Platelet-activating factor modulates phospholipid acylation in human neutrophils

The present study showed that platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), but not lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) rapidly (within 15 sec) stimulated the incorporation of both [1-¹⁴C]arachidonate and [1-¹⁴C]docosahexaenoate into phosphatidylinositol (PI) and phosphatidylcholine (PC) in human neutrophils. Concomitantly, it inhibited the formation of labeled phosphatidic acid from both fatty acids. The magnitude of stimulation (percentage of control) was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. It reached a maximum at 10⁻⁷ M and started to decline at 10⁻⁶ M. Extracellular Ca²⁺ was not essential for the action of PAF on phospholipid acylation. The distribution of labeled arachidonate in the molecular species of PC was not altered by PAF after 1 min incubation, suggesting that the increased formation of arachidonyl-PC during the early stage of neutrophil-PAF interaction was not originated from the added PAF. No measurable changes in the mass of each phospholipid were detected in neutrophils challenged by PAF from 15 sec to 2 min. The data suggest that the increased incorporated of extracellular fatty acids into PI and PC elicited by PAF may be secondary to increased deacylation of these phospholipids, and the magnitude of stimulation reflects the specificity of acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA.     

Leukotriene B4 promotes phospholipid acylation in human neutrophils

The present study was undertaken to test the hypothesis that leukotriene B₄ (LTB₄) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB₄ (1.5 to 150 nM) for 1 for 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferases catalyzing acylation of the respective lysophospholipids. LTB₄ stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid but not by arachidonic acid. LTB₄ and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB₄ or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB₄-activated neutrophils reflects activation of phospholipase A₂ and acyltransferases as well as ofde novo phospholipid synthesis.     

Platelet-activating factor acetylhydrolase activity in human tissues and blood cells

Human tissues, blood cells, and plasma have enzymes that catalyze the hydrolysis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The activities are not due to phospholipases A₂ that hydrolyze long chain acyl groups at thesn-2 position of glycerophospholipids, since they are calcium-independent and are specific for hydrolysis of short chain acyl groups. We examined the biochemical properties of these PAF acetylhydrolase activities (EC 3.1.1.47) in homogenates of human liver and spleen, in white blood cells (neutrophils and monocytes), and in erythrocytes. The data suggest that the plasma and intracellular PAF acetylhydrolase activities are likely due to different proteins. Second, the intracellular PAF acetylhydrolase activities in liver and spleen share several biochemical features that differentiate them from the activities in blood cells. Third, the activities in monocytes and neutrophils have properties that differentiate them from the activity present in human erythrocytes. Finally, the erythrocyte activity has unique properties that place it in a separate category of short chain acylhydrolases. In conclusion, there is a family of distinct enzymes that can be identified as PAF acetylhydrolases based on their calcium-independence and specificity for a short residue at thesn02 position of phospholipids. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor stimulates receptor-mediated fromation of reactive oxygen intermediates in human monocytes

Stimulation of production of reactive oxygen intermediates (ROI) was examined in human peripheral blood monocytes by luminol-dependent chemiluminescence. The dose-response cureve characterizing the dependence of ROI production on the concentration of platelet-activating factor (PAF) showed that stimulation occurred within a concentration range of 2×10⁻⁹M to 5×10⁻⁶M. Transformation of the dose-response curve to an Eadie-Hofstee plot indicated that the process is characterized by two K_m values. The K_m value corresponding to the high-affinity branch of the curve is 1.3±0.14 nM. In the same cells, the dissociation constant for the [³H]PAF/receptor complex was determined. The K_d value was 0.8±0.1 nM, which agreed quite well with the high-affinity K_m value obtained in the Eadie-Hofstee plot. The data indicate that stimulation of ROI generation is mediated through PAF binding at specific receptor sites at nanomolar PAF concentrations. Along with the specific receptor-mediated ROI generation, a nonspecific effect of PAF at a high concentration was demonstrated. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor in cardiovascular stress situations

Since the elucidation of its chemical structure two decades ago, platelet-activating factor (PAF) has emerged as an important mediator of various cardiovascular stress situations. Most notably, PAF was implicated as a key factor in the septic shock syndrome, based on the similarities between endotoxin and PAF biological effects, the elevation of circulating and tissue levels of PAF during endotoxemia, and the protective effect of PAF antagonists in the septic state. In addition, accumulating data suggest the involvement of PAF in the pathophysiological processes associated with ischemia, hemorrhage and trauma, where PAF exerts its effects directly on cells and blood elements or indirectly through interactions with other mediators such as cytokines and prostaglandins. Nevertheless, the relative contribution of PAF to the pathophysiological processes in endotoxemia is still unknown and should await further investigations. The primary aims of this chapter are: to delineate the effects of PAF on the cardiovascular system, to summarize the data which suggest the involvement of PAF in stress situations of the cardiovascular system, and to identify areas where future experimental efforts should be focused. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Synergistic induction of phospholipid metabolism by granulocyte-macrophage colony stimulating factor and steel factor in human growth factor-dependent cell line,M07e

Steel factor (SLF), the ligand for the c-kit protooncogene tyrosine kinase receptor, synergizes with several hematopoietic growth factors to produce greatly enhanced proliferation of normal human hematopoietic progenitor cells as well as that of the human growth factor-dependent myeloid cell line, M07e. The mechanisms of this phenomenon remain unknown. In an attempt to understand the cellular processes relevant to this phenomenon, we examined the effects of SLF and granulocyte-macrophage colony-stimulating factor (GM-CSF) on induced lipid metabolism in M07e cells. We find that both GM-CSF and SLF induced increased phosphatidylcholine (PC) turnover rates (biosynthesis and degradation) as measured by increased [³H]-choline labelling, with SLF being more potent stimulation. The labelling of aqueous intermediates of PC metabolism was also increased by cytokine stimulation, most notably phosphocholine. Simultaneous stimulation with GM-CSF plus SLF resulted in a true synergistic induction of PC, lysoPC, and phosphocholine labelling. GM-CSF and SLF each induced asymmetric labelling of various phospholipid classes as measured by incorporation of different [³H]-fatty acids. [³H]-myristic acid labelling of phosphatidylserine was most prominently induced (approximately 12-fold). Cytosolic choline kinase activity was also upregulated more than twofold over control by SLF, which might contribute to the increased phosphocholine labelling. These effects may have relevance to the intracellular mechanisms of the synergistic proliferative stimulation of SLF plus GM-CSF on M07e cells.     

Platelet-activating factor may mediate dexamethasone-induced gastric damage in the rat

The possible role of platelet-activating factor (PAF) in dexamethasone-induced gastric mucosal damage was studied in rats. PAF was measured by a platelet aggregation assay. The identity of the PAF-like product recovered from gastric tissues was ascertained by thin-layer chromatography and high-pressure liquid chromatography. Low levels of PAF were detected in the normal rat stomach, while in dexamethasone-treated animals PAF levels were significantly higher. Pretreatment of the animals with BN 52021, a specific PAF receptor antagonist, significantly attenuated dexamethasone-induced mucosal injury. These findings suggest that PAF may be a mediator of mucosal damage induced by glucocorticoids. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor may participate in signal transduction processes in rabbit leukocytes

The bacterial chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), induces the generation of platelet-activating factor (PAF), the mobilization of arachidonic acid and generation of superoxide anion (O₂ ⁻) in rabbit polymorphonuclear leukocytes (PMNs). The PAF receptor antagonists, WEB 2086 (10–100 μM) and CV 6209 (1–10 μM), reduced the mobilization of arachidonic acid and the O₂ ⁻ generation in response to fMLP but not that in response to A23187. Pretreatment of PMNs with the phospholipase A₂ inhibitor, chloroquine, or the serine protease inhibitor, tosyl-phenylalanine chloromethyl ketone, reduced the fMLP-stimulated generation of PAF and also reduced the generation of O₂ ⁻. The respiratory burst induced by a submaximal concentration of phorbol myristate acetate was not affected by these compounds. These data are consistent with the suggestion that endogenous PAF may contribute to the signal transduction cascade initiated by fMLP. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May, 1989.     

Platelet-activating factor detected in bronchoalveolar lavage fluids from an asthmatic patient

It recently has been recognized that platelet-activating factor (PAF) may be a mediator of asthma exacerbation. We had the opportunity to analyze bronchoalveolar lavage fluids from an asthmatic infant, which were characterized by neutrophil infiltration. The patient's lungs were washed on three occasions with saline during asthmatic attacks. PAF was found in each case on the basis of its ability to cause the immediate aggregation of washed rabbit platelets. The PAF detected was equivalent to 1–1.4 pmol of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, three quarters of which were recovered in cell-associated form. By contrast, we did not detect PAF in bronchoalveolar exudates from patients with larungeal stenosis or with respiratory distress syndrome. LysoPAF, the direct precursor as well as initial metabolite of PAF, was also analyzed after being converted to PAF by acetylation. There was a wide variation in the amount of lysoPAF present in individual patients, suggesting that lysoPAF levels cannot be taken as an indicator for the presence of PAF. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Compartmental study of rat renal phospholipid metabolism

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-¹⁴C]glycerol and [1-¹⁴C]arachidonic acid incorporation. By contrast, a higher amount of [1-¹⁴C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.     

Platelet-activating factor type activity in plasma from patients with septicemia and other diseases

The purpose of the present study was to determine whether increased levels of platelet-activating factor (PAF) type activity can be detected in plasma from patients with septicemia and other diseases. A level of PAF below 0.5 ng/mL of plasma was considered normal. We found that plasma from a patient with adverse anaphylactoidic reaction to intravenous analgetics contained 2.1 ng PAF/mL. In seven patients with septicemia, including urosepsis, endocarditis and peritonitis, and with positive blood culture, increased plasma PAF levels (1–20 ng PAF/mL) were observed. Other patients with clinical indications of septicemia had negative blood cultures and/or increased levels of C-reactive protein (CRP). Yet, in the plasma from these patients, no increased PAF levels were detected under the assay conditions used. Two patients with allergic asthma, requiring treatment with steroids, had no measurable plasma PAF. In the plasma from a patient with idiopathic thrombocytopenic purpura (ITP) only an “endogenous” inhibitor of PAF induced platelet aggregation was initially observed. In spite of this, the patient responded to treatment with the PAF antagonist WEB 2086 with a dramatic increase in platelet count (Lohmannet al., Lancet ii, 1147, 1988). Thereafter, also increased PAF levels (3.3 ng PAF/mL) were detected in plasma, although some “endogenous” inhibitor of PAF was still present. In conclusion, increased PAF levels in plasma from patients support a role of PAF in certain human disease states, such as in anaphylactoid reaction, sepsis and septic shock. The type, relevance and specificity of endogenous inhibitors of PAF deserve further study. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor stimulates expression of IL-1 beta mRNA in THP-1 cells

A human monocytic cell line (THP-1) was used to study the effects of PAF (platelet-activating factor) on the expression of IL-1 beta mRNA. THP-1 cells were incubated with 10 pM PAF in the presence or absence of 0.1 μg/mL endotoxin for 4 hr, after which cytoplasmic RNA was extracted and subjected to Northern hybridizations. PAF, alone and in combination with endotoxin, caused an increase in mRNA levels for IL-1 beta. The magnitude of the effects of PAF on IL-1 beta mRNA levels matched closely the effects seen at the level of protein synthesis, suggesting that the effects of PAF on IL-1 beta release may result largely from its effects on IL-1 beta mRNA levels.     

Effect of phagocytosis and lonophores on release and metabolism of arachidonic acid from human neutrophils

Challenge of human neutrophils prelabeled with [³H]arachidonate and [¹⁴C] palmitate or [¹⁴C]-stearate with opsonized zymosan or the Ca²⁺ ionophores A23187 or Ionomycin caused the release of [³H], but not [¹⁴C], fatty acid. With the ionophores, but not zymosan, considerable conversion of the [³H] arachidonate to hydroxyeicosatetraenoates occurred. Although various isomers were recovered, the 5-hydroxyeicosatetraenoate appeared to be the major product. In these experiments, no [¹⁴C] products were detected such as lysophospholipid, diglyceride or monoglyceride. Although no definitive statement can be made about the mechanism of release of arachidonate, our data are most easily interpreted as the result of the action of a phospholipase A₂.     

Platelet-activating factor and granulocyte-mediated oxidative stress. Strategy forin vivo oxyradical visualization

Platelet-activating factor (PAF) and leukotriene B₄ are potent lipid mediators of endothelium-granulocyte interaction which results in granulocyte-dependent increased microvascular permeability. The specific mechanism by which PAF induces granulocyte-mediated endothelial injury has not been fully investigated. Digital imaging photonic intensified microscopy has revealed that PAF effectively induces granulocyte-mediated oxidative stress on microvascular beds. The method has made it possible to visualize luminol-dependent photonic burst released from PAF-treated microvascular beds in the rat mesentery. The photonic activities clearly corresponded to the localization of sticking granulocytes in post-capillary venules. By contrast, no significant chemilumigenic response could be detected in the leukotriene B₄-induced activation of endothelium-granulocyte interactionin vivo. The present findings suggest that oxygen radicals are not prerequisites for granulocyte adhesion in the microcirculation and provide evidence for a dissociation ofin vivo granulocyte function between leukotactic and oxidative activation in leukotriene B₄-induced microvascular changes. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

原花青素经肠道微生态途径对脂质代谢的调节

目的研究原花青素(Proanthocyanidin,PC)经肠道微生态途径调节脂质代谢。方法取成年雄性SD大鼠和长爪沙鼠,经基础饲料适应性喂养1周后,采集尾静脉血,收集血清,检测血清总胆固醇(Total cholesterol,TC)水平,并按TC及体重分为基础对照组、模型对照组、低剂量组(25 mg/kg PC)、中剂量组(100 mg/kg PC)、高剂量组(150 mg/kg PC)、阳性对照组(非诺倍特80 mg/kg),基础对照组饲以普通基础饲料,其余各组均饲以高脂饲料,每天灌胃1次,大鼠连续灌胃8周,沙鼠连续灌胃2周。大鼠8周末、沙鼠2周末,经股动脉采血,分离血清,处死前3 d收集72 h粪便。采用全自动生化仪检测TC、甘油三酯(Triglyceride,TG)、高密度脂蛋白胆固醇(High-density lipopro-tein cholesterol,HDL-C)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol,LDL-C)、血总胆汁酸(Total bileacid,TBA)水平;循环酶法试剂盒测定粪TBA排出量水平;双抗体两步夹心ELISA法测定卵磷脂胆固醇酰基转移酶(Lecithin-cholesterol acyltransferase,LCAT)活性。解剖处死的大鼠和沙鼠,进行病理组织学分析。提取大鼠、沙鼠小肠、盲肠细菌基因组DNA,采用PCR-变性梯度凝胶电泳(Denatured gradient gel electrophoresis,DGGE)进行动物模型肠道微生态菌群多样性变化分析。结果模型对照组大鼠和沙鼠TC、TG水平明显高于基础对照组,差异有统计学意义(P<0.05),表明高脂模型建模成功。与模型对照组相比,各剂量组和阳性对照组大鼠、沙鼠TC、TG、LDL-C、血TBA水平均降低,LCAT活性、粪TBA排出量均明显升高,差异有统计学意义(P<0.05);各剂量组大鼠肝脏损伤、肝细胞肿胀、变性等病变程度明显减轻,肝组织病理变化明显改善。DGGE检测及图像分析显示,大鼠各剂量组肠道优势菌群多样性明显增加,随着PC干预剂量加大,中、高剂量组肠道菌群多样性明显减少;沙鼠低剂量组与模型对照组的肠道菌群结构基本相似,而中、高剂量组肠道菌群多样性明显减少,肠道优势菌群结构明显恢复。结论 100、150 mg/kg PC干预的实验动物模型中,肠道菌群结构多样性明显恢复,提示PC可通过肠道菌群这一靶标,进行脂质代谢的调节。     

Platelet-activating factor (PAF) induces contraction of saponin-skinned smooth muscle of coronary artery

The effect of platelet-activating factor (PAF) on the development of isometric tension by saponin-skinned coronary artery was studied. PAF caused two types of contractions of coronary smooth muscle cells (SMC):(i) rapid, transient (phasic) contractions of SMC were induced by inositol 1,4,5-trisphosphate dependent Ca²⁺ release from sarcoplasmic reticulum (SR); (ii) slow sustained (tonic) contractions were induced by increase in Ca²⁺-ensitivity of the contractile apparatus of SMC by protein kinase C activation. The present results support the hypothesis that, in SMC of coronary artery, PAF receptors are located not only on the plasma membrane, but also on the SR membranes. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Altered phospholipid metabolism in sodium butyrate-induced differentiation of C6 glioma cells

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP₃) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP₃ production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP₃ formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP₃ formation and its subsequent action, i.e., Ca²⁺ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.     

Lung lamellar bodies lack certain key enzymes of phospholipid metabolism

Palmitoyl CoA-glycerol-3-phosphate acyltransferase, phosphatidate phosphohydrolase, and phospholipase A were assayed in subcellular fractions of rat lung, including lamellar bodies, the putative site of storage and secretion of lung surfactant. The specific activity of each of these enzymes in lamellar bodies was relatively low and could be entirely accounted for by a small contamination of the lamellar bodies fraction by microsomes, as quantitated by the presence of the microsomal marker reduced triphosphopyridine nucleotide cytochromec reductase. These data indicate that lamellar bodies are not the site of synthesis of the lipid component of pulmonary surfactant by pathways involving these enzymes.