Journal of lipid mediators and cell signalling最新文献

The requirement of a trans double bond for the biological action of ceramide was assessed by comparing the apoptosis-inducing activity of various ceramide analogs. The cis isomer and an acetylene type derivative of sphingosine were chemically synthesized, and the 2-amino moiety was acylated with hexanoic acid. These cell-permeable ceramide derivatives were compared with N-hexanoyl sphingosine (C₆-Cer) or N-hexanoyl dihydrosphingosine (C₆-DH-Cer) in their activity to induce apoptosis of HL60. Either the cis isomer of C₆-Cer (C₆-cis-Cer) or a triple bond derivative (C₆-TRP-Cer) induced apoptosis when assessed by fluorescence microscopy of the morphological changes and electrophoretic analysis of DNA C₆-TRP-Cer yielded the highest percentage of apoptotic cells corresponding to three times that was induced by C₆-Cer. C₆-cis-Cer also showed stronger activity than C₆-Cer. The minimum amounts of C₆-TRP-Cer and C₆-cis derivative required to induce apoptosis were 0.1 and 0.5 μM, respectively, while 1 μM C₆-Cer was required to exhibit the activity. C₆-DH-Cer showed very low but significant activity above 10 μM. N-acetyl-sphingosine (C₂-Cer) induced more apoptotic cells than C₆-Cer, and C₂-TRP-Cer was much more potent than C₂-Cer. These observations suggest that the trans configuration of ceramide is not necessarily essential for the activity to induce apoptosis. In addition, distinctive activity of C₆- or C₂-TRP-Cer suggests that this ceramide analog might be useful for developing a new type of antitumor drug.

Platelet-activating fator (PAF) is present in the human bone marrow. We have investigated the effect of PAF and antagonists (BN 52021 and CV 3988) on the growth of human marrow stromal cells. PAF (1 μM) stimulates and PAF antagonists (0.1–1 μM) inhibit [³H]thymidine incorporation in cells grown in 5% serum. The catabolism of PAF by stromal cells was inhibited by CV 3988 suggesting the presence of specific PAF receptor on cells. PAF and antagonists (0.1 nM–10 μM) had no effect on cells cultured in high serum concentration (20%) or in low serum concentration (1%) with 0.5 ng/ml of basic fibroblast growth factor (bFGF). This study indicates for the first time that PAF modulates the serum-induced but not the bFGF-induced growth of marrow stromal cells. The interactions between PAF and stromal cells during inflammatory marrow events such as myelofibrosis deserve to be assessed.

Starting from a series of compounds which were known to be PAF antagonists, we have synthesized molecules that are good inhibitors of PLA₂s of groups I or II, with IC₅₀ in the micromolar range (Binisti et al., 1997). In this report we investigate the mechanism of inhibition of bovine and porcine pancreatic phospholipases A₂ (group I), and platelet lysate phospholipase A₂ (group II) by one of these compounds, 1-(4′-methoxybenzoyl)-2-n-tridecylpiperazine (PMS 832). We show that PMS 832 behaves as a reversible, competitive inhibitor, with K_i values of 4.1±1.2 and 1.5±0.4 μM for porcine pancreatic phospholipase A₂ and platelet lysate phospholipase A₂, respectively. PMS 832 failed to inhibit platelet activation induced by several agonists and was also found to be inactive towards phospholipase C from Bacillus cereus, indicating a high specificity for phospholipase A₂ inactivation. Thus, PMS 832 and its derivatives could serve as interesting tools to investigate the role of extracellular phospholipases A₂ in inflammatory processes, and may be useful in the development of new anti-inflammatory agents.

Freshly isolated human marrow mononuclear cells produce lipidic compounds such as PAF and leukotrienes. These lipidic molecules act on human marrow myelopoiesis and erythropoiesis by modulating the growth of committed progenitors (CFU-GM and BFU-E) in vitro. Nanomolar concentrations of leukotriene B₄ and C₄ stimulate the growth of human marrow CFU-GM. In contrast, micromolar concentrations of lipoxygenase inhibitors (NDGA and BW755C) decrease their growth suggesting a role for endogenous lipoxygenase metabolites in this process. Micromolar concentrations of prostaglandin E₂ up-regulate and down-regulate the growth of marrow BFU-E and CFU-GM, respectively. In contrast, the other cyclooxygenase metabolites have no effect. Recent studies indicate that nanomolar concentrations of PAF decrease the growth of CFU-GM and BFU-E from purified marrow CD34⁺ cells. Together these results indicate that lipidic mediators act on human myelopoiesis and erythropoiesis. However at this time the mechanisms and molecular signals mediating the effects of lipidic molecules on human marrow cells are unexplored.

14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid has been reported to induce adhesion of a monocyte cell line (U-937) to cultured endothelial cells. In this study, we identified a population of specific, high affinity binding sites for 14(R),15(S)-EET in U-937 cell surface with K_d of 13.84±2.58 nM and B_max of 3.54±0.28 pmol/10⁶ cells. The specific binding of [³H]-14,15-EET on U-937 cells is more effectively displaced by 14(R),15(S)-EET than the 14(S),15(R)-isomer thus indicating stereospecificity. The binding was sensitive to various protease treatments suggesting the binding site is protein in nature. 14,15-EET binding in U937 cells is attenuated by cholera toxin (CT) and dibutyryl cAMP. Mean binding site density (B_max) decreased 31.61% and 34.8% by the pretreatment with cholera toxin (200 μg/ml) and dibutyryl cAMP (300 nM), respectively, without affecting the dissociation constant. Under similar conditions, pertussis toxin (20–200 ng/ml) was less effective as compared to CT and dibutyryl cAMP. The down regulation of 14,15-EET binding caused by dibutyryl cAMP in U-937 cell was reversed by a specific protein kinase A (PKA) inhibitor, H-89, but not by the PKC inhibitor K252a. Thus, the results suggest that the specific binding site of 14,15-EET in U-937 cells is associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal transduction mechanism. We propose that the signal transduction mechanism of 14,15-EET begins with the binding of the receptor, which leads to the increase of intracellular cAMP levels and the activation of PKA, and finally with the down regulation of 14,15-EET receptor binding.

Evidence is accumulating suggesting that platelet-activating factor plays a role in inflammatory dermatoses. Mass spectrometric methods were used to examine the molecular species of sn-2 acetyl glycerophosphocholines (GPC) synthesized by primary cultures of human neonatal foreskin-derived keratinocytes. Ionophore-stimulated keratinocytes synthesize both 1-alkyl and 1-acyl sn-2 acetyl-GPC, and the relative amounts were as follows: hexadecyl>palmitoyl>octadecyl>stearoyl at the sn-1 position. PAF synthesis in the keratinocyte-derived cell line HaCaT was inhibited by dexamethasone, suggesting that the anti-inflammatory effects of glucocorticosteroids in inflammatory dermatoses might be in part related to the inhibition of the synthesis of mediators such as PAF.

Platelet-activating factor (PAF) is a powerful phospholipid-derived autacoid involved in many physiopathological mechanisms. Many PAF antagonists have been synthesized and evaluated as therapeutic candidates. In a previous report, we have described an electronic pharmacophore of PAF antagonists using the molecular electrostatic potential. In the present study, a molecular lipophilicity potential is used to compare the hydrophobic properties of 49 'heterocyclic sp² nitrogen' highly potent PAF antagonists, belonging to six structurally different series (nine hetrazepines, five pyrrolo[1,2-c]thiazoles, 14 carboxamides, nine dihydropyridines, nine pyridinel-thiazolidines and three imidazo[4,5-c]pyridines). Their common features consist of three hydrophilic (HYDz, HY14₃B and HYD3) and two lipophilic zones (LIP₃ and LIP₄), defining the lipophilic pharmacophore of the antagonists. This pharmacophore is also characterized by several zone-to-zone distances: HYD₃-HYD₂ = 1.3 ± 1.0 Å, HY₃B-HYD₂ = 7.8 ± 1.1, HYD₃-HY₃B = 5.1 ± 1.1 Å, LIP₄-LIP₃ = 5.4 ± 1.1 Å, LIP₃-HYD₂ - 11.3 ± 1.6 Å, LIP₃-HY₃B = 5.9 ± 1.0 Å, LIP₃-HYD₃ = 4.3 + 0.9 Å, LIP₄-HYD₂ = 14.7 ± 1.6 Å, LIP₄-HY₃B = 8.1 ± 1.2 Å and LIP₄-HYD₃ = 3.9 ± 1.1 Å. These results represent a new step in the determination of a global pharmacophore for PAF antagonists.

Platelet-activating factor (PAF) receptor in normal Golden Syrian hamster lung was characterized using radioligand binding studies and compared with guinea pig lung PAF receptor. [³H]WEB2086, a potent and specific PAF antagonist, was used as a radioligand for equilibrium binding, kinetic studies, competitive binding in receptor preparation (0–110 000 g fraction of lung homogenate) from hamster and guinea pig lungs. Binding of [³H]WEB 2086 to the receptor preparation was saturable, reversible and specific in both hamster and guinea pig lungs. Scatchard plot analysis of equilibrium binding data indicates a single binding site in hamster lung with the equilibrium dissociation constant (K_D) of 66.1 ± 36.7 nM (n = 4) and maximal binding (B_max) of 135.4 ± 63.1 fmol/mg, but two binding sites in guinea pig lung with a high affinity site (K_D = 1.7 ± 0.6 nM; B_max = 48.6 ± 2.6 fmol/mg) and a low affinity site (K_D = 83.8 ± 32 nM; B_max = 480.8 ± 158 fmol/mg). The heterogeneity of [³H]WEB2086 binding to guinea pig lung but not to hamster lung was also confirmed by dissociation kinetic studies, in which biphasic dissociation kinetic was shown in guinea pig and monophasic kinetic in hamster lung. Although the specific [³H]WEB 2086 binding to lungs of both species was displaced by PAF-C18 and antagonists L659989 and CL 184005 in a dose-dependent manner and not by lyso-PAF (a biologically inactive form of PAF), the potencies of the competitive inhibition were significantly different between the two species. The relative potencies ranked WEB2086 ≈ L659989 > PAF > CL184005 in hamster lung, whereas in guinea pig lung the potencies ranked PAF > WEB2086 ≈ L659 989 ≈ CL184005. The present study demonstrates for the first time the existence of PAF receptor in the hamster lung and its binding characteristics different from guinea pig lung suggest the possible existence of different PAF receptor subtypes in hamster lung.

Inhibitors of Ca²⁺-calmodulin (CaM)-dependent protein kinases strongly modify phospholipid metabolism. Two compounds, KN62 and KT5926 recognized as blockers of Ca²⁺ -CaM-dependent protein kinase II, induced a specific increase in phosphatidylserine (PtdSer) synthesis without noticeable changes in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) biosynthesis. The increase of PtdSer synthesis was dependent on the presence of Ca²⁺ in the incubation medium and was impaired in cells whose Ca²⁺ stores were depleted by pretreatment with CD3 mAb, thapsigargin or EGTA. The mechanism of the stimulation of PtdSer synthesis by these two compounds seems to involve an accumulation of Ca²⁺ into the endoplasmic reticulum, possibly due to an increased activity of the endoplasmic reticulum Ca²⁺ -ATPase. By contrast, ML-7 and ML-9, two inhibitors of the myosin light chain kinase (MLCK), another Ca²⁺-CaM-dependent kinase, were both capable of increasing PtdSer synthesis and decreasing PtdCho and PtdEtn synthesis, reproducing the effect previously described with CaM-antagonists. The increase of PtdSer caused by ML-7 and ML-9 was Ca²⁺-dependent while the inhibition of PtdCho and PtdEtn synthesis was not. The use of these four protein kinase inhibitors thus suggests the possible existence of two CaM-dependent pathways that differentially regulates phospholipid metabolism in T cells.

Lyso platelet-activating factor (PAF) is the precursor of PAF, an inflammatory phospholipid molecule present in human bone marrow. The present study shows that in healthy volunteers lyso PAF concentrations are significantly lower (P=0.0001, Mann-Whitney U-test) in bone marrow plasma (594 ± 67 ng/ml, n = 47) than in blood plasma (1448 ± 99 ng/ml, n = 31). Marrow plasma lyso PAF concentrations are similar in patients with lymphoid and nonlymphoid malignancies as compared with controls. Freshly isolated mononuclear marrow cells and cultures of marrow stromal cells contain lyso PAF. Experiments with [³H]lyso PAF indicate that human mononuclear bone marrow cells and marrow stromal cells actively acylate lyso PAF into a 1-alkyl analogue of phosphatidylcholine. Results of this investigation indicate: (1) that lyso PAF is present in human marrow cells and plasma; and (2) that marrow cells and stromal cells metabolize it, thus suggesting their role in the regulation of lyso PAF amounts in human bone marrow.