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PubMed1983

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.

Animals↗
PubMed1980

Bradykinin-stimulated release of arachidonate from phosphatidyl inositol in mouse fibrosarcoma cells.

We recently proposed a new pathway by which arachidonate is released from platelet phosphatidyl inositol after stimulation by either thrombin or calcium ionophore A23187. The initial step in arachidonate liberation involves hydrolysis of phosphatidyl inositol to form 1,2-diacyglycerol which is subsequently hydrolyzed by a diacyglycerol lipase to liberate arachidonate for the prostaglandin and lipoxygenase pathways. Whether this pathway is unique to platelets or accounts for arachidonate release from other tissues has not been previously studied. Thus we have now investigated arachidonate metabolism in mouse fibrosarcoma cells (HSDM1C1) grown in culture. These cells contain approximately 7.6% of their total phospholipid as phosphatidyl inositol in the resting state (range 6.5-8.3%). When bradykinin (12 microM) is added to the fibrosarcoma cells, there is a rapid depletion of membrane phosphatidyl inositol reaching 62 +/- 8% S.D. of baseline values by 15 seconds, falling to 36 +/- 6% by 15 minutes. The drop in membrane phosphatidyl inositol is accompanied by release of arachidonate and PGE2 into the culture medium. The time course of phosphatidyl inositol breakdown and PGE2 formation supports the idea that phosphatidyl inositol breakdown provides he arachidonate for prostaglandin synthesis in mouse fibrosarcoma cells. Crude extracts of HSDM1C1 cells contained sufficient phosphatidyl inositol-specific phospholipase C activity and diacylglycerol lipase activity to account for arachidonate release in these cells.

Arachidonic Acids↗
PubMed1988

Inositol lipids: receptor-stimulated hydrolysis and cellular lipid pools.

Our current knowledge of the process by which receptors stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has its origin in the discovery by Hokin & Hokin (J. biol. Chem. 263, 967 (1953] that some pancreatic secretagogues not only elicit exocrine secretion but also stimulate the metabolism of membrane phospholipids. Despite the recent elucidation of many aspects of this widespread signalling system, there is still little information on the control of the supply of its substrate, PtdIns(4,5)P2. In particular, some studies have suggested that inositol-lipid-mediated signalling involves much or all of the inositol lipid complement of the stimulated cells, whereas other observations have equally clearly implicated the receptor-activated hydrolysis of an inositol phospholipid pool that comprises only a small fraction of the total cellular complement of these lipids. These studies, which have largely employed radiochemical analyses using single isotopes, are briefly reviewed. In addition, we report the first information obtained by a new procedure for analysing the metabolic characteristics of the inositol lipids that are broken down during stimulation. This technique employs cells that are doubly labelled in the inositol moiety of their lipids (to isotopic equilibrium with 14C and only briefly with 3H) to search for functional metabolic heterogeneity among the inositol lipids of stimulated cells. Using this method, we have found that the inositol phosphates liberated in stimulated cells during brief stimulation of V1a-vasopressin receptors or prostaglandin F2 alpha receptors come from phospholipid that has a turnover rate typical of the bulk of the cellular inositol lipids.

Animals↗
PubMed2004

A role of Arabidopsis inositol polyphosphate kinase, AtIPK2alpha, in pollen germination and root growth.

Inositol polyphosphates, such as inositol trisphosphate, are pivotal intracellular signaling molecules in eukaryotic cells. In higher plants the mechanism for the regulation of the type and the level of these signaling molecules is poorly understood. In this study we investigate the physiological function of an Arabidopsis (Arabidopsis thaliana) gene encoding inositol polyphosphate kinase (AtIPK2alpha), which phosphorylates inositol 1,4,5-trisphosphate successively at the D-6 and D-3 positions, and inositol 1,3,4,5-tetrakisphosphate at D-6, resulting in the generation of inositol 1,3,4,5,6-pentakisphosphate. Semiquantitative reverse transcription-PCR and promoter-beta-glucuronidase reporter gene analyses showed that AtIPK2alpha is expressed in various tissues, including roots and root hairs, stem, leaf, pollen grains, pollen tubes, the flower stigma, and siliques. Transgenic Arabidopsis plants expressing the AtIPK2alpha antisense gene under its own promoter were generated. Analysis of several independent transformants exhibiting strong reduction in AtIPK2alpha transcript levels showed that both pollen germination and pollen tube growth were enhanced in the antisense lines compared to wild-type plants, especially in the presence of nonoptimal low Ca(2+) concentrations in the culture medium. Furthermore, root growth and root hair development were also stimulated in the antisense lines, in the presence of elevated external Ca(2+) concentration or upon the addition of EGTA. In addition, seed germination and early seedling growth was stimulated in the antisense lines. These observations suggest a general and important role of AtIPK2alpha, and hence inositol polyphosphate metabolism, in the regulation of plant growth most likely through the regulation of calcium signaling, consistent with the well-known function of inositol trisphosphate in the mobilization of intracellular calcium stores.

Arabidopsis↗
PubMed1989

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells.

We have shown previously that inositol-1,4,5-trisphosphate (IP(3)) stimulates an efflux of (45)Ca(2+) from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP(3) might serve as a second messenger for the mobilization of intracellular Ca(2+) in higher plant cells. To determine whether or not IP(3) and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-(3)H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [(3)H]inositol metabolites coeluted with inositol bisphosphate (IP(2)) and IP(3) when separated by anion exchange chromatography. However, we could not detect IP(2) or IP(3) when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP(2) and IP(3), were present in these cells. Thus, [(3)H] inositol metabolites other than IP(2) and IP(3) had coeluted on the anion exchange columns. The data indicate that either IP(3) is rapidly metabolized or that it is not present at a detectable level in the carrot cells.

Journal Article↗
PubMed1981

31P NMR study of the kinetics of binding of myo-inositol hexakisphosphate to human hemoglobin. Observation of fast-exchange kinetics in high-affinity systems.

The association and dissociation kinetics of the complexes of myo-inositol hexakisphosphate (P6-inositol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high-affinity systems with binding constants varying between 10(5) M-1 and 2 X 10(9) M-1. 31P NMR spectra of P6-inositol were recorded in the presence of hemoglobin as a function of the P6-inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the NMR time scale. This observation cannot be reconciled with a single-step binding mechanism of P6-inositol to hemoglobin. Analysis of the spectra revealed the occurrence of additional binding of P6-inositol to both Hb and HbCO. This binding was also observed in pH-state experiments performed at low ionic strength. 31P NMR experiments carried out with hemoglobin of which the alpha-chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6-inositol. A model is proposed which accounts for the enhancement of exchange kinetics in these high-affinity systems. In this model a rapid migration is assumed for P6-inositol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three-site exchange problem were calculated.

Carboxyhemoglobin↗
PubMed1989

Inositol lipid metabolism in rat hippocampal formation slices: basal metabolism and effects of cholinergic agonists.

Incubation of rat hippocampal formation slices under steady-state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady-state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5-bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.

Animals↗
PubMed1990

P2-, but not P1-purinoceptors mediate formation of 1, 4, 5-inositol trisphosphate and its metabolites via a pertussis toxin-insensitive pathway in the rat renal cortex.

1. The adenosine receptor (P1-purinoceptor) agonists N6-cyclopentyladenosine and N-5'-ethyl-carboxamidoadenosine at concentrations up to 10 mumols 1(-1) affected neither basal, nor noradrenaline- and angiotensin II-stimulated formation of inositol-1-phosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate in slices of rat renal cortex. 2. In contrast, adenine nucleotides (P2-purinoceptor agonists) markedly stimulated inositol phosphate formation. The observed rank order of potency adenosine-5'-O-(2-thiodiphosphate) (EC50 39 mumols 1(-1] greater than adenosine-5'-O-(3-thiotriphosphate) (587) greater than or equal to 5'-adenylylimidodiphosphate (App(NH)p, 899) greater than adenylyl-(beta, gamma-methylene)-diphosphate (4,181) was consistent with the interaction of the compounds with the P2Y-subtype of P2-purinoceptors. AMP and the ADP analogue (alpha, beta-methylene)-adenosine-5'-diphosphate were ineffective. ATP and ADP (less than or equal to 10 mmol 1(-1] did not produce a consistent increase, owing to their hydrolytic degradation in the incubation medium. 3. Whereas the inositol phosphate response to App(NH)p was linear only up to 5 min incubation, the time-dependent stimulation of noradrenaline declined at a slower rate. Following pre-exposure of the renal cortical slices to App(NH)p, renewed addition of App(NH)p caused no further enhancement in the accumulation of inositol phosphates, whilst noradrenaline was still capable of eliciting a response. This suggests that the apparent loss of responsiveness to App(NH)p is not due to substrate depletion or enzymatic inactivation, but most likely attributable to homologous desensitization of the purinoceptor. 4. Pretreatment of the animals with pertussis toxin caused a substantial reduction of functional Gi-protein, as indicated by the lack of [32P]-NAD incorporation in a membrane preparation of the renal cortex. Nevertheless, the increase in inositol phosphate formation induced by noradrenaline, angiotensin II, and App(NH)p was not significantly impaired. 5. We conclude that P2 gamma-purinoceptors are present in the renal cortex; these receptors stimulate formation of inositol phosphates via a pertussis toxin-insensitive pathway and undergo homologous desensitization. On the other hand, our results suggest that renal A,-adenosine receptors do not use stimulation of phosphoinositide breakdown as a transmembrane signalling system.

Adenine Nucleotides↗
PubMed1990

Pharmacological characterization of tachykinin-stimulated inositol phospholipid hydrolysis in peripheral tissues.

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of rat parotid gland, hamster urinary bladder and guinea-pig ileum longitudinal muscle. 2. In the presence of lithium, substance P and other naturally-occurring and synthetic tachykinins induced large, dose-dependent increases in [3H]-inositol monophosphate accumulation. 3. In slices of rat parotid gland, [pGlu6,L-Pro9]SP(6-11) was considerably more potent in stimulating inositol phospholipid hydrolysis than [pGlu6,D-Pro9]SP(6-11). 4. In contrast, in slices of hamster urinary bladder, [pGlu6,D-Pro9]SP(6-11) exhibited greater potency in evoking inositol phospholipid breakdown than [pGlu6,L-Pro9]SP(6-11). 5. The differential selectivity of these C-terminal fragments of substance P suggests that they may be useful tools for distinguishing between NK1 and NK2 receptors. 6. L-659,837 and L-659,874 antagonized eledoisin-stimulated inositol phospholipid hydrolysis in slices of hamster urinary bladder. Neither compound significantly reduced substance-P evoked inositol phospholipid breakdown in slices of rat parotid gland, or senktide-induced inositol phospholipid hydrolysis in slices of guinea-pig ileum. 7. L-659,837 and L-659,874 had no effect on the atropine-sensitive, carbachol-stimulated inositol phospholipid hydrolysis in slices of rat parotid gland. 8. These data further support the notion that L-659,837 and L-659,874 are potent and selective NK2 receptor antagonists.

Animals↗
PubMed1994

Agonist-induced desensitization of histamine H1 receptor-mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells.

1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II.

Alkaloids↗
PubMed1990

Role of inositol-1-phosphatase inhibition in the mechanism of action of lithium.

Li inhibition of noradrenergic adenylate cyclase may be due to inhibition by Li of agonist-induced increases in GTP binding to G-protein. Such inhibition by Li of G-protein function could have effects on phosphatidyl-inositol-mediated second messenger systems as well as on cyclic AMP-mediated systems. However, Sherman, Berridge and others have proposed that Li affects phosphatidylinositol metabolism by inhibiting inositol-1-phosphatase. We recently have been able to measure inositol-1-phosphatase in human red blood cells. Preliminary data on patients treated with Li compared with controls suggests that the enzyme is indeed inhibited in vivo in patients undergoing Li treatment. However, a series of experiments in rats on addition of inositol to Li treatment did not find that inositol could reverse Li effects. Chronic oral high dose inositol does not reverse Li-induced polyuria (measured by polydipsia), Li-induced weight loss or Li-induced depression of exploratory behavior. These results suggest that Li inhibition of inositol-1-phosphatase indeed occurs in vivo. However, the physiological significance of inositol-1-phosphatase inhibition is not yet established.

Animals↗
PubMed1994

Occurrence and Role of Di-myo-Inositol-1,1'-Phosphate in Methanococcus igneus.

Methanococcus igneus, a hyperthermophilic marine methanogen (optimum growth temperature of 88 degrees C) with a 25-min doubling time, synthesizes an unusual inositol phosphodiester which is present at high intracellular concentrations along with l-alpha-glutamate and beta-glutamate. Identification of this compound as a dimeric inositol phosphodiester (di-myo-inositol-1,1'-phosphate) was provided by two-dimensional nuclear magnetic resonance methods. The intracellular levels of all three negatively charged solutes (l-alpha-glutamate, beta-glutamate, and the inositol phosphodiester) increase with increasing levels of external NaCl, although the inositol compound shows much smaller increases with increasing NaCl levels than the glutamate isomers. The turnover of these solutes was examined by CO(2)-pulse-CO(2)-chase experiments. The results indicated that both the beta-glutamate and the inositol phosphodiester behaved as compatible solutes and were not efficiently metabolized by cells as was l-alpha-glutamate. At a fixed external NaCl concentration, lower ammonium levels increased the fraction of the inositol dimer present in extracts. The most pronounced changes in di-myo-inositol-1,1'-phosphate occurred as a function of cell growth temperature. While the organism grows over a relatively wide temperature range, the phosphodiester accumulated only when M. igneus was grown at temperatures of >/=80 degrees C. Thus, this unusual compound is a non-nitrogen-containing osmolyte preferentially synthesized at high growth temperatures.

Journal Article↗
PubMed1998

Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme.

Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1'-phosphate, an osmolyte found in hyperthermophilic archaeal. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K(m) = 0.091 +/- 0.016 mM; Vmax = 9.3 +/- 0.45 mumol of Pi min-1 mg of protein-1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

Amino Acid Sequence↗
PubMed1988

Toxic shock syndrome toxin-1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells.

Toxic shock syndrome toxin-1 (TSST-1) is a 22-kDa exotoxin produced by most Staphylococcus aureus strains responsible for toxic shock syndrome. TSST-1 is a mitogen for human T cells. The mechanism of T cell activation by TSST-1 was investigated. TSST-1 induced IL-2R expression, IL-2 synthesis, and proliferation in T cells in a monocyte-dependent fashion. Neither IL-1 nor IL-2, alone or in combination, substituted for monocytes in supporting TSST-1-induced mitogenesis. We investigated the mechanism by which TSST-1 induces initogenesis. TSST-1 failed to induce ADP-ribosylation of T cell membrane proteins. However, the toxin induced transient translocation of protein kinase C from cytosol to plasma membranes and also induced the mobilization of cellular Ca2+ stores in both PBMC and the Jurkat human tumor T cell line, suggesting that TSST-1 triggered inositol phospholipid turnover. This was directly demonstrated to be the case in both cellular preparations studied. TSST-1 induced the increased synthesis of the inositol phospholipid phosphatidyl inositol, phosphatidyl inositol-4 phosphate, and phosphoinositol inositol-4,5-bisphosphate, and induced the breakdown of inositol phospholipid as evidence by the accumulation of phosphatidic acid and inositol phosphates. We conclude that the action of TSST-1 involves the induction of inositol phospholipid turnover, protein kinase C activation, and mobilization of cellular Ca2+ stores. This effect is similar to that of mitogenic lectins and of anti-CD3 antibodies.

Adenosine Diphosphate Ribose↗
PubMed1992

Effects of high extracellular calcium concentrations on phosphoinositide turnover and inositol phosphate metabolism in dispersed bovine parathyroid cells.

We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, "receptor-like" mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca(2+)-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca(2+)-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5-10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29-36% decreases in PIP2 (within 5-10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic "Ca(2+)-mobilizing" hormones, the high Ca(2+)-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Ca(i)) in parathyroid cells.

Animals↗
PubMed1995

Preconditioning of perfused rat heart inhibits reperfusion-induced release of inositol(1,4,5)trisphosphate.

Preconditioning the heart by brief episodes of ischemia and reperfusion can be cardioprotective to injury from subsequent sustained ischemia and reperfusion. Such protective effects include the reduction of reperfusion-induced arrhythmias. We have previously reported a rapid rise in the second messenger inositol(1,4,5) trisphosphate with post-ischemic reperfusion, which has been implicated in the generation of reperfusion-induced arrhythmia. Given the possible importance of inositol(1,4,5)trisphosphate, studies were performed in isolated perfused rat hearts to determine if preconditioning could reduce the reperfusion-induced rise in inositol(1,4,5) trisphosphate. [3H]Inositol labeled hearts underwent 20 min global ischemia with or without 2 min reperfusion. This was preceded by preconditioning of two or three cycles of 2 or 5 min ischemia separated by 5 or 10 min reperfusion, or by time matched control perfusions. Two min reperfusion following 20 min ischemia in time matched controls caused an increase of [3H]inositol(1,4,5)trisphosphate from 20 +/- 3 cpm/mg protein to 52 +/- 5 cpm/mg protein (mean +/- S.E.M. n = 4, P < 0.01). Preconditioning of three cycles of 5 min ischemia and 5 min reperfusion inhibited the 2 min reperfusion-induced rise in [3H]inositol(1,4,5)trisphosphate (26 +/- 4 cpm/mg protein. P < 0.01 relative to non-preconditioned hearts), however protocols that involved either fewer ischemic cycles or shorter ischemic periods were ineffective. Preconditioning did not affect myocardial ATP levels or norepinephrine release with sustained ischemia and reperfusion. Inhibition of the reperfusion-induced rise in inositol(1,4,5) trisphosphate may provide an explanation for the inhibitory effects of preconditioning on reperfusion-induced arrhythmias.

Adenosine Triphosphate↗
PubMed1995

The release of intracellular Ca2+ in lacrimal acinar cells by alpha-, beta-adrenergic and muscarinic cholinergic stimulation: the roles of inositol triphosphate and cyclic ADP-ribose.

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1-4 phosphate groups, resulting in release of Ca2+ from intracellular stores. Stimulation with the alpha-adrenergic agonist phenylephrine, however, causes a release of Ca2+ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca2+ rise was completely inhibited by 100 microM ryanodine. Adrenaline (causing activation of both alpha- and beta-adrenergic receptors) induces a Ca2+ release with inositol phosphate synthesis identical to that occurring in the beta-adrenergic response. Thus, the signalling pathway for alpha-adrenergic stimulation occurs via a path different from that which releases Ca2+ via muscarinic cholinergic and beta-adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5'-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] cause release of Ca2+ from intracellular stores. The Ca2+ release evoked by cADP-ribose, but not by Ins(1,4,5)P3, was abolished by 100 microM ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca2+ signalling. When the intracellular free Ca2+ concentration ([Ca2+]i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca2+]i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.

Acetylcholine↗
PubMed1985

Evidence for the formation of inositol 4-monophosphate in stimulated human platelets.

Human platelets were prelabeled with [3H] inositol and exposed to thrombin or vasopressin. The radioactive inositol monophosphates were separated by high-performance liquid chromatography and identified by cochromatography with unlabeled standard substances. Radioactive inositol 1-monophosphate (Ins 1-P) and inositol 4-monophosphate (Ins 4-P) were detected in unstimulated platelets and accumulated in response to thrombin or vasopressin. Ins 4-P as well as Ins 1-P increased after the formation of inositol 1,4-bisphosphate (Ins 1,4-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). Lithium augmented the accumulation of Ins 1-P and Ins 1,4-P2 in stimulated platelets, and also of Ins 4-P in platelets stimulated by vasopressin, but not by thrombin. The results indicate that Ins 1,4-P2 formed in stimulated platelets is partly degraded to Ins 4-P. The significance of Ins 4-P as a marker molecule for the study of inositol phosphate metabolism in stimulated cells is discussed.

Blood Platelets↗