In Vivo Effect of Pancreatic Phospholipase A2 on the Arachidonic Acid Cascade

Summary Background. In acute pancreatitis, pancreatic phospholipase A₂ increases in systemic circulation. Yet the pathophysiological significance is controversial, because previous in vitro studies have shown that the enzyme has little cytotoxicity or ability to activate the arachidonic acid cascade by itself in contrast to other isozymes. Aim of the Study. The aims of this study are to examine the effect of pancreatic phospholipase A₂ on the arachidonic acid cascade in vivo; to explain the discrepancy, if present, between in vitro and in vivo findings; and to reassess the pathophysiological significance of circulating pancreatic phospholipase A₂. Methods. Pancreatic phospholipase A₂ was infused intravenously in guinea pigs, and changes in the arachidonic acid cascade, plasma lipoprotein, and cardiopulmonary function were investigated. Results. Plasma concentrations of 6-keto-prostaglandin F_1α, prostaglandin E₂, and thromboxane B₂ increased after intravenous (iv) infusion of pancreatic phospholipase A₂. Some of the plasma phospholipids such as phosphatidylcholine and phosphatidylethanolamine decreased, and free dihomo-γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid were detected in plasma. These changes were accompanied with decreases in blood pressure, heart rate, and base excess. Conclusion. Circulating pancreatic phospholipase A₂ activates the arachidonic acid cascade, probably by supplying free eicosanoid precursors from plasma lipoprotein to eicosanoid-producing cells. It is supposed to be a cause of systemic complications in acute pancreatitis.     

Tetanus toxin modulates serotonin transport in rat-brain neuronal cultures

As has been previously described, tetanus toxin (TeTx) and its H_C fragment inhibit the sodium-dependent 5-hydroxytryptamine (5-HT) uptake in rat-brain synaptosomes, probably through a kinase mechanism affecting the 5-HT transporter. Now, the inhibition of 5-HT uptake in neurons in primary culture by TeTx in a dose-dependent manner is described in this work. This effect is also produced by the nontoxic C-terminal fragment of the TeTx heavy chain (Hc-fragment), indicating that 5-HT uptake inhibition is a consequence of the toxin binding to the plasmatic membrane and not to its catalytic activity. This conclusion is supported by the fact that the 5-HT accumulation was not inhibited by the light chain of TeTx or the toxoid, and was even potentiated by botulinum neurotoxin A. These results correlate with the activation of phosphoinositide-phospholipase C activity in the cultures used in this study, this activity only being enhanced by TeTx and by its Hc-fragment. On the other hand, the use of tyrosine phosphorylation modulators indicates that both Na₃VO₄ and basic fibroblast growth factor (bFGF) produce an enhancement of 5-HT uptake in this system, which is also sensitive to TeTx inhibition. On the other hand, genistein alone is able to reduce the 5-HT transport in cultured neurons, and this effect did not appear to be additive to that elicited by TeTx. This result suggests that TeTx and genistein may share some events in their respective mechanisms of action. Furthermore, the incubation at different concentrations of 12-0-tetradecanoylphorbol 13-acetate (TPA) confirms the involvement of protein kinase C (PKC) in 5-HT transport modulation in rat-brain neuronal primary cultures. In summary, we shall demonstrate in this work that TeTx induces, through its Hc fragment, an inhibition of both basal and stimulated serotonin uptakes in primary neuronal cultures, in parallel to the activation of phosphoinositide-phospholipase C activity and PKC activation.     

白藜芦醇在体外对ADP诱导人血小板聚集的抑制作用及其机制

白藜芦醇(resveratrol，RESV)是一类广泛存在于葡萄、何首乌、花生等多种天然植物中的多酚类化合物。本研究观察RESV在体外对腺苷二磷酸(adenosine diphosphate，ADP)诱导健康志愿者血小板聚集、血小板膜结合纤维蛋白原(platelet membrane-bound fibrinogen，PFig)的抑制作用及其机制。应用血小板聚集仪、流式细胞仪及蛋白印迹技术(Western blotting)，研究RESV和磷脂酶Cβ抑制剂(phospholipase Cβ inhibitor，U73122)对ADP诱导的健康志愿者血小板聚集、PFig及血小板磷酸化磷脂酶Cβ3(phospho-phospholipase Cβ3，P-PLCβ3)和总磷脂酶Cβ3(total-phospholipase Cβ3，T-PLCβ3)蛋白表达的影响。与对照组相比，RESV(终浓度分别为25、50和100 μmol·L^-1)剂量依赖性地抑制ADP诱导的血小板聚集及Pfig、RESV与U73122对血小板聚集及PFig的抑制有叠加作用。RESV(终浓度为50 μmol·L^-1)还降低血小板P-PLCβ3蛋白的表达，降低血小板P-PLCβ3/T-PLCβ3的表达比率。RESV可能通过抑制健康志愿者血小板磷脂酶Cβ(phospholipase Cβ，PLCβ)的活性，降低了ADP诱导的血小板聚集及PFig，提示RESV有明确的抗血小板作用，具有新型抗血栓药物的研究前景。     

Asthma and leukotrienes: antileukotrienes as novel anti-asthmatic drugs

Antileukotriene drugs inhibit the formation or action of leukotrienes, which are potent lipid mediators generated from arachidonic acid in lung tissue and inflammatory cells. The leukotrienes were discovered in basic studies of arachidonic acid metabolism in leucocytes 20 years ago and were found to display a number of biological activities which may contribute to airway obstruction. Clinical studies with antileukotriene drugs have indeed demonstrated that leukotrienes are significant mediators of airway obstruction evoked by many common trigger factors in asthma. Moreover, treatment trials have established that this new class of drugs has beneficial anti-asthmatic properties, and several antileukotrienes have recently been introduced as new therapy of asthma. This communication presents an overview of the biosynthesis of leukotrienes, their biological effects and clinical effects of antileukotrienes in the treatment of asthama.     

Reduction of human recombinant type II phospholipase A2 and prostaglandin F2 alpha release by microtubule depolymerizing agents

1. The present study examines the effects of the microtubule depolarizing agent colchicine on secretory type II phospholipase A2 (PLA2) function in Chinese hamster ovary (CHO) cells that specifically overexpress human type II PLA2 and the effect of both colchicine and tubulazole on the release of type II PLA2 and prostaglandin (PG) F2 alpha from human placental explants. 2. Significant suppression by colchicine (0.01-10 mumol/L) of PLA2 activity (P < 0.00001), immunoreactive type II PLA2 (irPLA2; P < 0.00001) and PGF 2 alpha release (P < 0.01) was observed in medium from overexpressing CHO cells. These effects were significantly reduced (P < 0.0001) in the presence of 10 mumol/L taxol, an agent that prevents depolymerization of microtubules. The addition of 30 mumol/L arachidonic acid significantly reduced (P < 0.0001) the inhibition of PGF2 alpha production in CHO cell lines. 3. The addition of 1 mumol/L colchicine to human placental explants for 24 h significantly reduced irPLA2 (P < 0.00001) and PGF2 alpha production (P < 0.00001). Similarly, 1 mumol/L tubulazole significantly blocked irPLA2 (P < 0.001) and PGF2 alpha (P < 0.0001). 4. At 10 mumol/L, taxol significantly reduced irPLA2 inhibition by colchicine (n = 8; P < 0.05) and tubulazole (n = 8; P < 0.05). Similarly, taxol significantly reduced the reduction in PGF2 alpha production caused by colchicine (P < 0.001) and by tubulazole (P < 0.001). 5. These results suggest that integrity of the microtubule system is required for PLA2 function and the subsequent production of pro-inflammatory mediators.     

Serum phospholipase A2 in intensive care patients with peritonitis,multiple injury,and necrotizing pancreatitis

Summary To study the source and role of circulating phospholipase A₂ (PLA₂) catalytic activity we monitored the serum from patients with necrotizing pancreatitis (n=8), diffuse peritonitis (n=6), and multiple injuries (n=11). Immunoreactive PLA₂ serum protein concentration was analysed using a fluoroimmunoassay based on an antibody against human pancreatic PLA₂. Serum PLA₂ catalytic activity was analysed using a radiochemical method based on a substrate with tritiated palmitic acid in beta position. In necrotizing pancreatitis immunoreactive PLA₂ and PLA₂ catalytic activity both increased. Obviously, in necrotizing pancreatitis the major part of serum catalytic activity stems from the pancreas. In patients with diffuse peritonitis and multiple injuries, as a rule, immunoreactive phospholipase A₂ serum concentration appears to be within the normal range. In contrast, in these patients we demonstrated high serum catalytic PLA₂ activity comparable to that in necrotizing pancreatitis. The source of catalytic PLA₂ activity in peritonitis and multiple injuries seems not to be the pancreas. There was a correlation between pulmonary insufficiency and serum PLA₂ catalytic activity in patients with necrotizing pancreatitis, peritonitis, and multiple injuries.     

Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3′-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca²⁺-dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded β-barrel, reveals that the active site is located between the barrel wall and an α-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca²⁺ forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.     

Phospholipase production in morphological variants of Candida, albicans

The yeast Candida albicans is considered a dangerous opportunist in a compromised host. Both phases of growth are thought to be pathogenic, however, evidence suggests that the hyphal phase is the more virulent. It has been proposed that the increased virulence lies in the ability of hyphae to digest and penetrate host tissue, thus enabling access of fungal cells to the deeper tissues. However, this one characteristic does not sufficiently explain the organism's success as a pathogen. Recently, high-frequency, colonial morphology switching systems were described in C. albicans. We obtained some of these variants and tested them for the ability to produce extracellular phospholipase(s), a generally accepted mechanism of pathogenesis in many microorganisms. Using egg yolk agar plates, we showed that all variants produced the enzyme. However, one produced significantly more than the others.