Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

Polyphosphoinositide phospholipase C and evidence for inositol-phosphate-hydrolysing activities in the plasma-membrane fraction from light-grown wheat (Triticum aestivum L.) leaves

The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca²⁺ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca²⁺ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP₂) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca²⁺ the enzyme clearly favoured PIP. In addition, the PIP₂-PLC activity was increased by Mg²⁺ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P₃ was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP₂-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC deoxycholate - IDPase inosine 5-diphosphatase - InsP_s inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e. - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PLC phospholipase C This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance.     

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated<Emphasis Type="Italic"> Cupressus lusitanica</Emphasis> cell cultures

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P₃ phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P₃ occur simultaneously and that the Ins(1,4,5)P₃ level may be regulated by both PtdIns(4,5)P₂–PLC and Ins(1,4,5)P₃ phosphatases. Studies on the properties of PLC and Ins(1,4,5)P₃ phosphatases indicate that PLC activity toward PtdIns(4,5)P₂ was optimal at a lower Ca²⁺ concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P₃ phosphatase activity is inhibited by high Ca²⁺ concentration. This suggests that Ins(1,4,5)P₃ biosynthesis and degradation may be regulated by free cytosolic Ca²⁺. In addition, a relationship between Ins(1,4,5)P₃ signaling and accumulation of a phytoalexin (-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P₃ accumulation by neomycin or LiCl affects elicitor-induced production of -thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of -thujaplicin while thapsigargin alone induces -thujaplicin accumulation. These results suggest that Ca²⁺ released from intracellular calcium stores may mediate elicitor-induced accumulation of -thujaplicin via an Ins(1,4,5)P₃ signaling pathway, since it is widely accepted that Ins(1,4,5)P₃ can mobilize Ca²⁺ from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P₃ turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P₃ in elicitor-induced phytoalexin accumulation via a Ca²⁺ signaling pathway.Abbreviations Ins(1,4,5)P₃ Inositol-1,4,5-trisphosphate - Ins(1,4)P₂ Inositol-1,4-bisphosphate - Ins(4,5)P₂ Inositol-4,5-bisphosphate - Ins(1)P Inositol 1-phosphate - Ins(4)P Inositol 4-phosphate - PLC Phospholipase C - PtdIns Phosphatidylinositol - PtdIns(4,5)P₂ Phosphatidylinositol 4,5-bisphosphate - YE Yeast elicitor     

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K⁺ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K⁺ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532–1539). In contrast, transfer to darkness apparently closes open K⁺ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960–962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)P₃ levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P₃ production and K⁺-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P₃ production and K⁺ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P₃ levels and closes K⁺ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K⁺ channels in S. saman pulvinar cells.Abbreviations DiS-C₃-(5) 3,3-dipropylthiadicarbocyanine iodide - F measure change in Dis-C₃-(5) fluorescence - F_o initial Dis-C₃-(5) fluorescence - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate - rbc red blood cell Supported by grants from NSF (IBN 9206179 and MCB 9305154) and U.S.-Israel Binational Agricultural Research and Development Fund (IS-1670-90RC) to R.C.C. We thank the University of Connecticut Biotechnology Center for the use of a fluorescent spectrophotometer.     

Metabolism of 3- and 4-phosphorylated phosphatidylinositols in stomatal guard cells of Commelina communis L.

Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide-mediated signalling paradigm as purported to apply to plants. A non-equilibrium [³²P]P_i labelling strategy and enzymic and chemical dissection of labelled phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L. The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [³²P]P_i. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP₂) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90–97% of the [³²P]P_i label while the 4- and 5-monoester phosphates of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P₂ by successive 4- and 5-phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4- and 5-phosphorylated phosphatidylinositols in a single defined hormone-responsive plant cell type. The monoester phosphate in phosphatidylinositol 3-monophosphate (PtdIns3P) accounted for 83–95% of the ³²P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P₂] owing to the rapid attainment of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3,4)P₂, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P₂ was quicker than that of PtdIns(4,5)P₂, and in mass terms PtdIns(3,4)P₂ appeared to predominate over PtdIns(4,5)P₂. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P₂ show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P₂ in addition to those previously indicated for PtdIns(4,5)P₂ in stomatal guard cells.     

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.     

Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase.

Agonist-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], is considered the primary output signal of activated phosphoinositide (PI) 3-kinase. The physiological targets of this novel phospholipid and the identity of enzymes involved in its metabolism have not yet been established. We report here the identification of two enzymes which hydrolyze the 5-position phosphate of PtdIns(3,4,5)P3, forming phosphatidylinositol (3,4)-bisphosphate. One of these enzymes is the 75 kDa inositol polyphosphate 5-phosphatase (75 kDa 5-phosphatase), which has previously been demonstrated to metabolize inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. We have identified a second PtdIns(3,4,5)P3 5-phosphatase in the cytosolic fraction of platelets, which forms a complex with the p85/p110 form of PI 3-kinase. This enzyme is immunologically and chromatographically distinct from the platelet 43 kDa and 75 kDa 5-phosphatases and is unique in that it removes the 5-position phosphate from PtdIns(3,4,5)P3, but does not metabolize PtdIns(4,5)P2, Ins(1,4,5)P3 or Ins(1,3,4,5)P4. These studies demonstrate the existence of multiple PtdIns(3,4,5)P3 5-phosphatases within the cell.     

Inositol 1,4,5-trisphosphate as fertilization signal in plants: testcase Chlamydomonas eugametos

Within the first 2 h of sexual reproduction, gametes of the green alga Chlamydomonas eugametos agglutinate, fuse via their mating structures, de-agglutinate and swim off as vis-à-vis pairs. During this period, increases in intracellular inositol 1,4,5-trisphosphate levels and changes in polyphosphoinositide synthesis were associated with cell fusion. The protein-kinase-C inhibitor staurosporine (0.1–0.2 M) inhibited the de-agglutination of pairs and therefore prevented them swimming away, while earlier stages of mating such as agglutination or cell fusion were unaffected. The results suggest that inositol 1,4,5-trisphosphate and diacylglycerol are fertilization signals in C. eugametos. The idea that they could also be fertilization signals in higher plants is discussed in relation to in vitro embryogenesis.Abbreviations DAG diacylglycerol - InsP₃ inositol 1,4,5-trisphosphate - mt⁺/mt^– mating-type plus or minus - PKC protein kinase C - PtdOH phosphatidic acid - PtdInsP phosphatidylinositol - 4-phosphate PtdInsP₂ phosphatidylinositol 4,5-bisphosphate     

Metabolism of Inositol(1,4,5)trisphosphate by a Soluble Enzyme Fraction from Pea (Pisum sativum) Roots

Metabolism of the putative messenger molecule d-myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P₃] in plant cells has been studied using a soluble fraction from pea (Pisum sativum) roots as enzyme source and [5-³²P]Ins(1,4,5)P₃ and [2-³H]Ins(1,4,5)P₃ as tracers. Ins(1,4,5)P₃ was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P₂] whereas inositol(1,4)bisphosphate [Ins(1,4)P₂] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P₄. Dephosphorylation of Ins(1,4,5)P₃ to Ins(4,5)P₂ was dependent on Ins(1,4,5)P₃ concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P₃ to Ins(4,5)P₂ and Ins(1,4,5,X)P₄ was inhibited by 55 micromolar Ca²⁺. This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P₃ and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.     

Occurrence and functions of the phosphatidylinositol cycle in the myocardium

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by Ca²⁺ mobilizing signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the ₁-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca²⁺ transients initiated by Ptdlns(4,5)P₂ hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than Ptdlns(4,5)P₂ contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response. (Mol Cell Biochem116: 59–67, 1992)Abbreviations Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P₂ - Phosphatidylinositol 4-monophosphate PtdIns(4)P₄ - Phosphatidylinositol PtdIns - Inositol 1,4,5-triphosphate Ins(1,4,5)P₃ - Inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P₄ - Inositol 1-monophosphate Ins(1)P - Inositol 1,4-bisphosphate Ins(1,4)P₂ - Inositol Ins - Inositolphosphates InsP_n - Guanine 5'-triphosphate GTP - GTP binding protein G-protein - Phosphatidylinositolspecific phospholipase C PLC - Protein kinase C PKC - 1,2-Diacylglycerol DAG - Monoacylglycerol MAG - cytidyldiphoshate-diacylglycerol CDP-DAG - Sarcolemma SL - Sarcoplasmic reticulum SR - Stearic acid 18:0 - Polyunsaturated fatty acids PUFA - Arachidonic acid 20:4n-6 - Linoleic acid 18:2n-6 - Eicosapentaenoic acid 20:5n-3 - Docosahexaenoic acid 22:6n-3 - Phosphatidic acid PtdOH - Phospholipase D PLD - Phosphatidylcholine PtdChol     

The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [³H-]phosphatidylinositol 4,5-biphosphate ([³H-]PtdIns(4,5)P ₂) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca²⁺,14 mM Na-cholate and 20 AM [³H-]PtdIns (4,5)P ₂ (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg^–1·2.5 min^–1. The addition of Mg²⁺ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na⁺ and at a low Ca²⁺ concentration (0.3 M), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀0.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg^–1·2.5 min^–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg^–1·2.5 min^–1). Unlike PtdIns(4,5)P ₂, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca²⁺ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP ₂ inositol bisphosphate (unidentified isomer) - InsP ₃ inositol trisphosphate (unidentified isomer) - InsP _x any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

Histamine-Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis

Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [³H]Inositol 1,4,5-trisphosphate [[³H]Ins(1,4,5)P₃] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of ～140% of basal values. [³H]Inositol 1,4-bisphosphate [[³H]Ins(1,4)P₂] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [³H]inositol 1,3,4-trisphosphate [[³H]-Ins(1,3,4)P₃], [³H]inositol 1,3-bisphosphate, and [³H]-inositol 4-monophosphate ([³H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca²⁺, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [³H]Ins(1,4,5)P₃ was rapidly metabolized (t_1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t_1/2 values of 113, 133, 104, and 66 s for [³H]Ins(1,3,4)P₃, [³H]Ins(1,4)P₂, an unresolved mixture of [³H]inositol 1- and 3-monophosphate ([³H]Ins1/3P), and [³H]Ins4P, respectively. The calculated turnover rate of [³H]Ins(1,4,5)P₃ was sufficient to account for the turnover of the combination of both [³H]Ins(1,4)P₂ and [³H]Ins(1,3,4)P₃ but not that of [³H]Ins1/3P or [³H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca²⁺-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate.     

Antigen receptor-mediated regulation of sustained polyphosphoinositide turnover in a human T cell line. Evidence for a receptor-regulated pathway for production of phosphatidylinositol 4,5-bisphosphate

Stimulation of the human T cell line, Jurkat, by the addition of monoclonal antibodies reactive with the T cell antigen receptor complex (CD3/Ti) leads to sustained increases in levels of inositol 1,4,5-trisphosphate. To investigate the possibility that the production of polyphosphoinositides is regulated during CD3/Ti stimulation, we studied Jurkat cells whose inositol phospholipids had been labeled to steady state with [3H]inositol, as well as Jurkat cells during nonequilibrium labeling with [32P]orthophosphate. The addition of CD3 monoclonal antibodies led to a 4-5-fold increase in [3H]inositol trisphosphate that was sustained for greater than 20 min. Within 60 s of CD3/Ti stimulation, [3H] phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) decreased by 65 and 35%, respectively. This change in [3H]PtdIns(4,5)P2 persisted for greater than 20 min. The decrease in [3H]PtdIns4P, however, was transient, and, after 5 min, the levels of [3H]PtdIns4P were comparable in stimulated and unstimulated cells. To examine the rate of flux through inositol phospholipids, we measured the CD3/Ti-stimulated changes in the ratio, 32P cpm/3H cpm, in each inositol phospholipid. CD3/Ti stimulation led to accelerated fluxes through PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) that were maintained for greater than 20 min. After the initial 30 s, however, there was no detectable effect of anti-CD3 on flux through Ptsins4p. This observation suggested that, during CD3/Ti stimulation, production of PtdIns(4,5)P2 from PtdIns might occur via a small pool of PtdIns4P with a very high turnover. The existence of such a pool was established by determining that, in stimulated cells, the 32P-specific activity of the 1-position phosphate of PtdIns(4,5)P2 was 8-10-fold that of PtdIns4P. We conclude that, during the initial 60 s of CD3/Ti stimulation, there is a substantial depletion of cellular PtdIns(4,5)P2 and PtdIns4P. Thereafter, a CD3/Ti-regulated pathway generates PtdIns(4,5)P2 from PtdIns through a small, but highly labile, pool of PtdIns4P.     

Inositol phosphate signaling and gibberellic acid

To respond to physical signals and endogenous hormones, plants use specific signal transduction pathways. We and others have previously shown that second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] is used in abscisic acid (ABA) signaling, and that some mutants with altered Ins(1,4,5)P₃ have altered responses to ABA. Specifically, mutants defective in the myo-inositol polyphosphate 5-phosphatases (5PTases) 1 and 2 genes that hydrolyze 5-phosphates from Ins(1,4,5)P₃ and other PtdInsP and InsP substrates, have elevated Ins (1,4,5)P₃, and are ABA-hypersensitive. Given the antagonistic relationship between ABA and gibberellic acid (GA), we tested the response of these same mutants to a GA synthesis inhibitor, paclobutrazol (PAC). We report here that 5ptase1, 5ptase2 and 5ptase11 mutants are hypersensitive to PAC, suggesting a relationship between elevated Ins(1,4,5)P₃ and decreased GA signal transduction. These data provide insight into signaling cross-talk between ABA and GA pathways.Key words: inositol, phosphatidylinositol phosphate, paclobutrazol, gibberellic acid, inositol trisphosphate, paclobutrazol     

The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages.

The haemopoietic growth factors interleukin 3 (IL-3) and colony stimulating factor-1 (CSF-1) stimulate the survival and proliferation of murine normal bone-marrow-derived macrophages. To establish whether these growth factors elicit their effects via the hydrolysis of phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form the second messengers inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and diacylglycerol, macrophages were labelled with tracer quantities of [3H]inositol. Treatment of these cells with either IL-3 or CSF-1 did not alter the levels of PtdIns(4,5)P2 or Ins(1,4,5)P3. However, addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) which does not stimulate proliferation in macrophages caused a marked and rapid increase in the levels of Ins(1,4,5)P3, inositol bisphosphate and inositol monophosphate, and a decrease in the amount of PtdIns(4,5)P2. FMLP also evoked a rapid increase in intracellular cytosolic Ca2+ levels, as measured with quin 2 the Ca2+-sensitive fluorescent probe, whereas IL-3 and CSF-1 had no such effect. These results suggest that FMLP stimulates the hydrolysis of PtdIns(4,5)P2 to form the second messenger Ins(1,4,5)P3 which acts to increase the levels of cytosolic Ca2+, and that IL-3- and CSF-1-stimulated proliferation in macrophages is not associated with the formation of PtdIns(4,5)P2-derived second messengers.     

Measuring Brain Uptake and Incorporation into Brain Phosphatidylinositol of Plasma myo-[2H6]Inositol in Unanesthetized Rats: An Approach to Estimate In vivo Brain Phosphatidylinositol Turnover

The in vivo rate of turnover of phosphatidylinositol (PtdIns) in brain is not known. In brain, certain receptor-mediated signal transduction involves metabolism of PtdIns and a method to measure its turnover in awake animals is useful in studying the effect of lithium and other therapeutic agents. In a method described here, rats were infused subcutaneously with myo-[²H₆]inositol (Ins*) using an osmotic pump and, at 1 and 8 weeks, concentrations of free myo-inositol (Ins) and Ins* in plasma and brain were measured by GC-MS (chemical ionization). Also, PtdIns and PtdIns* together in brain were isolated, and Ins and Ins* from their headgroups were released enzymatically and specific activity of incorporated inositol was measured. The specific activity of inositol reached a steady state in plasma within 1 week of infusion, but not in brain even at 8 weeks. However, in brain, the specific activity of phosphatidylinositol was same as that of inositol at both time-points, suggestive of fast turnover of PtdIns. The animal experiment and the analytical methodology described here should be useful for measuring the rate of turnover of brain PtdIns in pathological and drug treatment conditions.     

An inositol 1,4,5-trisphosphate-6-kinase activity in pea roots

A soluble extract from pea (Pisum sativum L.) roots, when incubated with ATP and inositol 1,4,5-trisphosphate, produced an inositol tetrakisphosphate. The chromatographic properties of this inositol tetrakisphosphate, and of the products formed by its chemical degradation, identify it as inositol 1,4,5,6-tetrakisphosphate. No evidence was obtained for a 3-phosphorylation of inositol 1,4,5-trisphosphate. The importance of these observations with respect to inositol phosphates and calcium signalling in higher plants, is discussed.Abbreviations HPLC high-performance liquid chromatography - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - InsP₄ inositol tetrakisphosphate J.A.C. gratefully acknowledges support from the Agricultural and Food Research Council, U.K., Plant Molecular Biology Initiative.     

Phosphoinositide hydrolysis by guanosine 5''-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transmembrane Signaling via Phosphatidylinositol 4,5-Bisphosphate Hydrolysis in Plants

Recent investigations have confirmed the presence of the polyphosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP₂), as well as inositol phospholipid-specific phospholipase C in higher plant and microalgal cells. In addition, it has been shown that stimulation of some photosynthetic cell types by environmental or hormonal challenge is accompanied by degradation of the polyphosphoinositides. The products of phospholipase C-catalyzed PIP₂ hydrolysis, inositol 1,4,5-trisphosphate and diacylglycerol, appear to be capable of releasing organelle-bound Ca²⁺ and stimulating protein kinase C-like activity in vitro. However, a direct cause and effect relationship between stimulated PIP₂ breakdown and changes in intracellular calcium, protein phosphorylation, or cell function has not yet been unequivocally established. Despite a number of technical difficulties slowing progress in this field, it is likely that photosynthetic organisms will soon be shown to transmit physiologically significant extracellular signals across their plasma membranes by a PIP₂-mediated transduction mechanism.