Journal of lipid mediators and cell signalling最新文献

Platelet-activating factor (PAF) is present in human bone marrow leading us to investigate its effect on human bone marrow cell proliferation. While PAF (0.1 μM to 1 nM) stimulates the incorporation of [³H]thymidine by freshly isolated adherent human bone marrow cells, PAF has no effect on non adherent cells. A non-metabolizable PAF agonist is more potent than PAF to stimulate thymidine incorporation in adherent cells. The precise role of PAF in human haematopoiesis in vivo remains to be clarified.

The aim of the present study was to develop a new model of allergic late-phase reaction in the airways of concious guinea pigs (GPs) and to characterise it by pharmacological in tervention. GPs were pretreated with cyclophosphamide and sensitized with ovalbumin (OA) in Al(OH)₃. Weeklt inhalations of polymyxin B were performed before and during sensitization and continued throughout the study period. Under cover of 10 mg/kg i.p. mepyramine all GPs still exhibtied a pronounced immediate reaction (IR), peaking during the first 15 min after OA. Nine out of 15 GPs demonstrated, during screening, a reproducible (twice) second phase (late phase reaction (LPR) of decreased airflow and tidal volume (TV) peaking 4–8 h after OA. In a cross over study, methylprednisolone (MP) at 30 mg/kg p.o. (16 h and 1 h before OA) significantly inhibited the LPR at its peak (4–8 h) peak decrease of TV to % of basal; control 49.4 ± 3.7; MP 78.9 ± p < 0.01; n = 7). After another booster sensitization with 2 μg OA/GP under the same conditions, the Paf-antagonist WEB 2347 at 3 mg/kg p.o. (1 h before OA) inhibited the LPR at its peak agin (peak decrease of TV to % of basal: control 57.3 ± 3.5; WEB 2347 74.8 ± 7.6; p < 0.01; n = 6). In conclusion more than 50% of repeatedly ovalbumin sensitized (and polymyxin B-treated) unanaesthetized GPs developed a reproducible pulmonary late phase reaction (LPR). The LPR peaked at 4–8 h after antigen-exposure. The inhibitory effect by a glucocorticoid and the Paf-antagonist WEB 2347 suggests the inflammatory nature of the LPR and the involvement of platelet-activating factor (Paf) in this model.

This study examined the mttuence of human high density ltpoproteins (HDL) on the intracellular free calcium of cultured bovine aortic endothelial cells (BAECs). Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was determined by a fluorescent calcium indicator, Fura-2. It was found that, in the presence of 1 mmol/L extracellular calcium, HDL resulted in a biphasic elevation of [Ca²⁺]_i in BAECs, consisting of an initial, transient component followed by a lower, but more sustained component. Doses of HDL from 25 to 200 μg protein/ml induced marked concentration-dependent elevations of [Ca²⁺]_i in BAECs. The sustained component was abolished by deprivation of extracellular calcium or by pretreatment of endothelial cells with a calcium influx blocker, NiCl₂. HDL-induced elevation of [Ca²⁺]_i was attenuated in a concentration-dependent way by an inhibitor of calcium release, tetracaine. Repeated applications of HDL (100 μg protein/ml) markedly blunted the initial peak component of the calcium transient of BAECs. These results demonstrate that both intracellular and extracellular calcium pools are responsible for the biphasic elevation of [Ca²⁺]_i induced by HDL in cultured BAECs.

To investigate whether diacylglycerol (DAG) has a role in reversible platelet aggregation induced by low concentrations of platelet-activating factor (PAF), we attempted to use the DAG kinase inhibitor, R59022, to prevent rapid conversion of DAG to phosphatidic acid. However, we found that R59022 inhibited the binding of [³H]PAF to human and rabbit platelets and to rabbit platelet membranes. We then investigated whether exogenous, cell-penetrating DAGs (1,2-dihexanoyl-sn-glycerol (DHG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG)) act synergistically with low concentrations of PAF that alone induce only reversible aggregation. Platelets were isolated and labeled with [¹⁴C]serotonin. DHG (25–75 μM) caused slow, weak aggregation and some release of [¹⁴C]serotonin with human, but not rabbit, platelets. OAG (25–75 μM) did not aggregate either species' platelets. Phosphorylation of pleckstrin by DHG was more transient in rabbit platelets than previously observed with human platelets. Both DHG and OAG synergistically potentiated PAF-induced aggregation of human platelets, but, paradoxically, concurrently inhibited the PAF-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i); potentiation decreased upon incubation with DAGs before PAF addition. In contrast, DHG strongly inhibited PAF-induced aggregation of rabbit platlets; inhibition decreased upon preincubation. OAG, added with PAF, slightly potentiated aggregation of rabbit platelets; upon preincubation, OAG progressively inhibited. Effects of DHG and OAG on PAF-induced increases in [Ca²⁺]_i in rabbit platlets followed a similar pattern; thus, with rabbit platelets, inhibition of the [Ca²⁺]_i increase may at least partially account for inhibition of PAF-induced aggregation by exogenous DAGs. Results with human platelets are consistent with stimulation of protein kinase C by DAGs, and then metabolism of DAGs and/or negative feedback by DAGs, but results with rabbit platelets indicate both an unexpected species difference and a difference between the effects of DHG and OAG on PAF-induced platelet aggregation.

Tigers communicate with one another with the help of Marking Fluid (MF) which is a lipid-rich fluid sprayed upwards and backwards through the urinary channel of both the sexes. The volatile molecules of the MF are made to last longer with the help of lipid ‘fixatives’ the total amount of which is 1–2 mg/ml. This lipid comprises cholesterol ester, wax ester, triglyceride, free fatty acids, diglyceride, monoglyceride, free sterol and phospholipid as revealed by thin layer chromatography. The gas liquid chromatogram of fatty acid methyl esters of the total lipid of the MF, when compared with the fatty acids of groin and body fat of tiger (analysed by other workers), reveals differences in higher proportions of palmitoleic and myristic acids and of highly unsaturated fatty acids. The palmitoleic acid content of total lipid and triglyceride is high in comparison to the wax esters and cholesterol esters of the MF-fat. The unique feature of the alcohol part of the wax esters is a series of saturated straight chain primary alcohols of C₄ to C₂₀ and these are accompanied by the corresponding monoenoic unsaturates.

ADP-ribosylation factors are a family of ∼ 21 kDa GTP binding proteins which have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in mammals and yeast. Reversible membrane associations are thought to be an essential component in the physiological actions of ARF and are regulated by GTP binding. ARFs are unique among the superfamily of GTP binding proteins in having a strict dependence on phospholipids for nucleotide exchange. In addition, ARF proteins were found to bind phospatidylinositol 4,5-bisphosphate (PIP₂) specifically. PIP₂ was found to increase the rate of GDP dissociation and stabilize the nucleotide-free form of the protein. The previously described requirements for PIP₂ in the ARF stimulated phospholipase D (PLD) activity and ARF GTPase activating protein (ARF GAP) assays provide the basis for a model in which PIP₂ acts as a cofactor in one or more ARF pathways. There are potentially two distinct phospholipid binding sites each of which are coupled to the nucleotide binding site of ARFs.

The regulation of phospholipase D was studied in human neuroblastoma cells using phosphatidylethanol as a marker of the enzyme activity. Carbachol induced phospholipase D activity in SH-SY5Y cells. Muscarinic antagonists inhibited the response with potencies suggesting that muscarinic M₁ receptors are responsible for the activation. In permeabilized SH-SY5Y cells, both the carbachol- and GTPγS-induced Peth formation was inhibited by GDPβS, indicating that both responses are mediated via a G-protein. The protein kinase C inhibitors, bisindolylmaleimide and staurosporine significantly inhibited the carbachol-induced Peth formation whereas H7 had no effect. Thus, the cholinergic activation of phospholipase D in SH-SY5Y cells is probably mediated via a direct receptor-G-protein coupling but an involvement of protein kinase C cannot be excluded. Calmidazolium, a calmodulin antagonist, induced an increase in phosphatidylethanol formation in both SH-SY5Y and IMR-32 cells. This effect was inhibited by genistein and tyrphostin, indicating a tyrosine kinase dependent pathway for phospholipase D activation in neuroblastoma cells.

The receptor-mediated poly-phosphoinositide (PI) signalling pathway is known to play an important role in maintaining intracellular calcium homeostasis, which in turn, is critical for mediating neuronal function. In this study, we examined the effects of focal cerebral ischemia induced in rats by temporary occlusion of the middle cerebral artery (MCA) and both common carotid arteries (CCAs) on this signal transduction pathway. Results indicate that several parts of the pathway are altered, both during the early phase of focal cerebral ischemic insult and after recirculation. Cerebral ischemia induced a decrease in levels of phosphatidylinositol 4,5-biphosphate (PIP₂) in the ischemic MCA cortex, due partly to stimulated poly-PI hydrolysis and partly to the depletion of ATP required for resynthesis of this substrate. ATP depletion during ischemia was also attributed to a sustained decrease in inositol 1,4,5-triphosphate (IP₃) levels. On the other hand, the decline in IP₃ 3-kinase activity after 30 min of ischemic insult was not related to ATP depletion. During reperfusion upon prolonged ischemic insult, neither IP₃ level nor IP₃ 3-kinase activity were able to show recovery after reperfusion, despite that ATP levels recovered by 80%. In situ hybridization studies indicated a decrease in mRNA expression of IP₃ receptor but not IP₃ 3-kinase during the initial 4 h of reperfusion after a 45 min ischemic insult. Under this same condition, insulted cortical neurons started to show morphological changes between 4 and 8 h after reperfusion and extensive cell death could be observed by 16 h. Taken together, these results demonstrated early and delayed changes in the poly-PI signalling pathway due to focal cerebral ischemia. These effects are likely to cause impairment in neuronal function and underline the process of cerebral ischemic damage.