Neomycin does not interfere with the inositol phospholipid metabolism, but blocks binding of alpha-thrombin to intact human platelets.
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ABSTRACT:
SUBMITTER: Tysnes OB
PROVIDER: S-EPMC1150214 | biostudies-other | 1991 Jan
REPOSITORIES: biostudies-other
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Project description:High concentrations of neomycin (2-10 mM) inhibited aggregation, but not shape change, of intact platelets by collagen, ADP and the Ca2+ ionophore, A23187, the last two studies being carried out in the presence of the cyclo-oxygenase inhibitor indomethacin. In contrast, over the same range of concentrations neomycin inhibited both aggregation and shape change induced by thrombin. Under these conditions activation of platelets by collagen and by thrombin, but not by A23187 or by ADP, is believed to be dependent on the hydrolysis of membrane inositol phospholipids. These data therefore suggest that the inhibitory action of neomycin on intact platelets is not related to its previously reported inhibitory effect on phosphoinositide metabolism. The selective inhibition of thrombin-induced shape change indicates a second site of action of neomycin on intact platelets. On platelets rendered semi-permeable with saponin, neomycin and a second aminoglycoside antibiotic, streptomycin (each 0.06-2 mM), stimulated secretion and aggregation responses. These effects were inhibited by indomethacin and by EGTA. Activation of semi-permeabilized platelets by neomycin is associated with the formation of inositol phosphates and phosphatidic acid, indicating activation by phospholipase C. This effect is also inhibited by indomethacin, implying that it is secondary to the formation of prostaglandins and endoperoxides. These results are discussed in the context of the use of neomycin as a selective inhibitor of polyphosphoinositide metabolism.
| S-EPMC1147930 | biostudies-other
Project description:We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.
| S-EPMC1149337 | biostudies-other
Project description:Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3-38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.
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Project description:Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.
| S-EPMC2844139 | biostudies-literature
Project description:The activation of platelet V1-receptors by vasopressin (0.01-1 microM) induces the rapid formation of inositol phosphates, 1,2-diacylglycerol and phosphatidic acid, indicating inositol phospholipid hydrolysis by phospholipase C. Vasopressin immediately induces the formation of inositol bisphosphate and inositol trisphosphate. Accumulation of inositol 1-monophosphate and inositol 4-monophosphate occurs later after a time lag of 15 s. Low concentrations (10-100 nM) of vasopressin only activate phospholipase C, whereas high concentrations (1 microM) induce activation of phospholipase C and subsequently the production of arachidonate metabolites. Cyclo-oxygenase metabolites are associated with further activation of phospholipase C, release reaction and irreversible platelet aggregation. Vasopressin requires for its action extracellular Mg2+, but not Ca2+. The described platelet changes are not induced by 1-desamino-[8-D-arginine]vasopressin, a V2-receptor agonist, and are blocked by a specific V1-receptor antagonist. The results indicate that platelets possess a V1-receptor that is coupled to polyphosphoinositide hydrolysis by phospholipase C, leading to the formation of 1,2-diacylglycerol and inositol trisphosphate. Those compounds may act as second messengers for platelet responses induced by vasopressin, whereas endoperoxides and thromboxane A2 stimulated by vasopressin may serve as amplifiers for platelet activation.
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Project description:The fusion of chick embryonic myoblasts has been studied in tissue culture. Myoblasts are maintained at 0.1 microM-Ca2+ for 50 h. During this time they achieve fusion competence. Fusion is initiated by raising the medium Ca2+ concentration to 1.4 mM. A rapid breakdown of the polyphosphoinositides was detected within 3 min of Ca2+ addition. Rapid synthesis of phosphatidic acid was also detected at this time. Breakdown of phosphatidylinositol and synthesis of 1,2-diacylglycerol were also detected. Other phospholipids were unaffected. Sr2+ could replace Ca2+ in this process but Mg2+ could not and also inhibited the Ca2+ effect. The Ca2+-ionophore A23187 stimulated further apparent polyphosphoinositide breakdown in the presence of Ca2+. 6. The results are discussed with respect to myoblast fusion.
| S-EPMC1152212 | biostudies-other
Project description:At low concentrations of Mg2+, incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in plasma membranes isolated from human polymorphonuclear leucocytes was enhanced 2-4-fold by the polyamines spermidine and spermine. Polyamines had no effects on inositol phospholipid phosphorylation at high concentrations of Mg2+. At 1 mM-Mg2+, [32P]PIP2 synthesis was maximally enhanced by 2 mM-spermine and 5 mM-spermidine, whereas putrescine only slightly enhanced synthesis. Spermine decreased the EC50 (concn. for half-maximal activity) for Mg2+ in [32P]PIP2 synthesis from 5 mM to 0.5 mM. Spermine did not modulate the Km for ATP for [32P]PIP or [32P]PIP2 synthesis. Spermine also decreased the EC50 for PI in [32P]PIP synthesis. In contrast, spermine elevated the apparent Vmax, without affecting the EC50 for PIP, for [32P]PIP2 synthesis. Spermine and spermidine also inhibited the hydrolysis of [32P]PIP2 by phosphomonoesterase activity. Therefore polyamines appear to activate inositol phospholipid kinases by eliminating the requirements for super-physiological concentrations of Mg2+. Polyamine-mediated inhibition of polyphosphoinositide hydrolysis would serve to potentiate further their abilities to promote the accumulation of polyphosphoinositides in biological systems.
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Project description: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.
| S-EPMC1148994 | biostudies-other
Project description:In this study we have examined the implication of tyrosine kinase activities in aggregation, 5-hydroxytryptamine secretion and mainly phosphoinositide metabolism in response to human platelet stimulation by thrombin. Using the potent tyrosine kinase inhibitor tyrphostin AG-213, we have observed a significant inhibition of aggregation and 5-hydroxytryptamine release; however, this percentage inhibition was lower at high thrombin concentrations. On the other hand, tyrphostin treatment of metabolically 32P-labelled platelets significantly inhibited the thrombin-dependent accumulation of PtdIns(3,4)P2, which involves at least a PtdIns 3-kinase and/or a PtdIns3P 4-kinase, whereas the synthesis of phosphatidic acid (PtdOH), a good reflection of the phospholipase C (PLC) activation in platelets, was partially blocked. Inositol phosphate production was also inhibited by about 40% when tyrphostin-treated platelets were stimulated with thrombin. In addition, we show by Western-blot analysis that PLC gamma 1, as well as the regulatory subunit (p85) of the PtdIns 3-kinase, were present in the anti-phosphotyrosine immunoprecipitate isolated from thrombin-stimulated platelets. Furthermore, tyrphostin treatment clearly decreased the PLC gamma 1 and p85 contents in such an anti-phosphotyrosine immunoprecipitate. Our results provide the first evidence for a direct or indirect regulation of PtdIns(3,4)P2 accumulation and PLC gamma 1 activity by tyrosine phosphorylation during thrombin stimulation of human platelets.
| S-EPMC1134192 | biostudies-other
Project description:Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.
| S-EPMC1146811 | biostudies-other