The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.

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Guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint

Biochemical Journal ◽

10.1042/bj2710743 ◽

1990 ◽

Vol 271 (3) ◽

pp. 743-748 ◽

Cited By ~ 28

Author(s):

M Camps ◽

C F Hou ◽

K H Jakobs ◽

P Gierschik

Keyword(s):

Phospholipase C ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Anion Exchange Chromatography ◽

Major Product ◽

Exchange Chromatography ◽

High Protein ◽

High Protein Concentration ◽

Inositol Phosphate Accumulation ◽

Stimulation Of

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5′-[beta-thio]diphosphate. ATP, adenosine 5′-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

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Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates

Biochemical Journal ◽

10.1042/bj2240291 ◽

1984 ◽

Vol 224 (1) ◽

pp. 291-300 ◽

Cited By ~ 78

Author(s):

R A Akhtar ◽

A A Abdel-Latif

Keyword(s):

Smooth Muscle ◽

Time Course ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Ionophore A23187 ◽

Release Of Noradrenaline ◽

Adrenergic Blocker ◽

Rabbit Iris

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)

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The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection

Biochemical Journal ◽

10.1042/bj2640323 ◽

1989 ◽

Vol 264 (2) ◽

pp. 323-333 ◽

Cited By ~ 25

Author(s):

T Radenberg ◽

P Scholz ◽

G Bergmann ◽

G W Mayr

Keyword(s):

Mammalian Cells ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Detection System ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Phosphate Metabolism ◽

Qualitative And Quantitative ◽

Inositol Phosphate Metabolism ◽

Avian Erythrocytes

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called ‘metal-dye detection’ [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the ‘lowest-locant rule’ [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.

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The polyphosphoinositide phosphodiesterase of erythrocyte membranes

Biochemical Journal ◽

10.1042/bj1980133 ◽

1981 ◽

Vol 198 (1) ◽

pp. 133-140 ◽

Cited By ~ 419

Author(s):

C. Peter Downes ◽

Robert H. Michell

Keyword(s):

Inositol Phosphates ◽

Inositol Trisphosphate ◽

Aminoglycoside Antibiotic ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Erythrocyte Membranes ◽

Erythrocyte Ghosts ◽

Assay Procedure ◽

Serine Esterases ◽

Phosphatidylinositol 4

1. A new assay procedure has been devised for measurement of the Ca2+-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [32P]Pi. They are incubated under appropriate conditions for activation of the phosphodiesterase and the released32P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca2+. For both esters, optimum rates of release were seen at pH6.8–6.9. Mg2+did not provoke release of either ester. 3. Ca2+provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca2+. 4. In the presence of Mg2+, the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca2+and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg2+-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.

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Effects of PDGF on inositol phosphates, Ca2+, and contraction of mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1987.253.3.f458 ◽

1987 ◽

Vol 253 (3) ◽

pp. F458-F463

Author(s):

P. Mene ◽

H. E. Abboud ◽

G. R. Dubyak ◽

A. Scarpa ◽

M. J. Dunn

Keyword(s):

Mesangial Cells ◽

Inositol Phosphates ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Water Soluble ◽

Free Calcium ◽

Glomerular Mesangial Cells ◽

Cross Sectional ◽

Average Decrease ◽

Intracellular Probe

Platelet-derived growth factor (PDGF) is a potent mitogen and vasoactive polypeptide for aortic smooth muscle. Because contractile glomerular mesangial cells synthesize a PDGF-like molecule and may respond to PDGF released by infiltrating cells at the site of glomerular inflammation, we studied the effects of exogenous, highly purified PDGF on 1) contraction of cultured rat mesangial cells and 2) membrane phosphoinositide turnover and cytosolic free calcium ([Ca2+]i), as putative mechanisms of membrane signal transduction. PDGF, 10(-11) and 10(-10) M, contracted 56.1 +/- 5.2 and 72.9 +/- 6.4% of the cells, respectively, with an average decrease of cross-sectional area of 22.0 +/- 2.6 and 28.1 +/- 2.7% of basal, as assessed by image-analysis microscopy. PDGF also rapidly increased total water-soluble inositol phosphates, measured after anion-exchange chromatography on perchloric acid-extracted cells, and simultaneously raised [Ca2+]i, measured by the fluorescent intracellular probe fura-2, from basal levels of 83.1 +/- 6.8 to a peak of 229.4 +/- 20.0 nM. We conclude that PDGF stimulates contraction of rat mesangial cells via a phospholipase C-dependent pathway, with potential relevance to the control of glomerular hemodynamics and mesangial proliferation in immune-mediated glomerular disease.

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A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103262841 ◽

2004 ◽

Vol 9 (4) ◽

pp. 343-353 ◽

Cited By ~ 6

Author(s):

Elfrida R. Benjamin ◽

Sarah L. Haftl ◽

Dimitris N. Xanthos ◽

Gregg Crumley ◽

Mohamed Hachicha ◽

...

Keyword(s):

Anion Exchange ◽

Column Chromatography ◽

Large Volume ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Assay Method ◽

Signal To Noise ◽

Chromatography Method ◽

Phosphate Accumulation ◽

Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

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Purification and Characterization of Laccase fromChaetomium thermophilium and Its Role in Humification

Applied and Environmental Microbiology ◽

10.1128/aem.64.9.3175-3179.1998 ◽

1998 ◽

Vol 64 (9) ◽

pp. 3175-3179 ◽

Cited By ~ 160

Author(s):

Benny Chefetz ◽

Yona Chen ◽

Yitzhak Hadar

Keyword(s):

Municipal Solid Waste ◽

Solid Waste ◽

Laccase Activity ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Water Soluble ◽

Type I ◽

Light Spectrum ◽

Solid Media ◽

Wide Range

ABSTRACT Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process.C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

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Phosphate, inositol and polyphosphates

Biochemical Society Transactions ◽

10.1042/bst20150215 ◽

2016 ◽

Vol 44 (1) ◽

pp. 253-259 ◽

Cited By ~ 21

Author(s):

Thomas M. Livermore ◽

Cristina Azevedo ◽

Bernadett Kolozsvari ◽

Miranda S.C. Wilson ◽

Adolfo Saiardi

Keyword(s):

Inositol Phosphates ◽

Inositol Phosphate ◽

High Energy ◽

Inorganic Polyphosphate ◽

Covalent Bonds ◽

Polymeric Form ◽

Phosphodiester Bond ◽

Signalling Molecules ◽

Inositol Ring ◽

Phosphate Groups

Eukaryotic cells have ubiquitously utilized the myo-inositol backbone to generate a diverse array of signalling molecules. This is achieved by arranging phosphate groups around the six-carbon inositol ring. There is virtually no biological process that does not take advantage of the uniquely variable architecture of phosphorylated inositol. In inositol biology, phosphates are able to form three distinct covalent bonds: phosphoester, phosphodiester and phosphoanhydride bonds, with each providing different properties. The phosphoester bond links phosphate groups to the inositol ring, the variable arrangement of which forms the basis of the signalling capacity of the inositol phosphates. Phosphate groups can also form the structural bridge between myo-inositol and diacylglycerol through the phosphodiester bond. The resulting lipid-bound inositol phosphates, or phosphoinositides, further expand the signalling potential of this family of molecules. Finally, inositol is also notable for its ability to host more phosphates than it has carbons. These unusual organic molecules are commonly referred to as the inositol pyrophosphates (PP-IPs), due to the presence of high-energy phosphoanhydride bonds (pyro- or diphospho-). PP-IPs themselves constitute a varied family of molecules with one or more pyrophosphate moiety/ies located around the inositol. Considering the relationship between phosphate and inositol, it is no surprise that members of the inositol phosphate family also regulate cellular phosphate homoeostasis. Notably, the PP-IPs play a fundamental role in controlling the metabolism of the ancient polymeric form of phosphate, inorganic polyphosphate (polyP). Here we explore the intimate links between phosphate, inositol phosphates and polyP, speculating on the evolution of these relationships.

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Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

Journal of Applied Physiology ◽

10.1152/jappl.1989.66.1.504 ◽

1989 ◽

Vol 66 (1) ◽

pp. 504-508 ◽

Cited By ~ 3

Author(s):

T. Bainbridge ◽

R. D. Feldman ◽

M. J. Welsh

Keyword(s):

Adrenergic Receptor ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Second Messengers ◽

Receptor Activation ◽

Adrenergic Stimulation ◽

Cl Secretion ◽

Phosphate Accumulation ◽

Inositol Phosphate Accumulation ◽

Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

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Phosphoinositide breakdown in blowfly salivary glands

AJP Cell Physiology ◽

10.1152/ajpcell.1984.246.1.c141 ◽

1984 ◽

Vol 246 (1) ◽

pp. C141-C147 ◽

Cited By ~ 15

Author(s):

I. Litosch ◽

H. S. Lee ◽

J. N. Fain

Keyword(s):

Salivary Glands ◽

Phospholipase C ◽

Inositol Phosphates ◽

Ionophore A23187 ◽

Phosphoinositide Breakdown ◽

Transient Loss ◽

Phosphatidylinositol 4

In blowfly salivary glands, 5-hydroxytryptamine stimulated a rapid and sustained loss of [3H]inositol, [32P]phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. There was a corresponding increase in labeled inositol phosphates. In the absence of Ca2+, 5-hydroxytryptamine stimulated a rapid but transient loss of labeled phosphatidylinositol 4,5-bisphosphate. By 5 min, the amount of labeled phosphatidylinositol 4,5-bisphosphate recovered to control values. The divalent ionophore A23187 stimulated loss of labeled phosphatidylinositol 4,5-bisphosphate and increased the amount of labeled phosphatidylinositol. In homogenates, Ca2+ stimulated phosphatidylinositol 4,5-bisphosphate breakdown but not phosphatidylinositol breakdown. These results suggest that hormone-stimulated breakdown of labeled phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate occurs through a phospholipase C and is relatively independent of extracellular Ca2+. There is also a Ca2+-activated conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol.

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