Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.     

Regulation of inositol phosphate metabolism in chemoattractant-stimulated human polymorphonuclear leukocytes. Definition of distinct dephosphorylation pathways for IP3 isomers

The metabolism of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer was studied in myo-[3H]inositol labeled, chemoattractant-stimulated human polymorphonuclear neutrophils (PMNs), and in PMN lysates. It was determined that 1,4,5-IP3 is metabolized in vitro by two distinct pathways: 1) by sequential dephosphorylation to 1,4-IP2, 4-IP1, and inositol or 2) by ATP dependent conversion to 1,3,4,5-IP4, followed by dephosphorylation to form 1,3,4-IP3, 3,4-IP2, 3-IP1, and inositol. In PMNs stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), 1,4-IP2, 1,4,5-IP3, and IP4, were elevated by 5 s; whereas production of 1,3,4-IP3, 3,4-IP2, and IP1 occurred only after an initial lag (approximately 15 s). The predominant IP1 isomer formed in fMet-Leu-Phe-stimulated cells was 4-IP1. Production of 1,3,4-IP3 and 3,4-IP2 was markedly reduced (17 and 35% of control, respectively) in fMet-Leu-Phe-stimulated cells pretreated to prevent a rise in intracellular calcium ([Ca2+]i). PMNs were also stimulated with leukotriene B4 (LTB4) since this agent is a poor activator of the respiratory burst compared to fMet-Leu-Phe. Peak levels (5 s) of 1,4,5-IP3 were equivalent after stimulation with 0.1 microM fMet-Leu-Phe versus 0.1 microM LTB4 (320 +/- 38% versus 378 +/- 38% of control values, respectively; n = 5); however, at 30 s, 1,4,5-IP3 remained elevated only in fMet-Leu-Phe-stimulated cells. Similarly, elevation of [Ca2+]i was more prolonged in response to 0.1 microM fMet-Leu-Phe (greater than 3 min) versus LTB4 (1 min). Thus, signal transduction in PMNs may be modulated by both the duration of the initial 1,4,5-IP3 signal and by the metabolic pathway(s) utilized to convert this IP3 isomer to other, potentially active inositol phosphate products.     

Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate inhibit inositol-1,3,4,5-tetrakisphosphate 3-phosphatase in rat parotid glands

In assays containing a physiological concentration of inositol 1,3,4,5-tetrakisphosphate (1 microM), this isomer was attacked by both 3- and 5-phosphatases present in rat parotid homogenates and 100,000 X g supernatant and particulate fractions. As the concentration of cytosolic protein in the assay was decreased, the specific activity of the soluble 3-phosphatase increased significantly. In contrast, the specific activity of particulate 3-phosphatase was independent of protein concentration. At the lowest protein concentrations tested, the sum of soluble and particulate 3-phosphatase specific activities was 2.5-fold greater than that of the parent homogenate. These observations indicate that parotid cytosol contains a hitherto undescribed endogenous mechanism for inhibiting 3-phosphatase. The effects upon 3- and 5-phosphatase of a number of inositol polyphosphates were studied. Both activities were inhibited by inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate (IC50 approximately 50 microM). Inositol 3,4,5,6-tetrakisphosphate was a more potent inhibitor of 3-phosphatase (IC50 about 10 microM) and did not affect 5-phosphatase. Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate were very potent inhibitors of 3-phosphatase (IC50 values of 1 and 0.5 microM, respectively); these polyphosphates did not affect 5-phosphatase activity at concentrations of up to 10 microM. Inositol 1,3,4,5,6-pentakisphosphate was a competitive inhibitor of the 3-phosphatase, whereas inositol hexakisphosphate was a mixed inhibitor. These data lead to the proposal that the inositol 1,3,4,5-tetrakisphosphate 3-phosphatase is unlikely to be an important enzyme activity in vivo.     

Transient accumulation of inositol (1,3,4,5)-tetrakisphosphate in response to alpha 1-adrenergic stimulation in adult cardiac myocytes

To investigate the response to catecholamine stimulation of adult cardiac myocytes and the metabolism of inositol (1,4,5)-trisphosphate (1,4,5-IP3) and inositol (1,3,4,5)-tetrakisphosphate (IP4), we have employed a procedure developed in our laboratory to directly measure the mass of inositol phosphates after separation of individual isomers of inositol phosphates by high performance liquid chromatography. Control, unstimulated myocytes, contained low levels of inositol (1,4)-bisphosphate (1,4-IP2), inositol (1,3)-bisphosphate (1,3-IP2), inositol (3,4)-bisphosphate (3,4-IP2), inositol (1,3,4)-trisphosphate (1,3,4-IP3), 1,4,5-IP3 and IP4. Stimulation with norepinephrine for 30 seconds produced peak 1,4,5-IP3 and IP4 levels which rapidly returned to basal values by 60 seconds of norepinephrine stimulation. 1,4-IP2, 1,3-IP2 and 1,3,4-IP3 were increased markedly but only after stimulation with norepinephrine for 60 seconds. These results indicate a rapid yet transient increase in 1,4,5-IP3 and IP4 in response to norepinephrine stimulation and are the first quantitative measurements of the isomers of inositol phosphates in cardiac tissue.     

The Ca2+ release activities of D-myo-inositol 1,4,5-trisphosphate analogs are quantized

Comparison is made between several synthetic stereo and positional isomers of D-myo-inositol 1,4,5-trisphosphate (D-myo-1,4,5-IP3) with respect to their ability to mobilize calcium from the internal stores of saponin-permeabilized rat basophilic leukemia cells. D- and L-myo-Inositol 1,4,5-trisphosphates, D- and L-myo-inositol 2,4,5-trisphosphates, D- and L-chiro-inositol 1,3,4-trisphosphates, D,L-trans-1,2-cyclohexane-diol bisphosphate, D,L-myo-inositol 4,5-bisphosphate, L-glycerol 1,2-bisphosphate, glycerol 1,3-bisphosphate and D,L-(1R,3R,4R)-1-phosphoryloxymethyl-trans-3,4-cyclohexanediol bisphosphate were tested. The analogs, each of which contains a vicinal trans-1,2-diol-bisphosphate motif, displayed potencies that were distributed over a 10(4)-fold range of concentration and fell into 4 distinct classes of activity.     

Stereospecific recognition of inositol 1,4,5-trisphosphate analogs by the phosphatase, kinase, and binding proteins

A series of DL-inositol 1,4,5-trisphosphate (IP3) analogs, with a bulky substitutent on the 2nd carbon of the inositol ring, has been synthesized. These compounds exert biological activities with only minor reduction in potency, in several assay systems (Hirata, M., Watanabe, Y., Ishimatsu, T., Ikebe, T., Kimura, Y., Yamaguchi, K., Ozaki, S., and Koga, T. (1989) J. Biol. Chem. 264, 20303-20308). Two analogs with aminocyclohexanecarbonyl (designated as analog 206) or aminobenzoyl group (analog 209) were separated into individual optical isomers and examined for stereospecificity in recognition by IP3-5-phosphatase, IP3-3-kinase and IP3 binding activity. IP3-5-phosphatase activity of erythrocyte ghosts was competitively inhibited by L-209 with a lower Ki value than D-IP3, but with a higher Ki value by L-206. D-Isomers of both analogs at 100 microM failed to inhibit the hydrolysis of D-[3H]IP3. On the other hand, D-isomers but not L-isomers of both analogs were as potent as D-IP3 in the recognition by IP3-3-kinase of rat brain cytosol and only the D-isomer of analog 206 could serve as substrate for the kinase. Also D-isomers of both analogs were equipotent to D-IP3 in displacing [3H]IP3 binding to rat cerebellum microsomes. These observations suggest that the IP3 analogs we synthesized are stereospecifically recognized by three IP3-recognizable proteins, but the phosphatase recognizes opposite isomers. Such being the case, the second hydroxyl group of D-IP3 may be involved in the recognition by IP3-5-phosphatase, but not by IP3-3-kinase and binding sites.     

Metabolism of inositol-1,4,5-trisphosphate in the taste organ of the channel catfish, Ictalurus punctatus.

1. The metabolism of inositol-1,4,5-trisphosphate was studied in the taste organ (barbel) of the channel catfish, Ictalurus punctatus. 2. Homogenates of epithelial barbel scrapings were incubated with [3H]-1,4,5-IP3, whose dephosphorylation or phosphorylation was assayed under first-order conditions by measuring the production of either [3H]-1,4-IP2 (representing the activity of IP3-5-phosphatase) or [3H]-1,3,4,5-IP4 (representing the activity of IP3-3-kinase). 3. Both enzymes were predominantly cytosolic, magnesium-dependent and maximally active at pH 6.4. For IP3-phosphatase, Km = 6 microM and Vmax = 10.5 nmol/min/mg. For IP3-kinase, Km = 0.23 microM and Vmax = 0.05 nmol/min/mg. 4. Neither enzyme was significantly affected by the presence of taste stimuli (amino acids), GTP gamma S, cAMP or phorbol esters. 5. In the presence of physiological levels of free calcium (0.05-12 microM) IP3-phosphatase was moderately activated whereas IP3-kinase was moderately inhibited. 6. IP3-phosphatase was moderately activated by Mn2+, unaffected by LiCl, and strongly inhibited by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, CuCl2, FeCl3 and ZnSO4 7. IP3-kinase was strongly activated by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, FeCl3 and LiCl and inhibited by ZnSO4 and Mn2+. 8. IP3-kinase was significantly activated in a calcium-dependent manner by exogenously-added phosphatidylcholine and sphingomyelin, and to a lesser extent by diacylglycerol. IP3-phosphatase was unaffected by exogenously-added lipids. 9. IP3-phosphatase may participate in taste transduction since calculations based on the first-order rate constant (6.9 sec-1) indicate that it is capable of dephosphorylating basal levels of IP3 with a half-life of 0.1 sec.     

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.     

Formation and metabolism of [3H]inositol phosphates in AR42J pancreatoma cells. Substance P-induced Ca2+ mobilization in the apparent absence of inositol 1,4,5-trisphosphate 3-kinase activity

After 2 days of incubation of AR42J pancreatoma cells with 400 microM [3H]inositol, the specific radioactivity of [3H]phosphatidylinositol 4,5-bisphosphate and the specific radioactivity of [3H]inositol were similar, indicating that isotopic equilibrium had been achieved. The inositol 1,4,5-trisphosphate (1,4,5-IP3) level in cells was estimated to be approximately 2 microM and was increased by substance P receptor activation to about 25 microM. HPLC analysis of [3H]inositol phosphates indicated that only 1,4,5-IP3, inositol 1,4-bisphosphate, and inositol 4-monophosphate were increased upon receptor activation. There was no increase in inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4), or in any of its metabolites. Incubation of [3H]1,4,5-IP3 with a cell homogenate did not result in the formation of [3H]1,3,4,5-IP4. Therefore, it appears that 1,4,5-IP3 3-kinase is either not present or not functional under these assay conditions. Substance P increased cytosolic calcium levels in fura-2-loaded cells from about 600 nM to 2.5 microM. This increase in Ca2+ was partially attenuated in the absence of extracellular calcium, indicating that in AR42J cells, substance P stimulation appears to activate calcium signaling through both Ca2+ entry and intracellular Ca2+ release. These modes of Ca2+ mobilization occur without an increase in 1,3,4,5-IP4 or any of its metabolites.     

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline-inducible expression vector pUHD10-3. Antisense-transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5-phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent-soluble membrane fraction of the cell, as compared with antisense-transfected cells in the presence of tetracycline. Unstimulated antisense-transfected cells showed a concomitant 2-fold increase in Ins(1,4,5)P3 and 4-fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense-transfected cells (170 +/- 25 nM) was increased 1.9-fold, compared with cells transfected with vector alone (90 +/- 25 nM). Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7-fold faster rate, reached confluence at higher density and demonstrated increased [3H]thymidine incorporation compared with cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in Ins(1,4,5)P3 5-phosphatase activity is associated with cellular transformation.     

Stereospecific recognition sites for [3H]inositol(1,4,5)-triphosphate in particulate preparations of rat cerebellum

A very high density of stereospecific binding sites for inositol-(1,4,5)P3 have been identified in rat cerebellar membranes using [3H]inositol-(1,4,5)P3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (KD 23 nM). Incubations were carried out at 4 degrees and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [3H]-(1,4,5)P3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P3 was the only other phosphate to show relatively high affinity (KD 1500 nM). HPLC-pure Ins(1,3,4)P3 and Ins(1,3,4,5)P4 were substantially weaker and Ins(1,4)P2, Ins-2-P1, Ins-1-P1, Ins(1,2)-cyclic P1 and inositol were totally inactive at concentrations less than 50 microM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P3 can initiate the release of bound Ca2+.     

Ins(1,4,5)P3 metabolism and the family of IP3-3Kinases

The release of Ca2+ from intracellular stores is triggered by the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3). The regulation of this process is critically important for cellular homeostasis. Ins(1,4,5)P3 is rapidly metabolised, either to inositol (1,4)-bisphosphate (Ins(1,4)P2) by inositol polyphosphate 5-phosphatases or to inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) by one of a family of inositol (1,4,5)P3 3-kinases (IP3-3Ks). Three isoforms of IP3-3K have now been identified in mammals; they have a conserved C-terminal catalytic domain, but divergent N-termini. This review discusses the metabolism of Ins(1,4,5)P3, compares the IP3-3K isoforms and addresses potential mechanisms by which their activity might be regulated.     

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 absence of expression of the three isoenzymes of the inositol 1,4,5-trisphosphate 3-kinase does not prevent the formation of inositol pentakisphosphate and hexakisphosphate in mouse embryonic fibroblasts

The activation of phospholipase C leads to the formation of both I(1,4,5)P(3) and diacylglycerol (DAG). I(1,4,5)P(3) can be metabolized by dephosphorylation catalyzed by Type I I(1,4,5)P(3) 5-phosphatase and by enzymatic phosphorylation to various inositol phosphates. This last step is catalyzed by three mammalian isoenzymes that specifically phosphorylate the 3-phosphate position of the inositol ring Itpka, Itpkb and Itpkc and a less specific enzyme Ipmk (or inositol multikinase) that phosphorylates I(1,4,5)P(3) at the D-3 and D-6 positions. This study was performed in mice cells in order to understand the synthetic pathway of IP5 and IP6 following PLC stimulation and possible link with Itpk activity. Mouse embryonic fibroblasts (MEF) were prepared from Itpkb(-/-) Itpkc(-/-) mice. Western blot and RT-PCR analysis show that the cells do not express Itpka. In contrast, they do express Ipmk. The cells still produce IP5 and IP6. Our data show that the absence of expression of the three isoenzymes of Itpk does not prevent the formation of IP5 and IP6, at least in mouse embryonic fibroblasts. The nuclear Ipmk plays therefore a critical role in the metabolism of I(1,4,5)P(3) and production of highly phosphorylated IP5 and IP6.     

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

myo-inositol 1,4,5-trisphosphorothioate is a potent competitive inhibitor of human erythrocyte 5-phosphatase

The effect of the myo-inositol 1,4,5-trisphosphate (IP3) analogue, myo-inositol 1,4,5-trisphosphorothioate (IPS3) on the dephosphorylation of D-5-[32P]IP3 by the 5-phosphatase from human erythrocyte membranes has been investigated. DL-IPS3 was found to act as a competitive inhibitor with a Ki of 6 microM, making it the most potent inhibitor currently available for this enzyme. L-IP3 inhibited the enzyme with a Ki of 124 microM and was more potent than D-2,3-diphosphoglycerate (Ki 978 microM).     

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.     

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-triphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immmunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.     

Ca2+ dependence of inositol 1,4,5-trisphosphate 3-kinase activity in the cytosol fraction of pig coronary artery

1. The activity of inositol 1,4,5-trisphosphate 3-kinase in subcellular fractions of smooth muscles of the pig coronary artery was examined. 2. Incubation of [3H]inositol 1,4,5-trisphosphate (IP3) with muscle homogenates produced more polar 3H-radioactivity (probably as inositol 1,3,4,5-tetrakisphosphate, IP4) than IP3, in the Mg2+- and ATP-dependent manner, thereby indicating the presence of IP3 3-kinase activity in homogenates of the muscle. 3. Most of the kinase activity was present in the cytosol fraction. The enzyme activity was reversibly activated by Ca2+ with a half-maximal effective concentration of 2.5 x 10(-7) M. 4. The calmodulin antagonists, W-7 and chlorpromazine inhibited the Ca2+-activated enzyme activity.