Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum

Abstract: The effects of lithium on muscarinic cholinoceptor-stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [³H]inositol or [³H]cytidine prelabelling and inositol 1,4,5-trisphosphate [lns(1,4,5)P₃] and inositol 1,3,4,5-tetrakisphosphate [lns(1,3,4,5)P₄] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P₃ and lns(1,3,4,5)P₄ mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30-min incubation period. Carbachol-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations were inhibited by lithium in all brain regions studied in a time-and concentration-dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC₅₀ values for inhibition of agonist-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [³H]CMP-phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo-inositol to reverse these effects, as well as lithium-suppressed lns(1,4,5)P₃ accumulation, implicates myo-inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [³H]inositol monophosphate and [³H]CMP-phosphatidate levels and inhibitions of lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations. However, the inability of striatal slices to sustain carbachol-stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo-inositol may indicate differences in phosphoinositide signalling in this brain region.     

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.     

Histamine-Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis

Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [³H]Inositol 1,4,5-trisphosphate [[³H]Ins(1,4,5)P₃] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of ～140% of basal values. [³H]Inositol 1,4-bisphosphate [[³H]Ins(1,4)P₂] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [³H]inositol 1,3,4-trisphosphate [[³H]-Ins(1,3,4)P₃], [³H]inositol 1,3-bisphosphate, and [³H]-inositol 4-monophosphate ([³H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca²⁺, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [³H]Ins(1,4,5)P₃ was rapidly metabolized (t_1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t_1/2 values of 113, 133, 104, and 66 s for [³H]Ins(1,3,4)P₃, [³H]Ins(1,4)P₂, an unresolved mixture of [³H]inositol 1- and 3-monophosphate ([³H]Ins1/3P), and [³H]Ins4P, respectively. The calculated turnover rate of [³H]Ins(1,4,5)P₃ was sufficient to account for the turnover of the combination of both [³H]Ins(1,4)P₂ and [³H]Ins(1,3,4)P₃ but not that of [³H]Ins1/3P or [³H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca²⁺-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate.     

Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.     

Agonist-Stimulated Inositol Polyphosphate Formation in Cerebellum

The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second-messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], eight-fold; Ins(1,3,4,5)P4, 6.4-fold; Ins(1,4,5)P3, 75%; inositol 1,4-bisphosphate [Ins(1,4)P2], 0%; inositol 3,4-bisphosphate, 100%; inositol 1-phosphate/inositol 3-phosphate, 30%; and inositol 4-phosphate, 40%. [3H]Inositol 1,3-bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin-stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(1,4,5)P3 is metabolized primarily by Ins(1,4,5)P3-3'kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 microM), [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 levels were increased, even at 1 microM serotonin. Ketanserin (7 microM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 microM) also increased the levels of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3, but the profile of these increases was different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

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

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

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.     

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.     

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.     

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

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

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

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-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells

Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.     

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.     

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.     

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.     

Inositol 1,4,5-trisphosphate 3-kinase activity in frog skeletal muscle

Frog skeletal muscle contains a kinase activity that phosphorylates inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. The inositol 1,4,5-trisphosphate 3-kinase activity was mainly recovered in the soluble fraction, where it presented a marked dependency on free calcium concentration in the physiological range in the presence of endogenous calmodulin. At pCa 5, where the activity was highest, the soluble 3-kinase activity displayed a K_m for inositol 1,4,5-trisphosphate of 1.6 μM and a V_max value of 25.1 pmol mg⁻¹ min⁻¹. The removal rates of inositol 1,4,5-trisphosphate by 3-kinase and 5-phosphatase activities of the total homogenate under physiological ionic conditions were very similar, suggesting that both routes are equally important in metabolizing inositol 1,4,5-trisphosphate in frog skeletal muscle.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.