Journal of lipid mediators and cell signalling最新文献

Platelet activating factor (PAF), a potent lipid mediator, has been implicated in the pathogenesis of airways inflammation in bronchial asthma. Binding of PAF to its receptor (Nakamura et al., 1991) leads to changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) that is crucial to cell activation. Therefore, the aim of our study was to investigate whether PMNL of asthmatic patients stimulated with PAF(10⁻⁷ M) differ in relation to changes of [Ca²⁺]_i from cells of healthy subjects. PMNL from asthmatic patients revealed attenuated first response to PAF stimulation—increase in [Ca²⁺]_i (A[Ca²⁺]_i) was 1.3-fold lower in cells of asthmatics (P < 0.05) versus PMNL of healthy subjects. As determined in experiments with low extracellular calcium concentration, Ca²⁺ release from internal stores tended to be increased in asthmatics and hence the difference in total Ca²⁺ response was related to decrease in Ca²⁺ influx. Thus the contribution of Ca²⁺ from internal stores to the total first Ca²⁺ response upon PAF stimulation was two-fold higher (58 ± 18 vs. 29 ± 8%, P < 0.001) in PMNL of asthmatics compared with healthy subjects. Two subsequent Ca²⁺ responses evoked by stimulations with the agonist in 1 mM Ca²⁺ buffer did not differ between study groups. In low Ca²⁺ buffer PMNL of 50% of asthmatics responded to the second stimulation while cells of healthy subjects remained unresponsive. The altered Ca²⁺ responses in PMNL of asthmatic subjects may reflect previous contact with mediator(s) that occur in vivo which may be at least partially explained by the phenomenon of down regulation reported for PAF receptor upon cell stimulation.

Protein expression of microsomal GST-II and LTC₄ synthase was analyzed by Western blot. Correlation between a 17 kDa band and LTC₄ formation was observed for both enzymes. The expression of microsomal GST-II was several fold more efficient than the expression of LTC₄ synthase. In addition to catalyzing the biosynthesis of LTC₄, microsomal GST-II also produces another product, which has been subjected to mass spectrometric analysis. This analysis demonstrates that the novel product is an isomer of LTC₄.

Arachidonyltrifluoromethyl ketone (ATFMK), an analogue of arachidonic acid (AA), inhibits an 85 kDa cytosolic phospholipase A₂ enzyme. Exposure of HIT insulinoma cells to ATFMK induced a delayed, sustained, and irreversible increase in cytosolic [Ca²⁺] that required extracellular Ca²⁺ and a concentration-dependent inhibition of depolarization-induced increases in cytosolic [Ca²⁺] prior to onset of the delayed response to ATFMK. These results suggest a disruptive effect of ATFMK on calcium mobilization which may contribute to its effects on insulin secretion from β-cells.

The contribution of several vasoactive mediators such as histamine, serotonin, bradykinin, arachidonic acid metabolites and PAF to vascular permeability changes was determined in a rat model of acute endotoxemia. Lipopolysaccharide (10–40 mg/kg, i.v.) from E. coli 0127:138 (LPS) elicited an increase in Evans blue extravasation in trachea, thymus, seminal vesicle and stomach, whereas other organs remained unaffected. LPS (25 mg/kg)-induced extravasation was not inhibited by intravenous pretreatment with histamine (H₁) antagonist mepyramine (5 mg/kg) or bradykinin (B₂) antagonist HOE-140 (0.1 mg/kg), whereas other standard drugs selectively inhibited leakage in particular tissues, e.g. the cyclooxygenase inhibitor indomethacin (5 mg/kg) in trachea (78%) and seminal vesicle (64%), the serotonin and H₁ antagonist cyproheptadine (2 mg/kg) in trachea (88%) and stomach (56%) and the dual cyclooxygenase/lipoxygenase inhibitor phenidone (10 mg/kg) in seminal vesicle (87%). PAF antagonists lexipafant and UR-12460 (10 mg/kg), but not apafant, potently inhibited extravasation in trachea (59, 84%) and seminal vesicle (81, 78%) and in stomach only UR-12460 (52%), whereas all of them were ineffective in thymus. When extravasation was induced by PAF (4 μg/kg) a low dose (0.1 mg/kg) of the three PAF antagonists strongly reduced extravasation in thymus and seminal vesicle, whereas lexipafant and UR-12460 did so in trachea (82, 100%) and only lexipafant in stomach (100%). Mepyramine, cyproheptadine, HOE-140 and indomethacin did not inhibit the effect of PAF, whereas phenidone inhibited it by 58% in trachea. These results suggest that most of the LPS-induced increase in vascular permeability is mediated by secondary vasoactive mediators among which PAF plays a pivotal role, although their relative contribution may vary from tissue to tissue.

Yangambin, a new naturally-occuring platelet activating receptor (PAF) receptor antagonist competitively displaced [³H]-PAF from its high affinity binding sites on washed human platelets with a Ki value of 1.1 ± 0.3 μM (n = 3). Studies carried out in parallel demonstrated that SR 27417, a newly-developed PAF receptor antagonist also antagonized [³H]-PAF binding to these cells with a Ki value of 51 ± 2 pM. SR 27 417 (N-(2-dimethylamino ethyl)-N-(3-pyridinyl methyl) [4-(2,4,6-triisopropyl phenyl) thiazol-2-yl] amine) selectively and competitively inhibited the specific binding of [³H]-PAF on human polymorphonuclear leukocytes (Ki = 65 ± 5.2 pM) whereas high doses of yangambin remained ineffective. Yangambin inhibited PAF-induced aggregation of human platelets in vitro (IC₅₀ = 1.0 ± 0.2 μM) but had no effect PAF-induced oxidative burst in human polymorphonuclear leukocytes. In guinea pigs, yangambin inhibited PAF-induced thrombocytopenia but did not affect leukocytopenia whereas SR 27 417 afforded complete protection against both PAF-induced thrombocytopenia and leukocytopenia. In conclusion, yangambin discriminates between two different types of PAF receptors on platelets and polymorphonuclear leukocytes and can be considered as the first PAF receptor antagonist described to date exhibiting such an effect.

Hexadecylphosphocholine (HePC) reduced the growth of the human mammary tumor, MX-1, in the athymic nude mouse similar to the fish oil, MaxEPA. When used together, HePC and MaxEPA were additive towards reducing tumor growth. An unsaturated alkylphosphocholine mixture, ShisoPC, was not as effective as HePC in reducing tumor growth. MaxEPA reduced tumor PGE₂ levels greater than 90%, while HePC and the ShisoPC only reduced tumor PGE₂ 40–60% with HePC being slightly better than ShisoPC. MaxEPA markedly increased the cellular ω3 fatty acids and decreased 20:4ω6, the substrate for PGE₂. HePC did not alter the tumor fatty acid composition, but it significantly lowered the total fatty acid concentration of the tumor by about 47%. In addition, phosphatidylcholine and sphingomyelin decreased in tumors from animals treated with HePC, and alterations in other phospholipids also were noted. These data suggest that different mechanisms exist for HePC and fish oil in reducing tumor growth.

Phospholipase A₂ (PLA₂) activity is an important contributor to destructive cellular processes in the central nervous system. Two cytosolic forms of calcium dependent PLA₂ have been characterized in the gerbil brain and the neuronal cultures from rat brain. PLA₂ enzymatic activity in cell free extracts from cortical neuronal cultures is upregulated after cells are exposed to glutamate. Brief exposure to a calcium ionophore or phorbol 12-myristate 13-acetate (PMA) stably enhanced PLA₂ activity. Stable activation of the two cytosolic forms of PLA₂ occur prior to evidence of cell death and this activation is reversible. The larger molecular mass form was characterized as cPLA₂. The smaller form (∼ 14 kDa) was distinct from Group I and II PLA₂. Exposure to glutamate shifted the calcium activation curve of the smaller form to the left suggesting a novel mechanism of regulation of PLA₂. Glutamate-induced stable enhancement of PLA₂ activity, by processes involving calcium and protein kinase C activation, is a potential molecular switch likely mediating changes in synaptic function and contributing to excitotoxicity.

The effects of changing the composition of membrane lipids on protein kinase C (PKC) activation were studied in MCF-7 human breast cancer cells. The supply of linoleate or α-linolenate to MCF-7 cells altered cell membranes fatty acid composition but did not affect PKC activity. When the cells were additionally exposed to IGF-1, the same fatty acids caused a dramatic increase in membrane-bound PKC activity. We also found that the mitogenic response induced by IGF-1 was not enhanced in those conditions when PKC becomes activated by linoleate and α-linolenate. These data show that these fatty acids elicit a distinct route for the transmission of IGF-1 signal by inducing the PKC pathway. They suggest that linoleate and α-linolenate could control the biological response of MCF-7 cells to IGF-1.