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.     

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

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

Isolation of soluble metabolites of the phosphatidylinositol cycle from Samanea saman

An improved protocol for the separation of inositol phosphates by high performance liquid chromatography was used to resolve inositol phosphates from pulvini (motor organs) of the legume, Samanea saman. The pulvini contained inositol phosphate, inositol bisphosphate, and inositol trisphosphate isomers which co-migrated with those of mammalian red blood cells, and one or more other inositol metabolites which, to our knowledge, have not been previously noted in preparations of inositol phosphates. The finding of inositol phosphates in Samanea which comigrate with mammalian inositol phosphates supports the possibility that the phosphatidylinositol cycle may function in signal transduction in plants as well as in animals.Abbreviations HPLC high performance liquid chromatography - PI phosphatidylinositol     

Neural function: metabolism and actions of inositol metabolites in mammalian brain

In the nervous system, a variety of cell types respond to external stimuli through the inositol lipid signalling pathways. The stimulus-coupled sequence of intracellular events has been investigated in a homogeneous model system, the cloned mammalian neural cell line NG115-401L. The neural peptide bradykinin stimulates a rapid production of identified inositol phosphate isomers and an intracellular Ca2+ discharge followed by a persistent plasma membrane influx. The temporal sequence suggests that Ins(1,4,5)P3 or Ins(1,3,4,5)P4 or both may coordinate these events in a neuronal cell, as has been suggested in other cell types. Thapsigargin, an irritant and tumour-promoting plant product, produces calcium transients in the absence of inositol phosphate production, and may provide a new tool for investigating the interactions between inositol phosphates and changes in cellular calcium homeostasis. In the 401L line, high levels of radiolabelled InsP5 and InsP6 have been detected, which has led to the evaluation of their possible occurrence and actions in normal brain. Both InsP5 and InsP6 are produced from a radiolabelled myo-inositol precursor in intact mature brain in a region-specific manner. This suggests that both inositol polyphosphates may be end products of regionally regulated biosynthetic pathways. When microinjected into a nucleus of the brainstem, or iontophoretically applied to the dorsal horn of the spinal cord, both InsP5 and InsP6, but not Ins(1,3,4,5)P4 isomers, appear to be potent neural stimulants. These results suggest that the inositol lipid signalling pathways may generate both intracellular and extracellular signals in brain.     

Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology

Our laboratory studies the biology and enzyme regulation of inositol signal transduction pathways, which are activated in response to a wide range of stimuli. As a six-carbon cyclitol, inositol and its numerous phosphorylated derivatives efficiently generate combinatorial ensembles of signaling molecules. Through the cloning and characterization of inositol polyphosphate kinases (IPK), novel roles for inositol tetrakisphosphate (IP₄), inositol pentakisphosphate (IP₅), and inositol hexakisphosphate (IP₆) and inositol pyrophosphates (PP-IPs), have been identified. Studies have linked the IPKs and their inositide products to the regulation of nuclear processes including gene expression, chromatin remodeling, mRNA export, DNA repair and telomere maintenance. Analysis of IPK knockout animals has revealed a role for production of IPs in regulation of embryogenesis and organism development.The discoveries of the IPK proteins and their connection to nuclear signaling have generated significant interest in the field. Furthermore, they have provided interesting clues into the evolution of inositide-signaling pathways. Ipk2/IPMK and IPS/IP6K family members are conserved from yeast to man. In contrast, the IP₃ 3-kinase (ITPK) branch is observed in selected metazoans and not in plant or fungi. This may imply that Ipk2 and IPS activities evolved first among the group. The promiscuity of the Ipk2 protein further supports this notion and may provide the cell with a means to generate many IP species in a genetically economical fashion. Studies of yeast inositide signaling reveal that these simple eukaryotes do not have an IP₃ receptor in their genome and do not utilize diacylglycerol to activate protein kinase C. Thus, it appears that the canonical “text book” aspects of inositide-signaling pathways are not conserved throughout eukaryotic evolution. In light of the conservation of Ipk2/IPMK, Ipk1 and IPS/IP6K pathways from yeast to man it is interesting to speculate that a primordial role of phospholipase C-induced, IPK-dependent inositide signaling was to regulate nuclear processes. As calcium and PKC signaling evolved in metazoans, these may have greatly enhanced signaling capabilities. Recent studies demonstrating an essential role for IP₅, IP₆ and possibly PP-IP production in metazoan development highlight the importance of IPK signaling in cellular responses in metazoans. With these thoughts in mind, we eagerly await future studies aimed at further elucidating how these signaling codes participate in developmental processes and the control of gene expression, mRNA export, and DNA metabolism.     

Overexpression of D-myo-inositol-3-phosphate synthase leads to elevated levels of inositol in Arabidopsis

In this paper, we report on the generation of transgenic Arabidopsis plants containing elevated levels of the gene product encoding the enzyme catalysing the first committed step in inositol biosynthesis, D-myo-inositol-3-phosphate (Ins3P) synthase. These plants exhibit both an increase in Ins3P synthase activity and an increase in the level of free inositol of over four-fold compared to wild-type plants. Despite these changes, we could detect no significant difference in phenotype in the transgenic plants for a number of characteristics linked with putative functions of inositol and inositol-derived metabolites. Our results indicate that the proposed engineering of inositol metabolism to generate specific plant phenotypes (e.g. salt tolerance) may require the manipulation of several genes, and that Ins3P synthase activity can be manipulated to increase the pool size of free inositol.     

Beyond IP3: Roles for higher order inositol phosphates in immune cell signaling

Nearly 25 years ago the first function of an inositol phosphate, namely Ins(1,4,5)P3, was reported to act as a “second messenger” to mobilize calcium from the endoplasmic reticulum (ER). Since this discovery, many other inositol phosphates and the kinases and phosphatases that generate these inositol phosphates have subsequently been discovered. However, the function of these “higher order” inositol phosphates in biological processes, if any, has remained a mystery. Interest in higher order inositol phosphates, such as Ins(1,3,4,5)P4, was renewed this year following reports of novel roles for these molecules in distinct processes within the immune system ranging from T cell development, B cell development and tolerance induction, as well as neutrophil and mast cell function. In this review, we will touch upon recent advances in inositol phosphate function in mammalian cells. More specifically, we will highlight new studies that have identified novel functions for specific higher order inositol phosphates, such as Ins(1,3,4,5)P4, in the immune system.     

Structural Analysis of a Multifunctional, Tandemly Repeated Inositol Polyphosphatase

Mitsuokella multacida expresses a unique inositol polyphosphatase (PhyAmm) that is composed of tandem repeats (TRs). Each repeat possesses a protein tyrosine phosphatase (PTP) active-site signature sequence and fold. Using a combination of structural, mutational, and kinetic studies, we show that the N-terminal (D1) and C-terminal (D2) active sites of the TR have diverged and possess significantly different specificities for inositol polyphosphate. Structural analysis and molecular docking calculations identify steric and electrostatic differences within the substrate binding pocket of each TR that may be involved in the altered substrate specificity. The implications of our results for the biological function of related PTP-like phytases are discussed. Finally, the structures and activities of PhyAmm and tandemly repeated receptor PTPs are compared and discussed. To our knowledge, this is the first example of an inositol phosphatase with tandem PTP domains possessing substrate specificity for different inositol phosphates.     

IP3 3-kinase opposes NGF driven neurite outgrowth

The inositol (1,4,5) trisphosphate 3-kinases comprise a family of enzymes (A, B, and C) that phosphorylate the calcium mobilising molecule inositol (1,4,5) trisphosphate (IP(3)) to generate inositol (1,3,4,5) tetrakisphosphate. This molecule can function as a second messenger, but its roles are not completely understood. The A isoform of inositol (1,4,5) trisphosphate 3-kinase localises to filamentous actin within dendritic spines in the hippocampus and is implicated in the regulation of spine morphology and long term potentiation, however the mechanisms through which it signals in neuronal cells are not completely understood. We have used NGF driven neurite outgrowth from PC12 cells as a platform to examine the impact of signaling via inositol (1,4,5) trisphosphate 3-kinase activity in a neuronal cell. We have found that the catalytic activity of the enzyme opposes neurite outgrowth, whilst pharmacological inhibition of inositol (1,4,5) trisphosphate 3-kinase leads to a significant increase in neurite outgrowth, and we show that the reduction in neurite outgrowth in response to inositol (1,4,5) trisphosphate 3-kinase activity correlates with reduced ERK activity as determined by western blotting using phosphorylation-specific antibodies. Our findings suggest a novel neuronal signaling pathway linking metabolism of IP(3) to signaling via ERK.     

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.     

拟南芥中myo-肌醇-多磷酸合成与代谢及其信号

在酵母、真菌、动物和植物等真核生物中, 以myo-肌醇为基石通过不同位点的磷酸化形成各种myo-肌醇-多磷酸及其衍生物。过去10年的研究发现这些肌醇多磷酸参与了膜脂定向转运、蛋白结构稳定、离子通道调控、RNA转运以及DNA修复和染色质重塑等细胞生物学的基本进程。近些年在模式植物拟南芥(Arabidopsis thaliana)的研究中, 许多调控植物生长发育和环境胁迫应答的重要基因被发现, 并证实这些基因参与myo-肌醇-多磷酸的合成与代谢。该文概述了拟南芥中myo-肌醇-多磷酸合成与代谢的基因调控机理, 综述了不同肌醇多磷酸作为信号分子的功能, 提出肌醇多磷酸如同一类信息代码传递着植物细胞有序进程的基本指令。     

New frontiers of inositide-specific phospholipase C in nuclear signalling

Strong evidence has been obtained during the last 16 years suggesting that phosphoinositides, which are involved in the regulation of a large variety of cellular processes in the cytoplasm and in the plasma membrane, are present within the nucleus. A number of advances has resulted in the discovery that nuclear phosphoinositides and their metabolizing enzymes are deeply involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Even though nuclear inositol lipids generate second messengers such as diacyglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence functions such as pre-mRNA splicing and chromatin structure. This review aims at highlighting the most significant and up-dated findings about inositol lipid metabolism in the nucleus.     

Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases

Despite the high deposition of inositol hexakisphosphate (IP(6)), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP(6) synthesis occurs through the sequential phosphorylation of I(1,4,5)P(3) by two gene products, Ipk2 and Ipk1, a IP(3)/IP(4) dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2alpha and AtIpk2beta that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2alpha and AtIpk2beta are 70% identical to each other and 12-18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2alpha and AtIpk2beta have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P(3) to generate I(1,3,4,5,6)P(5) predominantly via an I(1,4,5,6)P(4) intermediate. While I(1,3,4,5)P(4) is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP(3) reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P(4) and I(1,2,3,4,6)P(5), which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP(6). Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP(4) and IP(5) production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class.     

Chronic Lithium-Induced Down-Regulation of MARCKS in Immortalized Hippocampal Cells: Potentiation by Muscarinic Receptor Activation

Abstract: Previous studies in our laboratory have demonstrated that exposure of rats to chronic lithium results in a significant reduction in the hippocampus of levels of the protein kinase C (PKC) phosphoprotein substrate MARCKS (myristoylated alanine-rich C kinase substrate), which persists after withdrawal and is not observed following acute administration. In an immortalized hippocampal cell line (HN33), we have determined that phorbol esters rapidly down-regulate PKC activity and lead to a subsequent PKC-dependent reduction in content of MARCKS protein. We now report that chronic exposure of HN33 cells to LiCl (1–10 m M ) produces a dose- and time-dependent down-regulation of MARCKS protein. The lithium-induced reduction in MARCKS is dependent on the concentration of inositol present in the medium and is reversed and prevented in the presence of elevated inositol concentrations. When HN33 cells were exposed to lithium at clinically relevant concentrations (1 m M ) under limiting inositol conditions, activation of muscarinic receptor-coupled phosphoinositide signaling significantly potentiated the lithium-induced down-regulation of MARCKS protein. It has been suggested that a major action of lithium in the brain is linked to its inositol monophosphatase inhibitory activity in receptor-mediated signaling through the inositol trisphosphate/diacylglycerol pathway, resulting in a relative inositol depletion. Our data provide evidence that this initial action of lithium may translate into a PKC-dependent long-term down-regulation of MARCKS protein expression in the hippocampus.     

3H]myoinositol incorporation into phospholipids in liver microsomes from humans with and without type II diabetes. The lack of synthesis of glycosylphosphatidylinositol, precursor of the insulin mediator inositol phosphate glycan

A class of inositol phosphate-containing oligosaccharides (IPG) derived from a membrane glycan-phosphatidylinositol precursor (GPI) has been identified as a possible mediator of insulin action. Saltiel's laboratory has recently communicated an in vitro assay for the synthesis of GPI in rat liver microsomes. Herein we have established this method in rat and human liver microsomes, it being our end point to evaluate if the pool of GPI was normal in diabetes and if failure of insulin to generate IPG from GPI could be involved in the mechanism of insulin resistance in Type II diabetes. However, subsequent to the detailed study of [3H]myoinositol incorporation into phospholipids in liver microsomes from our study subjects, we demonstrated by gas chromatography/mass spectrometry analysis that the material reported to be GPI is a mixture of lysophospholipids that does not contain hexosamine, ethanolamine, or amino acids.     

Simulations of inositol phosphate metabolism and its interaction with InsP(3)-mediated calcium release

Inositol phosphates function as second messengers for a variety of extracellular signals. Ins(1,4,5)P(3) generated by phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate, triggers numerous cellular processes by regulating calcium release from internal stores. The Ins(1,4,5)P(3) signal is coupled to a complex metabolic cascade involving a series of phosphatases and kinases. These enzymes generate a range of inositol phosphate derivatives, many of which have signaling roles of their own. We have integrated published biochemical data to build a mass action model for InsP(3) metabolism. The model includes most inositol phosphates that are currently known to interact with each other. We have used this model to study the effects of a G-protein coupled receptor stimulus that activates phospholipase C on the inositol phosphates. We have also monitored how the metabolic cascade interacts with Ins(1,4,5)P(3)-mediated calcium release. We find temporal dynamics of most inositol phosphates to be strongly influenced by the elaborate networking. We also show that Ins(1,3,4,5)P(4) plays a key role in InsP(3) dynamics and allows for paired pulse facilitation of calcium release. Calcium oscillations produce oscillatory responses in parts of the metabolic network and are in turn temporally modulated by the metabolism of InsP(3).     

Mammalian phosphoinositide-specific phospholipase C isoenzymes

Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.     

Changes in cellular levels of inositol polyphosphates during apoptosis

Accumulations of higher inositol polyphosphates, diphosphoinositol polyphosphates or pyrophosphates, have been implicated to mediate cellular apoptosis. Whether cellular levels of lower inositol phosphates (lower than inositol hexakisphosphates) change during apoptosis is not known, although these inositol phosphates are known to play crucial roles in a number of cellular signaling processes including calcium mobilization. Therefore, in this study, we have examined changes in cellular levels of inositol phosphates following metabolic labeling of these compounds by [³H]myo-inositol and induction of apoptosis. The levels of inositol mono- and bis-phosphates were increased, whereas the levels of inositol tris- and tetrakis-phosphates decreased significantly with an increasing rate of apoptosis induced by etoposide in a dose-dependent manner. NaF treatment, which increased the rate of apoptosis in a time- and dose-dependent manner, also increased the levels of inositol mono- and bis-phosphates and drastically reduced the levels of inositol tris- and tetrakis-phosphates. Prior treatment with antimycin A, a strategy used to reverse the NaF-induced accumulations of higher InsPs, partially reduced the effects of NaF on apoptosis as well as the levels of lower InsPs. Taken together, our results suggest that cellular levels of lower InsPs are altered during apoptosis.     

Ca2+ dynamics in a population of smooth muscle cells: modeling the recruitment and synchronization

Many experimental studies have shown that arterial smooth muscle cells respond with cytosolic calcium rises to vasoconstrictor stimulation. A low vasoconstrictor concentration gives rise to asynchronous spikes in the calcium concentration in a few cells (asynchronous flashing). With a greater vasoconstrictor concentration, the number of smooth muscle cells responding in this way increases (recruitment) and calcium oscillations may appear. These oscillations may eventually synchronize and generate arterial contraction and vasomotion. We show that these phenomena of recruitment and synchronization naturally emerge from a model of a population of smooth muscle cells coupled through their gap junctions. The effects of electrical, calcium, and inositol 1,4,5-trisphosphate coupling are studied. A weak calcium coupling is crucial to obtain a synchronization of calcium oscillations and the minimal required calcium permeability is deduced. Moreover, we note that an electrical coupling can generate oscillations, but also has a desynchronizing effect. Inositol 1,4,5-trisphosphate diffusion does not play an important role to achieve synchronization. Our model is validated by published in vitro experiments obtained on rat mesenteric arterial segments.     

Inositol phospholipid turnover in platelets of schizophrenic patients.

Abnormalities in blood cell membrane phospholipid composition and metabolism from schizophrenic patients have been reported by many groups of investigators. Among membrane phospholipids, inositol phospholipids are of special importance as they are involved in transduction system that generates second messengers such as inositol trisphosphate and diacylglycerol. Our studies on platelet inositol phospholipid turnover suggest a significant increase in platelet phosphatidylinositol 4,5-bisphosphate levels, an increased production of inositol trisphosphate in neuroleptic-treated and neuroleptic-free schizophrenic patients platelets and a reduced calcium release by thrombin in neuroleptic-treated schizophrenic patients platelets. The enhanced production of inositol trisphosphate may be due to an increase in its precursor phosphatidylinositol 4,5-bisphosphate with an associated desensitisation of the intracellular inositol trisphosphate receptor by neuroleptics, which may explain the diminished calcium response to thrombin in schizophrenic patients platelets.