Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation.

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.     

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.     

Structures and metabolism of inositol tetrakisphosphates and inositol pentakisphosphate in bovine adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II stimulates rapid increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), followed by slower increases in two additional inositol tetrakisphosphate (InsP4) isomers. One of these InsP4 isomers was previously identified as Ins-1,3,4,6-P4 and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4 isomer, purified from cultured bovine adrenal cells labeled with [3H]inositol and stimulated by angiotensin II, revealed that the polyol produced by periodate oxidation, borohydrate reduction, and dephosphorylation was [3H]iditol. This finding is consistent with precursor structures of either Ins-1,4,5,6-P4 or Ins-3,4,5,6-P4 (= L-Ins-1,4,5,6-P4) for the third InsP4 isomer. The [3H]iditol was readily converted to [3H]sorbose by the stereospecific enzyme, L-iditol dehydrogenase, indicating that it originated from Ins-3,4,5,6-P4. Chicken erythrocytes labeled with [3H]inositol also contained high levels of Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4, as well as InsP5, but only small amounts of Ins-1,3,4,5-P4. Both [3H]Ins-1,3,4,6-P4 and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5 in permeabilized bovine glomerulosa cells. In addition, InsP5 itself was slowly dephosphorylated to Ins-1,4,5,6-P4, indicating that its structure is Ins-1,3,4,5,6-P5. These results demonstrate that the higher inositol phosphates are metabolically interrelated and are linked to the receptor-regulated InsP3 response by the conversion of Ins-1,3,4-P3 through Ins-1,3,4,6-P4 to Ins-1,3,4,5,6-P5. The source of Ins-3,4,5,6-P4, the other precursor of InsP5, is not yet known but its elevation in angiotensin II-stimulated glomerulosa cells suggests that its formation is also influenced by agonist-regulated processes.     

Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells

Absolute concentrations of inositol phosphate isomers (InsP(s] were quantified in the myeloid cell line HL-60 using the metal-dye detection technique. Stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) led to distinct alterations in at least seven different inositol phosphate species. Whereas the intracellular concentrations of the tetrakisphosphate isomers (InsP4(s] were found below the micromolar range, inositol 1,3,4,5,6-pentakis- and hexakisphosphate levels were about two orders of magnitude higher (36 and 54 +/- 2 microM (mean +/- S.D.), respectively). The three InsP4(s) showed distinct kinetic pattern upon receptor activation, the transient elevation of inositol 1,3,4,5-tetrakisphosphate being faster both in onset and in redecrease than inositol 1,3,4,6-tetrakisphosphate. Whereas the two latter isomers reached maximally 2.75 and 2.9 +/- 0.2 microM, respectively, 1 min after stimulation, inositol 3,4,5,6-tetrakisphosphate remained elevated (3.5 +/- 0.4 microM) up to 5 min after fMLP. Unexpected changes in highly phosphorylated InsP(s) were observed, notably a rise in inositol 1,3,4,5,6-pentakisphosphate and in inositol hexakisphosphate to 52 +/- 3 and 60 +/- 1 microM, respectively. In terms of mass, the increases in highly phosphorylated inositols are by far highest among all InsP(s). Combining radiotracer method with mass determination it was observed that the specific radioactivity of various InsP(s) was different and changed markedly upon fMLP stimulation, in spite of a prolonged labeling period leading to apparent isotopic steady state. The data presented demonstrate agonist-induced elevations of highly phosphorylated InsP(s) and suggest that inositol 1,4,5-trisphosphate, product of receptor-activated phospholipase C, is metabolized rather via phosphorylation than only by dephosphorylation pathways.     

Ca influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P₄-induced increase in DT cells depended upon extracellular Ca²⁺ and was enhanced by membrane hyperpolarization. Identical [Ca²⁺]_i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P₄ and Ins(1,3,4,5)P₄. These results suggest that Ins(3,4,5,6)P₄ may serve as a second messenger for continuous Ca²⁺ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.     

The regulation of the phosphorylation of inositol 1,3,4-trisphosphate in cell-free preparations and its relevance to the formation of inositol 1,3,4,6-tetrakisphosphate in agonist-stimulated rat parotid acinar cells

High performance liquid chromatography analysis of supernatants from acid-quenched [3H]inositol-labeled parotid acinar cells revealed an inositol pentakisphosphate and three inositol tetrakisphosphates. Two of the latter were identified as the 1,3,4,5 and 1,3,4,6 isomers, whereas the third was probably a mixture of unknown proportions of the 3,4,5,6/1,4,5,6 enantiomeric pair. Methacholine (100 microM) produced a 40-50-fold increase in the levels of inositol trisphosphate (mainly the 1,3,4 isomer) and inositol 1,3,4,5-tetrakisphosphate, but inositol 1,3,4,6-tetrakisphosphate only increased 5-fold. Levels of inositol 3,4,5,6/1,4,5,6-tetrakisphosphate and inositol pentakisphosphate were unaffected by agonist stimulation. Thus, in parotid cells, an agonist-induced increase in both inositol trisphosphate and inositol 1,3,4,6-tetrakisphosphate formation does not result in an increase in the rate of formation of inositol pentakisphosphate. Following the addition of 100 microM atropine to methacholine-stimulated parotid cells, the levels of [3H]inositol 1,3,4,5-tetrakisphosphate fell rapidly, returning to basal levels within 5 min. Inositol trisphosphate was metabolized more slowly and was still elevated 20-fold above basal 5 min after the addition of atropine. Inositol 1,3,4,6-tetrakisphosphate was metabolized much more slowly (t1/2 approximately 15 min). Inositol 1,3,4-trisphosphate metabolism was examined in parotid homogenates as well as in 100,000 x g cytosolic and particulate fractions. Inositol 1,3,4-trisphosphate was both dephosphorylated and phosphorylated. Two inositol tetrakisphosphate products were formed, namely the 1,3,4,6 and 1,3,4,5 isomers. Over 90% of both kinase and phosphatase activities were found in the cytosolic fractions. The ratio of activities of kinase to phosphatase decreased as the levels of inositol 1,3,4-trisphosphate substrate were increased from 1 nM to 10 microM. These data led to the conclusion that the kinetic parameters of the inositol 1,3,4-trisphosphate kinases and phosphatases are such that in stimulated cells, dephosphorylation of inositol 1,3,4-trisphosphate is greatly favored. Inositol 1,3,4-trisphosphate kinase activity was potently inhibited by inositol 3,4,5,6-tetrakisphosphate (IC50 = 0.1-0.2 microM), which leads us to propose that inositol 3,4,5,6-tetrakisphosphate is an endogenous inhibitor of the kinase.     

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.     

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.     

Product-precursor relationships amongst inositol polyphosphates. Incorporation of [32P]Pi into myo-inositol 1,3,4,6-tetrakisphosphate, myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 3,4,5,6-tetrakisphosphate and myo-inositol 1,3,4,5,6-pentakisphosphate in intact avian erythrocytes.

Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.     

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.     

Metabolism of inositol-1,3,4,6-tetrakisphosphate to inositol pentakisphosphate in adrenal glomerulosa cells

Angiotensin II stimulates rapid formation of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) in bovine adrenal glomerulosa cells. In addition to being rapidly metabolized to lower inositol phosphates, Ins-1,4,5-P3 is converted to Ins-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and Ins-1,3,4-P3 which is in turn phosphorylated to a further Ins-P4 isomer, namely Ins-1,3,4,6-P4. In bovine adrenocortical cytosol [3H]Ins-1,3,4,5-P4 and [3H]Ins-1,3,4-P3 were converted to Ins-1,3,4,6-P4 and inositol pentakisphosphate (Ins-P5) in a metabolic sequence suggesting that unlike Ins-1,3,4,5-P4, Ins-1,3,4,6-P4 is a direct precursor of Ins-P5. Consistent with this assumption, [3H]Ins-1,3,4,6-P4 was converted to Ins-P5 in electropermeabilized adrenal glomerulosa cells. These findings demonstrate that Ins-1,3,4,6-P4 is an intermediate link between InsP3 metabolism and the higher inositol phosphates detected in several tissues.     

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.     

Agonist-induced regulation of inositol tetrakisphosphate isomers and inositol pentakisphosphate in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II (AII) rapidly stimulates the formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and causes marked long-term changes in the levels of highly phosphorylated inositols. Glomerulosa cells prelabeled with [3H]inositol for 48 h and exposed to AII for 10 min showed prominent increases in inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and smaller increases in two additional tetrakisphosphates, Ins-1,3,4,6-P4 and another (Ins-3,4,5,6-P4) eluting in the position of Ins-3,4,5,6-P4 and its stereoisomer, Ins-1,4,5,6-P4, on anion exchange liquid chromatography. A concomitant decrease in InsP5 indicates that an increase in Ins-1,4,5,6-P4, the breakdown product of InsP5, is probably responsible for the initial rise in Ins-3,4,5,6-P4 during 10 min stimulation by AII. During prolonged stimulation by AII, Ins-1,3,4,5-P4 began to decline from its high, stimulated level after the first hour but the level of Ins-1,3,4,6-P4 remained elevated for several hours. There were also progressive increases in the levels of Ins-3,4,5,6-P4 and InsP5 during stimulation for up to 16 h with AII. Treatment of adrenal cells for 16 h with the cyclic AMP-mediated secretagogue, adrenocorticotropic hormone (ACTH), slightly increased basal levels of Ins-1,3,4,6-P4, Ins-3,4,5,6-P4, and InsP5, and enhanced the subsequent AII-stimulated increases in the two additional tetrakisphosphate isomers but not of inositol trisphosphates or Ins-1,3,4,5-P4. This change in the pattern of the higher inositol phosphate response to AII was manifested within 2 h after exposure to ACTH, and was mimicked by treatment with 8-bromo cyclic AMP or forskolin. Treatment with 50 microM cycloheximide abolished the ACTH-induced increases in inositol polyphosphate responses during AII stimulation, but had no effect on the responses of untreated cells to AII. The conversion of [3H]Ins-1,3,4-P3 to [3H]Ins-1,3,4,6-P4, a reaction linking the receptor-mediated InsP3 response to higher inositol phosphates, was enhanced in permeabilized cells that were pretreated for 16 h with either ACTH or AII. These results demonstrate that the reactions by which Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4 are formed and converted to InsP5 are influenced by agonist-stimulated regulatory processes that include both calcium-dependent and cyclic AMP-dependent mechanisms of target cell activation. They also reveal changes consistent with agonist-induced conversion of InsP5 to its dephosphorylated metabolite, Ins-1,4,5,6-P4, during short-term stimulation by AII.     

A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.     

Identification of Inositol 1,3,4-Trisphosphate 5-Kinase and Inositol 1,3,4,5-Tetrakisphosphate 6-Kinase in Immature Soybean Seeds

In extracts of immature soybean (Glycine max [L.] Merr.) seeds inositol tetrakisphosphate was formed from [³H]inositol 1,3,4-trisphosphate but not from [³H]inositol 1,4,5-trisphosphate. Inositol 1,3,4-trisphosphate kinase was purified to a specific activity of 3.55 min⁻¹ mg⁻¹ by polyethylenimine clarification and anion-exchange chromatography. The partially purified enzyme converted [³H]inositol 1,3,4-trisphosphate to inositol 1,3,4,5-tetrakisphosphate as the major product and inositol 1,3,4,6- and/or 1,2,3,4-tetrakisphosphate as the minor product. Subsequent experiments revealed a separate inositol 1,3,4,5-tetrakisphosphate 6-kinase activity, which could link these enzymes to inositol hexakisphosphate synthesis via the previously reported inositol 1,3,4,5,6-pentakisphosphate 2-kinase. The apparent K_m values for inositol 1,3,4-trisphosphate kinase were 200 ± 0 nm for inositol 1,3,4-trisphosphate and 171 ± 4 μm for ATP, and the reaction was not reversible. The kinetics were such that no activity could be detected using unlabeled inositol 1,3,4-trisphosphate and [γ-³²P]ATP, which suggested that other kinases may have been observed when less purified fractions were incubated with radiolabeled ATP. Inositol 1,3,4-trisphosphate kinase was nonspecifically inhibited more than 80% by various inositol polyphosphates at a concentration of 100 μm.     

Rapid formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands may both result indirectly from receptor-stimulated release of inositol 1,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate.

Addition of 1 mM-carbachol to [3H]inositol-labelled rat parotid slices stimulated rapid formation of [3H]inositol 1,3,4,5-tetrakisphosphate, the accumulation of which reached a peak 20 s after stimulation, and then declined rapidly towards a new steady state. The initial rate of formation of inositol 1,3,4,5-tetrakisphosphate was slower than that for inositol 1,4,5-trisphosphate. The radioactivity in [3H]inositol 1,3,4,5-tetrakisphosphate fell quickly in carbachol-stimulated and then atropine-blocked parotid slices, suggesting that it is rapidly metabolized during stimulation. Parotid homogenates rapidly dephosphorylated inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and, less rapidly, inositol 1,3,4-trisphosphate. Inositol 1,3,4,5-tetrakisphosphate was specifically hydrolysed to a compound with the chromatographic properties of inositol 1,3,4-trisphosphate. The only 3H-labelled phospholipids that we could detect in parotid slices labelled with [3H]inositol for 90 min were phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Parotid homogenates synthesized inositol tetrakisphosphate from inositol 1,4,5-trisphosphate. This activity was dependent on the presence of ATP. We suggest that, during carbachol stimulation of parotid slices, the key event in inositol lipid metabolism is the activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. The inositol 1,4,5-trisphosphate thus liberated is metabolized in two distinct ways; by direct hydrolysis of the 5-phosphate to form inositol 1,4-bisphosphate and by phosphorylation to form inositol 1,3,4,5-tetrakisphosphate and hence, by hydrolysis of this tetrakisphosphate, to form inositol 1,3,4-trisphosphate.     

The interconversion of inositol 1,3,4,5,6-pentakisphosphate and inositol tetrakisphosphates in AR4-2J cells.

Data from several cell types have indicated that activation of hormone receptors promotes the metabolism of inositol 1,3,4,5,6-pentakisphosphate (IP5) to inositol 3,4,5,6-tetrakisphosphate ((3,4,5,6)IP4). However, to date, metabolism of IP5 by cell-free preparations has resulted in the formation of only inositol 1,4,5,6-tetrakisphosphate ((1,4,5,6)IP4). Thus, the metabolic relationships of IP5 with various inositol tetrakisphosphate (IP4) isomers have been investigated in both intact cells and cell homogenates of the rat pancreatoma cell line, AR4-2J. The steady-state concentration of IP5 was estimated to be 65 microM, while the combined concentration of (3,4,5,6)IP4 and (1,4,5,6)IP4 was approximately 1.0 microM. AR4-2J cell homogenates converted (1,3,4,6)IP4, (3,4,5,6)IP4, and (1,4,5,6)IP4 to IP5. (1,4,5,6)IP4 previously has not been demonstrated to be a precursor of IP5. To alter steady-state levels of inositol phosphates that were maintained by phosphorylation-dephosphorylation cycles, intact cells were treated with 10 microM antimycin A which reduced ATP levels by > 90% within 10 min. Following 2 h of treatment with antimycin A, there was a 6-fold increase in both (3,4,5,6)IP4 and (1,4,5,6)IP4, presumably derived from IP5. Experiments with cell-free systems determined that IP5 was dephosphorylated to (1,4,5,6)IP4 by a predominantly particulate Mg(2+)-independent, Li(+)-insensitive IP5 3-phosphatase. However, in the presence of 5 mM MgATP, IP5 also was metabolized to (3,4,5,6)IP4. Therefore, our data demonstrate novel and complex relationships between IP5, (3,4,5,6)IP4, and (1,4,5,6)IP4.     

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.     

Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells.

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.     

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.