyl and di-O-alkyl glycerol inositol phosphates. Lyso-1-O-alkyl phosphatidylinositols could be cleaved by treatment of PI-specific phosphalipase C. The di-O-alkyl-phospho inositols of these parasites being the first dialkylglycerol lipids reported from eukaryotic membranes raises the possibility of chemotherapy for leishmaniasis and trypanosomiasis based upon functional impairment of alkyl ether lipids. Tritrichomonas foetus contains two major glycophosphosphingolipids, designated TF1 and TF2, which are metabolically labelled with [3H]myo-inositol and H3 32PO4. Both lipids contained ceramides. The major ceramide contains the 18:0 and 18:1 bases and 16:0 N-acyl group. The major glycolipid fraction (TF1) contains fucose linked to inositol diphosphate; one of the phosphates being linked to the ceramide moiety, and the other to ethanolamine. TF1 appears to be a novel class of glycophosphosphingolipid, which may be a part of a membrane anchor.     

Ca(2+)-inositol phosphate chelation mediates the substrate specificity of beta-propeller phytase

Inositol phosphates are recognized as having diverse and critical roles in biological systems. In this report, kinetic studies and TLC analysis indicate that beta-propeller phytase is a special class of inositol phosphatase that preferentially recognizes a bidentate (P-Ca(2+)-P) formed between Ca(2+) and two adjacent phosphate groups of its natural substrate phytate (InsP(6)). The specific recognition of a bidentate chelation enables the enzyme to sequentially hydrolyze one of the phosphate groups in a bidentate of Ca(2+)-InsP(6) to yield a myo-inositol trisphosphate (InsP(3)) and three phosphates as the final products. A comparative analysis of (1)H- and (13)C NMR spectroscopy with the aid of 2D NMR confirms that the chemical structure of the final product is myo-Ins(2,4,6)P(3). The catalytic properties of the enzyme suggest a potential model for how the enzyme specifically recognizes its substrate Ca(2+)-InsP(6) and produces myo-Ins(2,4,6)P(3) from Ca(2+)-InsP(6). These findings potentially provide evidence for a selective Ca(2+)-InsPs chelation between Ca(2+) and two adjacent phosphate groups of inositol phosphates.     

Preparation of inositol phosphates from sodium phytate by enzymatic and nonenzymatic hydrolysis

Procedures for preparing myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates from sodium phytate were established. Hydrolysis was achieved by autoclaving or enzymatic treatment; the inositol phosphates were separated by anion-exchange chromatography and were identified by fast atom bombardment-mass spectrometry. Enzymatic hydrolysis was more specific than autoclaving for isomer formation, whereas autoclaving was more efficient for producing the bis- and trisphosphates, which did not accumulate in significant amounts under the conditions of enzymatic hydrolysis. Sodium salts of the inositol phosphates were more powdery and less hygroscopic than the potassium salts. The procedures were satisfactory for producing gram quantities of each inositol phosphate, amounts adequate for animal studies of effects on mineral bioavailability.     

myo-inositol metabolites as cellular signals

The discovery of the second-messenger functions of inositol 1,4,5-trisphosphate and diacylglycerol, the products of hormone-stimulated inositol phospholipid hydrolysis, marked a turning point in studies of hormone function. This review focuses on the myo-inositol moiety which is involved in an increasingly complex network of metabolic interconversions, myo-Inositol metabolites identified in eukaryotic cells include at least six glycerophospholipid isomers and some 25 distinct inositol phosphates which differ in the number and distribution of phosphate groups around the inositol ring. This apparent complexity can be simplified by assigning groups of myo-inositol metabolites to distinct functional compartments. For example, the phosphatidylinositol 4-kinase pathway functions to generate inositol phospholipids that are substrates for hormone-sensitive forms of inositol-phospholipid phospholipase C, whilst the newly discovered phosphatidylinositol 3-kinase pathway generates lipids that are resistant to such enzymes and may function directly as novel mitogenic signals. Inositol phosphate metabolism functions to terminate the second-messenger activity of inositol 1,4,5-trisphosphate, to recycle the latter's myo-inositol moiety and, perhaps, to generate additional signal molecules such as inositol 1,3,4,5-tetrakisphosphate, inositol pentakisphosphate and inositol hexakisphosphate. In addition to providing a more complete picture of the pathways of myo-inositol metabolism, recent studies have made rapid progress in understanding the molecular basis underlying hormonal stimulation of inositol-phospholipid-specific phospholipase C and inositol 1,4,5-trisphosphate-mediated Ca2+ mobilisation.     

Inositol phosphates in the environment

The inositol phosphates are a group of organic phosphorus compounds found widely in the natural environment, but that represent the greatest gap in our understanding of the global phosphorus cycle. They exist as inositols in various states of phosphorylation (bound to between one and six phosphate groups) and isomeric forms (e.g. myo, D-chiro, scyllo, neo), although myo-inositol hexakisphosphate is by far the most prevalent form in nature. In terrestrial environments, inositol phosphates are principally derived from plants and accumulate in soils to become the dominant class of organic phosphorus compounds. Inositol phosphates are also present in large amounts in aquatic environments, where they may contribute to eutrophication. Despite the prevalence of inositol phosphates in the environment, their cycling, mobility and bioavailability are poorly understood. This is largely related to analytical difficulties associated with the extraction, separation and detection of inositol phosphates in environmental samples. This review summarizes the current knowledge of inositol phosphates in the environment and the analytical techniques currently available for their detection in environmental samples. Recent advances in technology, such as the development of suitable chromatographic and capillary electrophoresis separation techniques, should help to elucidate some of the more pertinent questions regarding inositol phosphates in the natural environment.     

Chlorpromazine induces accumulation of inositol phosphates in C6 glioma cells

The capacity to modify the incorporation of [2-3H]myo-inositol into inositides and inositol phosphates was different for three psychotropic cationic amphiphilic drugs. Chlorpromazine, desmethylimipramine and propranolol were able to increase the labeling of inositol-containing lipids, but only chlorpromazine dramatically increased the incorporation into inositol phosphate, -bisphosphate and -trisphosphate. The increase was 10- to 50-fold in 60 min as compared with controls. This effect is not due to stimulation of lipid labeling, because in chase experiments radioactivity in inositol phosphates increased to a greater extent than in their parent lipids. It is possible that the alteration of phosphoinositide catabolism is related to the neuroleptic activity of the drug.     

Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphoinositide metabolism.

The purpose of this study was to investigate whether the increased contractile responsiveness of aortae from male rats with 12-14 week streptozotocin-induced diabetes to noradrenaline is associated with alterations in phosphoinositide metabolism. The contractile response to noradrenaline (10 microM) in both the presence and absence of extracellular calcium was significantly enhanced in aortae from diabetic rats. No significant differences were found between control and diabetic arteries in the basal incorporation of 32P and [3H]myo-inositol into phosphoinositides, or in the basal accumulation of [32P]phosphatidic acid and [3H]inositol phosphates. However, noradrenaline (10 microM) caused significantly greater breakdown of [32P]phosphatidylinositol 4,5-bisphosphate and formation of [32P]phosphatidic acid and [3H]inositol phosphates in diabetic aortae than in control preparations. The production of [3H]inositol phosphates induced by noradrenaline was selectively reduced by the alpha 1-adrenoceptor antagonist, prazosin, in both control and diabetic tissues. These results indicate that phosphoinositide metabolism in response to noradrenaline via stimulation of alpha 1-adrenoceptors is enhanced in aortae from chronic streptozotocin-diabetic rats. The increase in inositol 1,4,5-trisphosphate and 1,2-diacylglycerol production that presumably results could be responsible, at least in part, for the enhanced contractile response of aortae from diabetic rats to noradrenaline.     

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.