scholarly journals The phosphoinositides exist in multiple metabolic pools in rabbit platelets

1986 ◽  
Vol 238 (2) ◽  
pp. 411-417 ◽  
Author(s):  
J D Vickers ◽  
J F Mustard
Keyword(s):  
Phosphatidic Acid ◽  
Specific Radioactivity ◽  
Rabbit Platelets ◽  
Phosphatidylinositol 4 ◽  
Stimulation Of

The labelling of the phosphoinositides and phosphatidic acid in washed rabbit platelets incubated with [32P]phosphate or [3H]glycerol was studied in the presence of isotope and after unincorporated isotope had been removed. With both isotopes the increase in the specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) lagged behind that of phosphatidylinositol 4-phosphate (PIP) but the specific radioactivity remained higher after unincorporated isotope had been removed. This result was consistent with the presence of a second pool of PIP2, which interconverted slowly with the pool of PIP2 which was in direct equilibrium with PIP, proposed to explain the increase in specific radioactivity of PIP2 which accompanies the decrease in amount of PIP2 at 10 s in ADP-stimulated platelets. In platelets labelled with [3H]glycerol, the specific radioactivity of PIP2 became higher than that of PIP and the specific radioactivity of PIP became higher than that of phosphatidylinositol (PI). These results were interpreted to indicate that there were two pools of PIP; of these the pool with the higher specific radioactivity was the precursor of PIP2. Similarly, two pools of PI were proposed. The presence of pools of the phosphoinositides with different specific radioactivities necessitates the measurement of chemical amount of these compounds when studying the effect of stimulation of the platelets, since changes in labelling may not accurately reflect changes in the amount of the phosphoinositides.

Changes In Triphosphatidylinositol Metabolism During PGE1- Induced Shape Change Of Washed Rabbit Platelets

1981 ◽  
Author(s):  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Phosphatidic Acid ◽  
Shape Change ◽  
Specific Radioactivity ◽  
Inositol Phospholipids ◽  
Rabbit Platelets ◽  
Turn Over ◽  
Camp Levels ◽  
Camp Formation ◽  
Platelet Shape ◽  
Stimulation Of

Since the inositol phospholipids are present in small amounts in platelets and turn over rapidly during platelet shape change, aggregation and release, they are thought to have a functional rather than structural role in platelets. We have previously reported that within 10 sec of stimulation of prelabeled, washed rabbit platelets with ADP, the amount of triphosphatidylinositol (TPI) is significantly reduced while the specific radioactivity of its [32p]phosphate is increased. One explanation of this result is that ADP- stimulation may divert ATP required for phosphorylation of diphosphatidylinositol (DPI) to TPI, leading to a decrease in the amount of TPI. PGE1 (10 μM) causes conversion of ATP to cAMP and induces a transient platelet shape change. The shape change may be due to the reduction in ATP with a concomitant fall in TPI. We have therefore studied whether PGE1-stimulation of washed rabbit platelets prelabeled with [32P] causes a change in TPI. Within 10 sec the amount of TPI in PGE1-treated platelets was reduced from 2.22 nmoles/ 109 platelets to 1.98 nmoles/109 platelets (p<0.05) although neither the [32P] labeling (51.1 × 103 dpm/109 platelets) nor specific radioactivity (24.1 × 103 dpm/nmole) were significantly changed. These results are compatible with the theory that diversion of ATP by PGE1-stimulation of cAMP formation from ATP, may reduce the amount of TPI. A similar effect was observed previously with ADP-stimulation. PGE1 caused no change in the [32p] labeling of phosphatidic acid (PA) (ADP caused a 290% increase) and caused only a small increase in its specific radioactivity (16% compared to 270% with ADP). If the rates of turnover of TPI and PA which are reflected in their specific radioactivities are Ca2+- dependent, Ca2+ sequestration due to increased cAMP levels induced by PGE1 would, after the initial effects, terminate these changes. The results further support the suggestion that reduction in the amount of TPI may be involved in platelet shape change and initiation of aggregation.

PLATELET-FIBRIN CLOTS FORMED BY THROMBIN SELECTIVELY RETAIN PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIF2)

1987 ◽  
Author(s):  
John D Vickers ◽  
Raelene L Kinlough-Rathbone ◽  
J Fraser Mustard
Keyword(s):  
Phosphatidic Acid ◽  
Human Platelets ◽  
Large Decrease ◽  
Clot Retraction ◽  
Rabbit Platelets ◽  
Specific Association ◽  
Phosphatidylinositol 4 ◽  
Chloroform Methanol ◽  
Stimulation Of ◽  
Fibrin Clots

It is established that stimulation of human platelets with thrombin for 60 s in the absence of fibrinogen increases the amount of PIPp compared with unstimulated controls (4.7 ± 0.24 nmol/109 plat, vs 3.83 ± 0.14 nmol/109 plat., p<0.01, n=8). However, stimulation with thrombin for 60 s in the presence of fibrinogen causes a large decrease in the amount of PIP2 that can be extracted with acidified chloroform/methanol compared with unstimulated controls (1.62 ± 0.39 nmol/109 plat, vs 3.84 ± 0.44 nmol/109 plat., p<0.001, n=6). Stimulation of rabbit platelets with thrombin in the presence of fibrinogen also decreases the amount of extractable PIP2 (60% at 60 s, p<0.001, n=8). Similar decreases in amount can not be demonstrated for phosphatidylinositol 4-phosphate, phosphatidylinositol, phosphatidic acid or phosphatidylcholine under the same conditions, indicating that the decrease is specific for PIP2. With rabbit platelets, polymerized fibrin formed by reptilase, which does not stimulate platelets or induce clot retraction, does not cause the decrease in extractable PIP, (3.06 ± 0.05 nmol/109 plat, were extracted compared with 3.182 ± 0.07 nmol/109 plat, without reptilase). However, stimulation of rabbit platelets with ADP in the presence of polymerizing fibrin formed by reptilase causes | larger decrease in extractable PIP2 (to 2.54 ± 0.19 nmol/109 plat., p<0.05, n=4) than is caused Dy ADP and fibrinogen alone (to 2.87 ± 0.06 nmol/10 plat., p<0.05, n=4). Inhibition by glycyl-L-prolyl-L-arginyl-L-proline of polymerization of fibrin formed by the action of thrombin prevents the large.decrease in the amount of extractable PIP2 (4.37 ± 0.30 nmol/10 plat, were extracted) from human platelets. These results indicate that the interaction of polymerizing fibrin with stimulated platelets is required for the decrease in PIP2. The decrease in extractable PIP2 seen with polymerizing fibrin can not be explained by increased degradation of PIP2 to IP3 or PIP. Thus, when human or rabbit platelets are stimulated with thrombin in the presence of fibrinogen, an association of polymerizing fibrin with the stimulated platelets occurs that leads to decreased extractability of PIP2. This may mean that PIP2 forms a specific association with pratelet proteins that are involved in clot retraction.

scholarly journals Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1972 ◽  
Vol 128 (3) ◽  
pp. 531-541 ◽  
Author(s):  
Janet M. Stein ◽  
C. N. Hales
Keyword(s):  
Phosphatidic Acid ◽  
Specific Radioactivity ◽  
Phospholipid Composition ◽  
Fat Cells ◽  
Fat Pad ◽  
Fat Cell ◽  
32P Incorporation ◽  
The Individual ◽  
Adrenergic Blocking ◽  
Stimulation Of

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [32P]Pi into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [32P]Pi into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [32P]Pi into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.

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.

scholarly journals Insulin provokes co-ordinated increases in the synthesis of phosphatidylinositol, phosphatidylinositol phosphates and the phosphatidylinositol–glycan in BC3H-1 myocytes

1988 ◽  
Vol 256 (1) ◽  
pp. 185-188 ◽  
Author(s):  
R V Farese ◽  
D R Cooper ◽  
T S Konda ◽  
G Nair ◽  
M L Standaert ◽  
...  
Keyword(s):  
Insulin Action ◽  
De Novo ◽  
Specific Radioactivity ◽  
Growth Cycle ◽  
Head Group ◽  
Isotopic Equilibrium ◽  
Phosphatidylinositol Phosphates ◽  
Phosphatidylinositol 4 ◽  
Stimulation Of

BC3H-1 myocytes were cultured in the presence of [3H]inositol or [3H]glucosamine during their entire growth cycle to ensure that all lipids containing inositol and glucosamine were labelled to isotopic equilibrium or maximal specific radioactivity. After such labelling, a lipid (or group of lipids), which was labelled with both inositol and glucosamine, was observed to migrate between phosphatidylinositol 4-phosphate and phosphatidylinositol (PI) in two different t.l.c. systems. Insulin provoked rapid, sizeable, increases in the inositol-labelling of this lipid (presumably a PI-glycan), and these increases were similar to those observed in PI and PI phosphates. Our results indicate that insulin provokes co-ordinated increases in the net synthesis de novo of PI and its derivatives, PI phosphates and the PI-glycan, in BC3H-1 myocytes. This increase in synthesis of PI may serve as the mechanism for replenishing the PI-glycan during stimulation of its hydrolysis by insulin. Moreover, increases in the content of the PI-glycan may contribute to increases in the generation of head-group ‘mediators’ during insulin action.

scholarly journals Changes in the platelet phosphoinositides during the first minute after stimulation of washed rabbit platelets with thrombin

1984 ◽  
Vol 219 (1) ◽  
pp. 25-31 ◽  
Author(s):  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Phosphatidic Acid ◽  
De Novo ◽  
Initial Response ◽  
Initial Increase ◽  
Dihydroxyacetone Phosphate ◽  
Low Concentration ◽  
Early Change ◽  
Rabbit Platelets ◽  
Partial Release ◽  
Stimulation Of

Experiments with washed platelets from rabbits demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml) that causes maximal aggregation and partial release of granule contents does not significantly decrease the amount of phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)P2] at 10s; this contrasts with ADP stimulation. The amount of PtdIns (4,5)P2 was significantly decreased by a higher concentration of thrombin (0.3 unit/ml). Increased turnover of the PtdIns (4,5)P2 at 60s was indicated by changes in labelling with [3H]glycerol in platelets stimulated with both concentrations of thrombin. An unexpected observation with the lower thrombin concentration was a significant increase in the amount of phosphatidylinositol (PtdIns) at 10s. This contrasts with data from other laboratories, which indicate that thrombin causes a significant decrease in PtdIns . At 60s, with the lower concentration of thrombin, PtdIns was significantly decreased. With the higher concentration of thrombin there was a significant decrease in the amount of PtdIns at 10s, in keeping with the data from other laboratories. The initial increase in PtdIns may not have been observed by other investigators because higher concentrations of thrombin were used. The reaction involved in this initial increase in the amount of PtdIns does not appear to be increased degradation of PtdIns4P or PtdIns (4,5)P2, since their total amount was unchanged at 10s. The magnitude of the increase in PtdIns is such that more than the existing pool of phosphatidic acid would have to be converted into PtdIns to account for the increase. It is suggested that synthesis of phosphatidic acid de novo from dihydroxyacetone phosphate and glycerol 3-phosphate might be the source of phosphatidic acid, which leads to increased PtdIns at 10s with the lower concentration of thrombin. Thus it appears that the initial response of platelets to thrombin does not require an early change in PtdIns (4,5)P2 and may involve stimulation of synthesis de novo of PtdIns via phosphatidic acid.

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.

Interaction of Vasopressin with Human Blood Platelets: Dependency on Mg2+

1986 ◽  
Vol 56 (03) ◽  
pp. 260-262 ◽  
Author(s):  
Isabella Roos ◽  
Fabrizia Ferracin ◽  
Alfred Pletscher
Keyword(s):  
Phosphatidic Acid ◽  
Human Blood ◽  
Arginine Vasopressin ◽  
Specific Binding ◽  
Blood Platelets ◽  
Bivalent Cations ◽  
Platelet Membranes ◽  
Maximal Rise ◽  
Human Blood Platelets ◽  
Stimulation Of

SummaryArginine-vasopressin (AVP) in the presence of Mg2+ but not in the absence of bivalent cations led to accumulation of [32P]-phosphatidic acid ([32P]-PA) in human blood platelets. Mg2+ also enhanced the specific binding of [3H]-AVP to intact platelets. The concentrations of the cation which enabled AVP to cause half maximal rise of [32P]-PA and those inducing half maximal [3H]-AVP-binding were of the same order. It is concluded that the stimulation of phosphatidyl inositide breakdown by AVP in presence of Mg2+ is at least partially due to a Mg2+-induced enhancement of specific AVP-binding to the platelet membranes.

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