A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster

Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.     

Regulation of nuclear processes by inositol polyphosphates

Inositide signaling pathways represent a multifaceted ensemble of cellular switches capable of regulating a number of processes, for example, intracellular calcium release, membrane trafficking, chemotaxis, ion channel activity and several nuclear functions. Over 30 inositide messengers are found in eukaryotic cells that may be grouped into two classes: (1) inositol lipids, phosphatidylinositols or phosphoinositides (PIPs) and (2) water-soluble inositol polyphosphates (IPs). This review will focus on inositol polyphosphate kinases (IPK) and inositol pyrophosphate synthases (IPS) responsible for the cellular production of IP(4), IP(5) IP(6) and PP-IPs. Of interest, IPK and IPS proteins localize, in part, within the nucleus and their activities are necessary for proper regulation of gene expression, mRNA export, DNA repair and telomere maintenance. The breadth of nuclear processes regulated and the evolutionary conservation of the genes involved in their synthesis have sparked renewed interest in inositide messengers derived from sequential phosphorylation of inositol 1,4,5-trisphosphate.     

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.     

Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae

The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.     

The function of inositol high polyphosphate binding proteins

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5-trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5-tetrakisphosphate (IP₄), inositol 1,3,4,5,6-pentakisphosphate, or inositol hexakisphosphate] enables us to speculate on the physiological function of these compounds. In this article we focus on two major issues: (1) the roles of inositol high polyphosphates in vesicular trafficking, especially exocytosis, and (2) pleckstrin homology domaincontaining IP4 binding proteins involved in the Ras signaling pathway.     

Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes

[3H]Inositol hexakisphosphate (InsP6) binds with a heterogeneous distribution to frozen sections of unfixed rat brain and is displaced by unlabelled InsP6. The pattern of binding correlates with binding to neuronal cell bodies. [3H]InsP6 binding to cerebellar membranes has been further characterised, is reversible, and saturable, and exhibits high specificity for inositol polyphosphates. The IC50 for competition by unlabelled InsP6 is approximately 100nM, whereas inositol 1,3,4,5,6 pentakisphosphate (Ins(13456)P5), inositol 1,3,4,5 tetrakisphosphate (Ins(1345)P4), and inositol 1,4,5 trisphosphate (Ins(145)P3) bind with an affinity at least one order of magnitude lower. [3H]InsP6 binding is clearly distinct from previously characterised Ins(145)P3 (ref. 1, 2) and Ins(1345)P4 (ref. 3) binding, both in terms of pharmacology and brain distribution.     

Metabolism of inositol 1,4,5-trisphosphate in guinea-pig hepatocytes.

Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.     

Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): implications for the phytic acid biosynthetic pathway

Phytic acid, myo-inositol-hexakisphosphate (InsP(6)), is a storage form of phosphorus in plants. Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about its biosynthetic pathway in plants. Recent work has suggested two pathways. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of 1D-myo-inositol 3-phosphate (Ins(3)P). The second is a phospholipase C (PLC)-mediated pathway, in which inositol 1,4,5-tris-phosphate (Ins(1,4,5)P(3)) is sequentially phosphorylated to InsP(6). We identified 12 genes from rice (Oryza sativa L.) that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include 1D-myo-inositol 3-phosphate synthase (MIPS), inositol monophosphatase (IMP), inositol 1,4,5-tris-phosphate kinase/inositol polyphosphate kinase (IPK2), inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1), and inositol 1,3,4-triskisphosphate 5/6-kinase (ITP5/6K). The quantification of absolute amounts of mRNA by real-time RT-PCR revealed the unique expression patterns of these genes. Outstanding up-regulation of the four genes, a MIPS, an IPK1, and two ITP5/6Ks in embryos, suggested that they play a significant role in phytic acid biosynthesis and that the lipid-independent pathway was mainly active in developing seeds. On the other hand, the up-regulation of a MIPS, an IMP, an IPK2, and an ITP5/6K in anthers suggested that a PLC-mediated pathway was active in addition to a lipid-independent pathway in the anthers.     

Cross-linking membrane IgM induces production of inositol trisphosphate and inositol tetrakisphosphate in WEHI-231 B lymphoma cells

The addition of anti-IgM to the immature B lymphoma cell line WEHI-231 resulted in breakdown of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). These reactions have recently been demonstrated in mature resting B cells stimulated with anti-IgM, as well. In addition to Ins(1,4,5)P3, inositol tetrakisphosphate (InsP4) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) were rapidly generated in WEHI-231 cells upon stimulation of the antigen receptor with anti-IgM. These two inositol polyphosphates are probably generated from Ins(1,4,5)P3 by phosphorylation to yield InsP4 and removal of the 5-phosphate from InsP4 to yield Ins(1,3,4)P3. It is possible that these inositol polyphosphates play a second messenger role in mediating the biologic effects of antigen-receptor signaling. It had previously been shown that anti-IgM also causes an increase in cytoplasmic free calcium. Therefore, the relationship between Ca2+ elevation and phosphoinositide breakdown was investigated. Although elevation of cytoplasmic Ca2+ with ionophores can trigger phosphoinositide breakdown, this required levels of Ca2+ well beyond those normally seen in response to anti-IgM. Thus, the Ca2+ elevation seen in response to anti-IgM cannot be the event controlling phosphoinositide breakdown. WEHI-231 cells have been shown to have a calcium storage compartment that releases Ca2+ in the presence of Ins(1,4,5)P3; therefore, it is likely that anti-IgM stimulates phosphoinositide breakdown as a primary event and this leads to the elevation of cytoplasmic Ca2+.     

Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate

In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.     

Inositol 1,3,4,5-tetrakisphosphate and not phosphatidylinositol 3,4-bisphosphate is the probable precursor of inositol 1,3,4-trisphosphate in agonist-stimulated parotid gland.

When [3H]inositol-prelabelled rat parotid-gland slices were stimulated with carbachol, noradrenaline or Substance P, the major inositol trisphosphate produced with prolonged exposure to agonists was, in each case, inositol 1,3,4-trisphosphate. Much lower amounts of radioactivity were present in the inositol 1,4,5-trisphosphate fraction separated by anion-exchange h.p.l.c. Analysis of the inositol trisphosphate head group of phosphatidylinositol bisphosphate in [32P]Pi-labelled parotid glands showed the presence of phosphatidylinositol 4,5-bisphosphate, but no detectable phosphatidylinositol 3,4-bisphosphate. Carbachol-stimulated [3H]inositol-labelled parotid glands contained an inositol polyphosphate with the chromatographic properties and electrophoretic mobility of an inositol tetrakisphosphate, the probable structure of which was determined to be inositol 1,3,4,5-tetrakisphosphate. Since an enzyme in erythrocyte membranes is capable of degrading this tetrakisphosphate to inositol 1,3,4-trisphosphate, it is suggested to be the precursor of inositol 1,3,4-trisphosphate in parotid glands.     

Formation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate from inositol 1,3,4,5-tetrakisphosphate and their pathways of degradation in RBL-2H3 cells

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.     

Separation of multiple isomers of inositol phosphates formed in GH3 cells.

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.     

Chemoattractant and guanosine 5''-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.     

Effects of transformation with the v-src oncogene on inositol phosphate metabolism in rat-1 fibroblasts. D-myo-inositol 1,4,5,6-tetrakisphosphate is increased in v-src-transformed rat-1 fibroblasts and can be synthesized from D-myo-inositol 1,3,4-trisphosphate in cytosolic extracts

Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.     

Subsecond and second changes in inositol polyphosphates in GH4C1 cells induced by thyrotropin-releasing hormone.

It has been demonstrated previously that thyrotropin-releasing hormone (TRH) induces changes in inositol polyphosphates in the GH3 and GH4C1 strains of rat pituitary cells within 2.5-5.0 s. TRH also causes a rapid rise in cytosolic free calcium concentration ([Ca2+]i) in these cells which is due largely to redistribution of cellular calcium stores. Therefore, it has been concluded that TRH acts to release sequestered calcium in these cells via enhanced generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. If this conclusion were correct, TRH-enhanced accumulation of Ins(1,4,5)P3 should occur at least as rapidly as the increase in [Ca2+]i. We have shown previously that the rise in [Ca2+]i induced by TRH occurs within about 400 ms; thus, it was important to investigate the subsecond time-course of changes in inositol phosphates caused by TRH. Using a rapid mixing device, we have measured changes in inositol polyphosphates on a subsecond time scale in GH4C1 cells prelabelled with myo-[2-3H]inositol. Although TRH did alter inositol polyphosphate metabolism within 500 ms, the changes observed did not reveal a statistically significant increase in Ins(1,4,5)P3 within time intervals of less than 1000 ms. Thus, we have been unable to demonstrate that a TRH-induced rise in Ins(1,4,5)P3 precedes or occurs concomitantly with the rise in [Ca2+]i in GH4C1 cells. Although these results do not disprove the current view that Ins(1,4,5)P3 mediates the action of TRH on intracellular calcium redistribution, we conclude that caution should be exercised in this, and possibly other cell systems, in accepting the dogma that all of the rapid, agonist-induced redistributions of intracellular calcium are mediated by Ins(1,4,5)P3.     

Kinetics of inositol 1,4,5-trisphosphate and inositol cyclic 1:2,4,5-trisphosphate metabolism in intact rat parotid acinar cells. Relationship to calcium signalling

Stimulation of rat parotid acinar cells by the muscarinic cholinergic receptor agonist methacholine results in the formation of inositol 1,4,5-trisphosphate [1,4,5)IP3) and inositol cyclic 1:2,4,5-trisphosphate [c1:2,4,5)IP3) which, after 40 min, accumulate to a ratio of 1:0.57. The turnover rates of these inositol trisphosphates have been determined in cholinergically stimulated rat parotid cells by measuring the degradation of the 3H-labeled compounds following receptor blockade. (1,4,5)IP3 is rapidly metabolized, with a half-time of 7.6 s; (c1:2,4,5)IP3 declines much more slowly with a half-time of almost 10 min. Because the formation and metabolism of (c1:2,4,5)IP3 are so slow, (c1:2,4,5)IP3 gradually accumulates upon prolonged receptor activation. Inositol trisphosphate turnover was compared to the receptor-mediated changes in cytoplasmic Ca2+ concentration, as measured by the fluorescent Ca2+ indicator, fura-2. The Ca2+ signal decays upon termination of inositol phosphate formation and returns to base line within 30 s. Thus, while (c1:2,4,5)IP3 may have some yet unknown biological effects on Ca2+ homeostasis, its metabolism seems far too slow to be the primary regulator of cytosolic Ca2+ levels under long term stimulatory conditions. The rate at which the Ca2+ signal decays is, however, somewhat slowed after prolonged agonist stimulation. Furthermore, the capacity of the cells to mobilize intracellular Ca2+ in response to a second agonist stimulation is slightly delayed when the duration of the first stimulus is prolonged. The results suggest that the regulation of cytoplasmic Ca2+ levels may be more complicated than initially realized and could depend on the combined actions of more than one inositol polyphosphate.     

Inositol polyphosphate multikinase regulates inositol 1,4,5,6-tetrakisphosphate

The human inositol phosphate multikinase (IPMK, 5-kinase) has a preferred 5-kinase activity over 3-kinase and 6-kinase activities and a substrate preference for inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) over inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). We now report that the recombinant human protein can catalyze the conversion of inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P4) to Ins(1,3,4,5,6)P5 in vitro; the reaction product was identified by HPLC to be Ins(1,3,4,5,6)P5. The apparent Vmax was 42 nmol of Ins(1,3,4,5,6)P5 formed/min/mg protein, and the apparent Km was 222 nM using Ins(1,3,4,6)P4 as a substrate; the catalytic efficiency was similar to that for Ins(1,4,5)P3. Stable over-expression of the human protein in HEK-293 cells abrogates the in vivo elevation of Ins(1,4,5,6)P4 from the Salmonella dublin SopB protein. Hence, the human 5-kinase may also regulate the level of Ins(1,4,5,6)P4 and have an effect on chloride channel regulation.     

Formation of inositol polyphosphates in cultured human sweat duct cells in response to cholinergic stimulation

Inositol phosphate formation in response to cholinergic stimulation was studied in cultured human sweat duct cells, prelabelled with myo-[2-3H]inositol. Formation of inositol mono-, bis-, tris- and tetrakisphosphates was increased after 15 min stimulation by 30 microM carbachol. Formation of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate was significantly increased within 1 min at carbachol concentrations between 10 microM and 100 microM. No detectable increase in inositol 1,4,5-trisphosphate formation was observed at 15 s or 1 min, but an increase was observed after 15 min at a carbachol concentration of 30-100 microM. The data are consistent with an involvement of inositol polyphosphates in the biphasic response of ion transport, to cholinergic stimulation in these cells (see Pederson, P.S. (1986) 6th Professional Conference "Broken Arrow 1986". Genetic and Eptihelial Dysfunction in Cystic Fibrosis (Riordan, J.R. and Buchwalds, M., eds.), Alan Liss, New York and Pedersen, P.S. (1987) Med. Sci. Res. 15, 769-770) and suggest a different pattern of metabolism from exocrine acinar cells.