scholarly journals Accumulation of the inositol phosphates in thrombin-stimulated, washed rabbit platelets in the presence of lithium

1984 ◽  
Vol 224 (2) ◽  
pp. 399-405 ◽  
Author(s):  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Platelet Activation ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Important Change ◽  
Early Event ◽  
Rapid Degradation ◽  
Rabbit Platelets ◽  
Phosphatidylinositol 4 ◽  
Partial Release ◽  
Stimulation Of

Experiments with washed rabbit platelets demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml), that causes maximal aggregation and partial release of amine granule contents, also causes increased accumulation of [3H]inositol-labelled inositol trisphosphate (InsP3) in the presence of 20 mM-Li+. This concentration of Li+ was found to inhibit the degradation of inositol phosphates by phosphomonoesterases. This result indicates that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is degraded early after platelet stimulation with thrombin, although in a previous study we had found no decrease in amount. In the absence of Li+, the labelling of inositol bisphosphate (InsP2) increased more rapidly than that of InsP3, consistent with rapid degradation of InsP3 by phosphomonoesterase. After 30s the increase in InsP2 was augmented by Li+. This increase in InsP2 could have been due to increased degradation of phosphatidylinositol 4-phosphate or inhibition of breakdown of InsP2 to InsP with a lesser inhibition of breakdown of InsP3 to InsP2. The effect on InsP3 and InsP2 of stimulation of the platelets with 1.0 unit of thrombin/ml was comparable with the effect of the lower concentration of thrombin. Inositol phosphate (InsP) labelling did not increase in response to 0.1 unit of thrombin/ml, but increased when the platelets were stimulated with 1.0 unit of thrombin/ml. Whether the increase in InsP was due to increased degradation of phosphatidylinositol or a greater rate of breakdown of InsP2 to InsP than InsP to inositol cannot be determined in these experiments. These results indicate that degradation of PtdIns(4,5)P2 is an early event in platelet activation by thrombin and that formation of inositol phosphates and 1,2-diacylglycerol rather than a decrease in PtdIns(4,5)P2 may be the important change.

Eicosapentaenoic Acid Interferes with U46619-Stimulated Formation of Inositol Phosphates in Washed Rabbit Platelets

1989 ◽  
Vol 62 (04) ◽  
pp. 1116-1120 ◽  
Author(s):  
N Chetty ◽  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
J F Mustard
Keyword(s):  
Signal Transduction ◽  
Fatty Acid Composition ◽  
Eicosapentaenoic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Dense Granule ◽  
Detectable Change ◽  
Membrane Phospholipids ◽  
Rabbit Platelets ◽  
Stimulation Of

SummaryEicosapentaenoic acid (EPA) inhibits platelet responsiveness to aggregating agents. To investigate the reactions that are affected by EPA, we examined the effect of preincubating aspirintreated rabbit platelets with EPA on stimulation of inositol phosphate formation in response to the TXA2 analogue U46619. Stimulation of platelets with U46619 (0.5 μM) caused aggregation and slight release of dense granule contents; aggregation and release were inhibited by preincubation of the platelets with EPA (50 μM) for 1 h followed by washing to remove unincorporated EPA. Incubation with EPA (50 μM) for 1 h did not cause a detectable increase in the amount of EPA in the platelet phospholipids. When platelets were prelabelled with [3H]inositol stimulation with U46619 of control platelets that had not been incubated with EPA significantly increased the labelling of mos1tol phosphates. The increases in inositol phosphate labelling due to U46619 at 10 and 60 s were partially inhibited by premcubat10n of the platelets with 50 μM EPA. Since the activity of cyclo-oxygenase was blocked with aspirin, inhibition of inositol phosphate labelling in response to U46619 indicates either that there may be inhibition of signal transduction without a detectable change in the amount of EPA in platelet phospholipids, that changes in signal transduction require only minute changes in the fatty acid composition of membrane phospholipids, or that after a 1 h incubation with EPA, activation of phospholipase C is affected by a mechanism that is not directly related to incorporation of EPA.

scholarly journals Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol

1988 ◽  
Vol 251 (1) ◽  
pp. 279-284 ◽  
Author(s):  
M L Rand ◽  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
J F Mustard
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Thromboxane A2 ◽  
Second Messenger ◽  
Inhibitory Effect ◽  
Thromboxane B2 ◽  
Rabbit Platelets ◽  
Platelet Functions ◽  
Stimulation Of

Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.

Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

1989 ◽  
Vol 66 (1) ◽  
pp. 504-508 ◽  
Author(s):  
T. Bainbridge ◽  
R. D. Feldman ◽  
M. J. Welsh
Keyword(s):  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Adrenergic Stimulation ◽  
Cl Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

scholarly journals Effects of pertussis toxin on growth factor-stimulated inositol phosphate formation and DNA synthesis in Swiss 3T3 cells

1988 ◽  
Vol 249 (3) ◽  
pp. 917-920 ◽  
Author(s):  
C W Taylor ◽  
D M Blakeley ◽  
A N Corps ◽  
M J Berridge ◽  
K D Brown
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Phosphoinositide Hydrolysis ◽  
Polypeptide Growth Factors ◽  
Swiss 3T3 ◽  
Swiss 3T3 Cell ◽  
Stimulation Of

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C. Production of inositol phosphates in response to platelet-derived growth factor, fibroblast growth factor and prostaglandin F2 alpha were unaffected by the toxin while bombesin- and vasopressin-stimulated formation of inositol phosphates were inhibited by only 27 and 23% respectively. These results argue against a major role for a pertussis toxin-sensitive G protein in coupling any of these mitogen receptors to activation of a phosphoinositidase C. Furthermore, the results suggest that the widespread inhibitory effects of pertussis toxin on mitogen-stimulated DNA synthesis may be unrelated to the toxin's limited actions on phosphoinositide hydrolysis.

Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state

1993 ◽  
Vol 264 (1) ◽  
pp. H126-H132
Author(s):  
V. Pijuan ◽  
I. Sukholutskaya ◽  
W. G. Kerrick ◽  
M. Lam ◽  
C. van Breemen ◽  
...  
Keyword(s):  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Rat Aorta ◽  
Release Mechanism ◽  
Maximal Increase ◽  
Contractile State ◽  
Rapid Stimulation ◽  
Relationship Of ◽  
Stimulation Of

Rapid stimulation of Ins(1,4,5)P3 production in rat aorta by NE: correlation with contractile state. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H126-H132, 1993.--The isomeric composition of inositol phosphates generated in response to norepinephrine (NE) stimulation and the relationship of inositol phosphate production to release of intracellular Ca2+ as measured by contraction were characterized in rat aorta prelabeled with [3H]inositol. NE stimulated a rapid and transient increase in labeled D-myo-inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] levels. A maximal increase in labeled Ins(1,4,5)P3 occurred within 15 s of stimulation followed by a decline to control levels at 5 min. D-Myo-inositol 1,3,4-trisphosphate [Ins-(1,3,4)P3] and D-myo-inositol 1-monophosphate [Ins(1)P] levels also increased rapidly in response to NE. In contrast to the transient production of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1)P production was maintained in the presence of NE. Half-maximal stimulation of Ins(1,4,5)P3 production and Ca2+ release occurred at 0.3 microM NE, and maximal effects were obtained with 10 microM NE. The concentration-response curve and time course for production of Ins(1,4,5)P3 correlated with the neurotransmitter-induced Ca2+ release from intracellular stores, indicating that the level of Ins(1,4,5)P3 regulated the Ca(2+)-release mechanism. In the continued presence of NE, the intracellular pools did not completely refill with Ca2+ despite the return of Ins-(1,4,5)P3 levels to basal at 5 min. These results demonstrate that NE stimulates a rapid increase in Ins(1,4,5)P3 that correlates with contraction in Ca(2+)-free buffer. The reuptake of Ca2+ into intracellular stores is regulated by a mechanism that may not involve Ins(1,4,5)P3.

scholarly journals Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands

1983 ◽  
Vol 216 (3) ◽  
pp. 633-640 ◽  
Author(s):  
C P Downes ◽  
M M Wusteman
Keyword(s):  
Molecular Mechanisms ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Muscarinic Agonist ◽  
Parotid Glands ◽  
Muscarinic Agonists ◽  
Inositol 1 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Rat Parotid ◽  
Phosphatidylinositol 4

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.

scholarly journals Antigen-stimulated metabolism of inositol phospholipids in the cloned murine mast-cell line MC9

1986 ◽  
Vol 234 (1) ◽  
pp. 205-212 ◽  
Author(s):  
M W Musch ◽  
M I Siegel
Keyword(s):  
Mast Cell ◽  
Inositol Phosphates ◽  
Cell Clone ◽  
Inositol Trisphosphate ◽  
Early Event ◽  
Ionophore A23187 ◽  
Murine Mast Cell ◽  
Sugar Phosphates ◽  
Mast Cell Line ◽  
Stimulation Of

Cells of the murine mast-cell clone MC9 grown in suspension culture were sensitized with an anti-DNP (dinitrophenol) IgE and subsequently prelabelled by incubating with [32P]Pi. Stimulation of these cells with DNP-BSA (bovine serum albumin) caused marked decreases in [32P]polyphosphoinositides (but not [32P]phosphatidylinositol) with concomitant appearance of [32P]phosphatidic acid. Whereas phosphatidylinositol monophosphate levels returned to baseline values after prolonged stimulation, phosphatidylinositol bisphosphate levels remained depressed. Stimulation of sensitized MC9 cells with DNP-BSA increased rates of incorporation of [32P]Pi into other phospholipids in the order: phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine. In sensitized cells prelabelled with [3H]inositol, release of inositol monophosphate, inositol bisphosphate and inositol trisphosphate, was observed after stimulation with DNP-BSA. When Li+ was added to inhibit the phosphatase activity that hydrolysed the phosphomonoester bonds in the sugar phosphates, greater increases were observed in all three inositol phosphates, particularly in inositol trisphosphate. The IgE-stimulated release of inositol trisphosphate was independent of the presence of extracellular Ca2+. In addition, the Ca2+ ionophore A23187 caused neither the decrease in [32P]polyphosphoinositides nor the stimulation of the release of inositol phosphates. These results demonstrate that stimulation of the MC9 cell via its receptor for IgE causes increased phospholipid turnover, with effects on polyphosphoinositides predominating. These data support the hypothesis that hapten cross-bridging of IgE receptors stimulates phospholipase C activity, which may be an early event in stimulus-secretion coupling of mast cells. The results with the Ca2+ ionophore A23187 indicate that an increase in intracellular Ca2+ alone is not sufficient for activation of this enzyme.

Carbamoylcholine regulation of polyphosphoinositide synthesis and hydrolysis in cultured, dispersed, digitonin-permeabilized mouse pancreatic islet cells

1997 ◽  
Vol 136 (5) ◽  
pp. 539-545 ◽  
Author(s):  
Andrew M Kardasz ◽  
Peter Thams ◽  
Kirsten Capito ◽  
Carl J Hedeskov
Keyword(s):  
Pancreatic Islet ◽  
Kinase Activity ◽  
Inositol Phosphates ◽  
Islet Cells ◽  
Pancreatic Islet Cells ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Mouse Pancreatic Islet ◽  
Phosphatidylinositol 4 ◽  
Stimulation Of

Abstract Continuing formation of inositol phosphates during stimulation of pancreatic β-cells by hormones and neurotransmitters requires the continued synthesis of the polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2) from phosphatidylinositol (PI). In the present study we have investigated how this pathway and the activity of phosphoinositide-specific phospholipase C (PI-PLC) are regulated by carbamoylcholine (CCh), Ca2+, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), GTPγS and NaF in 44-h [3H]inositol-labelled, dispersed and digitonin-permeabilized mouse pancreatic islet cells. CCh stimulated not only PI-PLC (G-protein-mediated) but also, by an as yet unknown mechanism, significantly enhanced PI 4-kinase activity, estimated as the PIP:PI ratio, by 100%, and further increased the flux from PI to PIP and PIP2. GTPγS and NaF mimicked the effects of CCh on PI-PLC but had no effect on the levels of PIP and PIP2. TPA raised the PIP:PI ratio by 75%. In addition TPA counteracted the CCh stimulation of PI-PLC. There was no effect of 10−6 mol/l Ca2+ on the levels of PIP and PIP2. Experiments with quinacrine and adenosine confirmed that PI-PLC and PI 4-kinase could be regulated independently of each other. In conclusion, these data point to differential regulation of polyphosphoinositide synthesis and breakdown. European Journal of Endocrinology 136 539–545

Inositol phosphate formation and [Ca2+]i in secretagogue-stimulated rabbit gastric mucous cells

1991 ◽  
Vol 260 (1) ◽  
pp. G133-G141 ◽  
Author(s):  
U. Seidler ◽  
A. Pfeiffer
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Mucous Cell ◽  
Parietal Cells ◽  
Secretory Cells ◽  
Rapid Decline ◽  
Mucous Cells ◽  
Chief Cells ◽  
Gastric Mucous ◽  
Stimulation Of

The formation of inositol phosphates and the changes in free intracellular Ca2+ ([Ca2+]i) in isolated rabbit gastric mucous cells during cholinergic stimulation were examined and the potential role of inositol phosphate turnover and [Ca2+]i in gastric mucus secretion evaluated. Rabbit chief and parietal cells were studied for comparison. The formation of [3H]inositol phosphates in mucous, chief, and parietal cells was stimulated in a time- and concentration-dependent fashion by acetylcholine (ACh). The ACh-induced initial [Ca2+]i peak was maximally (10(-4) M ACh) 199 +/- 8% of basal in mucous cells, 427 +/- 20% in chief, and 455 +/- 31% in parietal cells and was followed by a lower-level plateau in mucous and parietal cells but by a more rapid decline in chief cells. As in parietal and chief cells, the initial [Ca2+]i peak occurred in mucous cells in the absence of external Ca2+. ACh stimulated a mucous cell membrane Ca2(+)-entry mechanism in addition to release of Ca2+ from intracellular stores. The concentration-response relationships for the production of [3H]-inositol phosphates, the initial rise in [Ca2+]i, and the stimulation of glycoprotein secretion by ACh were virtually identical. Suppression of the [Ca2+]i rise by the intracellular Ca2(+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the secretory response. As with many other secretory cells, gastric mucous cells possess cholinergic receptors that upon stimulation mediate the hydrolysis of phosphoinositides, a release of Ca2+ from intracellular stores, and a stimulation of Ca2+ influx through the plasma membrane.

scholarly journals Muscarinic-receptor stimulation enhances polyphosphoinositide breakdown in guinea-pig ileum smooth muscle

1984 ◽  
Vol 223 (2) ◽  
pp. 527-531 ◽  
Author(s):  
M C Sekar ◽  
B D Roufogalis
Keyword(s):  
Guinea Pig ◽  
Muscarinic Receptor ◽  
Longitudinal Muscle ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Exchange Chromatography ◽  
Guinea Pig Ileum ◽  
Receptor Stimulation ◽  
Inositol 1 ◽  
Stimulation Of

Muscarinic-receptor stimulation by 0.1 mM-carbachol in longitudinal muscle of the guinea-pig ileum increases the incorporation of [3H]inositol into inositol-containing phospholipid. This effect was blocked by 16 microM-atropine. After 60 min incubation, carbachol increased the accumulation of total inositol phosphates 20-fold in the presence of 10 mM-Li+. Less than 20% of the total inositol phosphate corresponded to inositol 1-phosphate by ion-exchange chromatography, whereas of the remainder about two-thirds corresponded to inositol bisphosphate and one third to inositol trisphosphate. It is concluded that stimulation of muscarinic receptors in guinea-pig ileum enhances breakdown of polyphosphoinositides, suggesting that this may be a primary event associated with Ca2+ mobilization in the guinea-pig ileum.

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