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.     

Degradation of inositol 1,3,4,5-tetrakisphosphates by porcine brain cytosol yields inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate

Inositol 1,3,4,5-tetrakisphosphates (Ins(1,3,4,5)P4), 32P-labelled in positions 4 and 5 were prepared enzymatically, using [4-32P]-phosphatidylinositol 4-phosphate (PtdInsP) and [5-32P]phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates, respectively. Degradation studies of Ins(1,3,4,5)P4, using an enriched phosphatase preparation from porcine brain cytosol, led to the formation of two inositol trisphosphate isomers which were identified as inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). This novel degradation pathway of Ins(1,3,4,5)P4 to Ins(1,4,5)P3 provides an additional source for the generation of Ins(1,4,5)P3, involving a 3-phosphatase.     

A salt-activated inositol 1,3,4,5-tetrakisphosphate 3-phosphatase at the inner surface of the human erythrocyte membrane.

The localization of the human erythrocyte membrane Ins(1,3,4,5)P4 3-phosphatase was investigated by saponin permeabilization of resealed 'isoionic' erythrocyte ghosts. This enzyme is active at the inner face of the plasma membrane, at the same site as a specific 5-phosphatase that degrades both Ins (1,4,5)P3 and Ins(1,3,4,5)P4. In the presence of EDTA, Ins(1,4,5)P3 was the only product of Ins(1,3,4,5)P4 metabolism. However, when Mg2+ was present both the 5-phosphatase and the 3-phosphatase attacked Ins (1,3,4,5)P4, directly forming Ins(1,3,4)P3 and Ins(1,4,5)P3;some Ins(1,4)P2 was also formed as a product of 5-phosphatase attack on the liberated Ins(1,4,5)P3. The Ins(1,3,4,5)P4 3-phosphatase was potently activated by KCl, thus making the route of metabolism of Ins(1,3,4,5)P4 by erythrocyte ghosts strikingly sensitive to variations in ionic strength: at 'cytosolic' K+ and Mg2+ levels, 3-phosphatase activity slightly predominated over 5-phosphatase. Ins(1,3,4,5)P4 3-phosphatase was potently inhibited by Ins-(1,3,4,5,6)P5 and InsP6 at levels lower than those often observed within cells. This leaves open the question as to whether the cellular function of inositol polyphosphate 3-phosphatase is to participate in a physiological cycle that interconverts Ins(1,3,4,5)P4 and Ins(1,4,5)P3 or to metabolize other inositol polyphosphates in the cytosol compartment of cells.     

Purification and characterization of receptors for myoinositol trisphosphate and myoinositol tetrakis phosphate from Entamoeba histolytica

The microsomal fraction from the log phase of Entamoeba histolytica cells contains Ins(1,4,5)P3 and Ins(1,3,4,5)P4 binding activity. The binding proteins/receptors for both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were purified and found to be specific for each ligand. The molecular masses for native proteins for InsP3 and InsP4 are 138 kDa and 130 kDa respectively having subunits of 69 kDa and 64 kDa respectively. That these proteins are associated with Ca2+ release was confirmed by including these proteins separately in proteoliposomes and adding InsP3 and InsP4 in both the cases.     

Formation and metabolism of inositol 1,4,5-trisphosphate in human platelets.

1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.     

T-cell receptor-mediated metabolism of inositol polyphosphates in Jurkat T-lymphocytes. Identification of a D-myo-inositol 1,2,3,4,6-pentakisphosphate-2-phosphomonoesterase activity, a D-myo-inositol 1,3,4,5,6-pentakisphosphate-1/3-phosphatase activity and a D/L-myo-inositol 1,2,4,5,6-pentakisphosphate-1/3-kinase activity.

Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor/CD3 complex by an anti-CD3 antibody (OKT3) induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate (InsPn) isomers. Quantitative mass analysis by anion-exchange HPLC and a recently developed postcolumn dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591) revealed basal intracellular concentrations between less than 5 pmol/10(9) cells for Ins(1,3,4,5)P4 and 6380 +/- 355 pmol/10(9) cells for InsP6. Time course analysis of samples from stimulated Jurkat T-cells showed an increase of Ins(1,3,4,5)P4 to 1125 +/- 125 pmol/10(9) cells within 10 min and remained elevated over more than 30 min. Moreover, increases of the intracellular concentrations of Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture), Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5 and InsP6 were observed. In contrast, the concentration of Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 (determined as the enantiomeric mixture) decreased after stimulation. Using cytosolic extracts from Jurkat T-lymphocytes incubated with purified Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5, or Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 three enzyme activities were observed. Ins(1,3,4,5,6)P5 was dephosphorylated by a phosphatase removing a phosphate group at the 1 and/or 3 position resulting in the formation of Ins(1,4,5,6)P4 and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture). Ins(1,2,3,4,6)P5 was metabolized by a specific phosphatase that cleaved the phosphate group at the 2 position, thereby generating the product Ins(1,3,4,6)P4. On the other hand, Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 was phosphorylated by a 1/3-kinase activity to InsP6. Together novel receptor-mediated metabolic pathways of inositol polyphosphates were demonstrated in human T-lymphocytes, and corresponding enzyme activities for the inositol pentakisphosphate metabolism were found in cell lysates.     

Regulation of innate immunity by inositol 1,3,4,5-tetrakisphosphate

Many neutrophil functions are mediated by PtdIns(3,4,5)P3 that exerts its role by mediating protein translocation via binding to their PH-domains. Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) binds the same PH domain, competes for its binding to PtdIns(3,4,5)P3, and thus negatively regulates PtdIns(3,4,5)P3 signaling. In neutrophils, chemoattractant stimulation triggers rapid elevation in Ins(1,3,4,5)P4 level. Depletion of Ins(1,3,4,5)P4 by deleting InsP3KB, the major enzyme producing Ins(1,3,4,5)P4 in neutrophils, augments PtdIns(3,4,5)P3 downstream signals, leading to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. InsP3KB gene is also expressed in hematopoietic stem/progenitor cells. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population is expanded and the proliferation of GMP cells is accelerated. As results, neutrophil production in the bone marrow is enhanced and peripheral blood neutrophil count is elevated. Ins(1,3,4,5)P4 also plays a role in maintaining neutrophil survival. Depletion of Ins(1,3,4,5)P4 leads to accelerated neutrophil spontaneous death. Finally, InsP3KB and Ins(1,3,4,5)P4 are essential components in bacterial killing by neutrophils. Despite of the augmented neutrophil recruitment, the clearance of bacteria in the InsP3KB knockout mice is significantly impaired. Collectively, these findings establish InsP3KB and its product Ins(1,3,4,5)P4 as essential modulators of neutrophil function and innate immunity.     

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.     

Inositol tetrakisphosphate-induced sequestration of Ca2+ replenishes an intracellular pool sensitive to inositol trisphosphate

In a permeable neoplastic rat liver epithelial (261B) cell system, inositol 1,3,4,5-tetrakisphosphate--Ins(1,3,4,5)P4--induces sequestration of Ca2+ released by inositol 2,4,5-trisphosphate--Ins(2,4,5)P3; a non-metabolized inositol trisphosphate (InsP3) isomer--and Ca2+ added exogenously in the form of CaCl2. Studies were performed to identify the Ca2+ pool filled after Ins(1,3,4,5)P4 treatment. Both Ins(2,4,5)P3 and inositol 1,4,5-trisphosphate--Ins(1,4,5)P3--dose-dependently release Ca2+ from permeable 261B cells--Ins(1,4,5)P3 having a threefold greater potency--but differ in that Ca2+ released by Ins(1,4,5)P3 is readily sequestered, while the Ca2+ released by Ins(2,4,5)P3 is not. Maximal release of Ca2+ by 6 microM Ins(2,4,5)P3 blocked the action of Ins(1,4,5)P3, demonstrating that these two isomers influence the same intracellular Ca2+ pool through a shared membrane receptor. Addition of 2 microM Ins(2,4,5)P3 to discharge partially the Ca2+ pool reduced the amount of Ca2+ released by a maximal dose of Ins(1,4,5)P3 (2 microM). Ins(1,3,4,5)P4 combined with Ins(2,4,5)P3 produced a Ca2+ release and sequestration response, which replenished the InsP3-sensitive pool as indicated by a recovery of full Ca2+ release by 2 microM Ins(1,4,5)P3. Induction of Ca2+ sequestration by Ins(1,3,4,5)P4 occurred dose-dependently, with a half-maximal response elicited at a dose of 0.9 microM. Further studies of the effect of Ins(1,3,4,5)P4 apart from the influence of Ins(2,4,5)P3 using a model in which the Ca2+ levels are raised by an exogenous addition of CaCl2 showed that Ins(1,4,5)P3 released twice the amount of Ca2+ from the storage pool following Ins(1,3,4,5)P4-induced Ca2+ sequestration. These results demonstrate that the Ca2+ uptake induced by Ins(1,3,4,5)P4 preferentially replenishes the intracellular Ca2+ storage sites regulated by Ins(1,4,5)P3 and Ins(2,4,5)P3.     

Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate.

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Inositol phosphates: proliferation, metabolism and function

After the initial discovery of receptor-linked generation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) it was generally assumed that Ins(1,4,5)P3 and its proposed breakdown products inositol(1,4)bisphosphate (Ins(1,4)P2) and Ins1P, along with cyclic inositol monophosphate, were the only inositol phosphates found in significant amounts in animal cells. Since then, three levels of complexity have been introduced. Firstly, Ins(1,4,5)P3 can be phosphorylated to Ins(1,3,4,5)P4, and the subsequent metabolism of these two compounds has been found to be intricate and probably different between various tissues. The functions of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are almost certainly to regulate cytosolic Ca2+ concentrations, but the reasons for the labyrinth of the metabolic pathways after their deactivation by a specific 5-phosphatase remain obscure. Secondly, inositol pentakis- and hexakisphosphates have been found in many animal cells other than avian erythrocytes. It has been shown that their synthesis pathway is entirely separate from the inositol phosphates discussed above, both in terms of many of the isomers involved and probably in the subcellular localization; some possible functions of InsP5 and InsP6 are discussed here. Thirdly, cyclic inositol polyphosphates have been reported in stimulated tissues; the evidence for their occurrence in vivo and their possible physiological significance are also discussed.     

Inositol polyphosphates are not increased by overexpression of Ins(1,4,5)P3 3-kinase but show cell-cycle dependent changes in growth factor-stimulated fibroblasts.

NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.     

Distinct binding sites for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in bovine parathyroid glands

We utilized high specific activity, [32P]-labelled ligands to measure the binding of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 to membranes prepared from bovine parathyroid glands. [32P]Ins(1,3,4,5)P4 bound rapidly and reversibly to parathyroid membranes, and the binding data could be fitted by the interaction of the ligand with two sites, one with Kd = 6.8 x 10(-9) M and Bmax = 26 fmol/mg protein and a second, lower affinity site, with Kd = 4.1 x 10(-7) M and Bmax = 400 fmol/mg protein. InsP5 was 10-20 fold less potent than InsP4, and Ins(1,3,4)P3 and Ins(1,4,5)P3 were nearly 1000-fold less potent in displacing [32P]Ins(1,3,4,5)P4. [32P]Ins(1,4,5)P3, on the other hand, bound to a single class of sites with Kd = 7.6 x 10(-9) M and Bmax = 34 fmol/mg. While the binding of [32P]Ins(1,4,5)P3 increased markedly on raising pH from 5 to 8, the binding of [32P]Ins(1,3,4,5)P4 decreased by 75% over this range of pH. Thus, [32P]-labelled Ins(1,3,4,5)P4 and Ins(1,4,5)P3 may be used to identify distinct binding sites which may represent physiologically relevant intracellular receptors for InsP3 and InsP4 in parathyroid cells.     

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Antiproliferative plant and synthetic polyphenolics are specific inhibitors of vertebrate inositol-1,4,5-trisphosphate 3-kinases and inositol polyphosphate multikinase

Inositol-1,4,5-trisphosphate 3-kinases (IP3K) A, B, and C as well as inositol polyphosphate multikinase (IPMK) catalyze the first step in the formation of the higher phosphorylated inositols InsP5 and InsP6 by metabolizing Ins(1,4,5)P3 to Ins(1,3,4,5)P4. In order to clarify the special role of these InsP3 phosphorylating enzymes and of subsequent anabolic inositol phosphate reactions, a search was conducted for potent enzyme inhibitors starting with a fully active IP3K-A catalytic domain. Seven polyphenolic compounds could be identified as potent inhibitors with IC50 < 200 nM (IC50 given): ellagic acid (36 nM), gossypol (58 nM), (-)-epicatechin-3-gallate (94 nM), (-)-epigallocatechin-3-gallate (EGCG, 120 nM), aurintricarboxylic acid (ATA, 150 nM), hypericin (170 nM), and quercetin (180 nM). All inhibitors displayed a mixed-type inhibition with respect to ATP and a non-competitive inhibition with respect to Ins(1,4,5)P3. Examination of these inhibitors toward IP3K-A, -B, and -C and IPMK from mammals revealed that ATA potently inhibits all kinases while the other inhibitors do not markedly affect IPMK but differentially inhibit IP3K isoforms. We identified chlorogenic acid as a specific IPMK inhibitor whereas the flavonoids myricetin, 3',4',7,8-tetrahydroxyflavone and EGCG inhibit preferentially IP3K-A and IP3K-C. Mutagenesis studies revealed that both the calmodulin binding and the ATP [corrected] binding domain in IP3K are involved in inhibitor binding. Their absence in IPMK and the presence of a unique insertion in IPMK were found to be important for selectivity differences from IP3K. The fact that all identified IP3K and IPMK inhibitors have been reported as antiproliferative agents and that IP3Ks or IPMK often are the best binding targets deserves further investigation concerning their antitumor potential.     

Inositol 1,4,5-trisphosphate concentrations increase after adherence in the macrophage-like cell line J774.1.

Several properties of macrophages change when suspended cells become adherent. To determine the intracellular signals involved in these changes, concentrations of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] were monitored during adherence of J774.1 cells, a macrophage-like cell line. When cells grown in suspension were allowed to adhere to a glass surface, there was a transient increase in InsP3 that reached a peak between 100 and 120 s after plating. Inositol mono- and bis-phosphate concentrations were also elevated 100 and 120 s after plating. Analysis of isomer distribution showed significant 3-fold increases in Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] at 100 s after plating. These values were maintained at 120 s, with the additional appearance of a 4-fold increase in inositol 1,3,4-trisphosphate. The adherence-induced generation of Ins(1,4,5)P3 was decreased, and Ins(1,3,4,5)P4 formation was blocked, in Ca2+-free medium. However, doubling intracellular [Ca2+] by addition of the Ca2+ ionophore ionomycin (1 microM) did not increase Ins(1,4,5)P3 in suspended cells. Adherence of J774.1 cells to fibronectin-coated glass also induced an increase in InsP3.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.