Phosholipase A2 Research Articles

The expression of secretory phosholipase A2 in the serum and the impact of intervention therapy of meconium aspiration syndrome

目的 了解胎粪吸入综合征(meconium aspiration syndrome，MAS)患儿血清中分泌型磷脂酶A2(secretory phospholipase A2，sPLA2)的表达趋势及临床意义，及给予肺泡表面活性物质(pulmoalveolar surfactant，PS)干预治疗的影响，为临床诊治提供帮助。 方法 选取2013年1月至2016年1月我院收治的重度MAS患儿(需气管插管机械通气)54例，28例入院后2 h内给予PS的MAS患儿作为PS干预组，26例未使用PS的MAS患儿作为观察组，同时选取无肺损伤的非感染性疾病患儿32例为对照组。分别于入院后第0、1、3、7、10天用ELISA法检测3组患儿血清中sPLA2的表达情况。 结果 对照组各时间点sPLA2有轻度表达，组内各时间点比较差异无统计学意义(P>0.05)；PS干预组sPLA2表达量在第3天达高峰，第0、1、3天高于对照组，差异有统计学意义(P 0.05)；观察组sPLA2各时间点表达量均较对照组、PS干预组增高，第0、1、3、7天与对照组、PS干预组相比差异有统计学意义(P 0.05)。 结论 sPLA2参与了MAS的发生发展过程，给予外源性PS有一定治疗作用，但存在局限性，给予sPLA2抑制剂可能为治疗MAS提供帮助。

Zoanthid mucus as new source of useful biologically active proteins

Palythoa caribaeorum is a very common colonial zoanthid in the coastal reefs of Brazil. It is known for its massive production of mucus, which is traditionally used in folk medicine by fishermen in northeastern Brazil. This study identified biologically active compounds in P. caribaerum mucus. Crude mucus was collected during low tides by the manual scraping of colonies; samples were maintained in an ice bath, homogenized, and centrifuged at 16,000 g for 1 h at 4 °C; the supernatant (mucus) was kept at −80 °C until use. The enzymatic (proteolytic and phospholipase A2), inhibitory (metallo, cysteine and serine proteases), and hemagglutinating (human erythrocyte) activities were determined. The results showed high levels of cysteine and metallo proteases, intermediate levels of phosholipase A2, low levels of trypsin, and no elastase and chymotrypsin like activities. The mucus showed potent inhibitory activity on snake venom metalloproteases and cysteine proteinase papain. In addition, it showed agglutinating activity towards O+, B+, and A+ erythrocyte types. The hemostatic results showed that the mucus prolongs the aPTT and PT, and strongly inhibited platelet aggregation induced by arachidonic acid, collagen, epinephrine, ADP, and thrombin. The antimicrobial activity was tested on 15 strains of bacteria and fungi through the radial diffusion assay in agar, and no activity was observed. Compounds in P. caribaeorum mucus were analyzed for the first time in this study, and our results show potential pharmacological activities in these compounds, which are relevant for use in physiopathological investigations. However, the demonstration of these activities indicates caution in the use of crude mucus in folk medicine. Furthermore, the present or absent activities identified in this mucus suggest that the studied P. caribaeorum colonies were in thermal stress conditions at the time of sample collection; these conditions may precede the bleaching process in zoanthids. Hence, the use of mucus as an indicator of this process should be evaluated in the future.

The Association between Periodontal Status, Serum Lipid Levels, Lipoprotein Associated Phosholipase A2 (Lp-PLA2) in Chronic Periodontitis Subjects and Healthy Controls.

Lipoprotein associated phospholipase A2 (Lp-PLA2) has been explored as a potential biomarker for Cardiovascular Disease (CVD). The human Lp-PLA2 is a serine-dependent, Ca2+- independent enzyme. It gives way to oxidised free fatty acids and lysophosphatidyl choline by hydrolysing oxidised phospholipids, that leads to smooth muscle cell apoptosis, inflammatory cell chemotaxis and endothelial cell dysfunction in CVD. Owing to its role in inflammation, it may influence the development and progression of periodontitis as well. To compare the demographic variables, Gingival Index(GI), Bleeding On Probing (BOP), Probing Pocket Depth (PPD), and Clinical Attachment Level (CAL) with serum lipid profile and Lp-PLA2 level in Chronic Periodontitis (CP) subjects. A total of 75 subjects were selected and divided into three groups; based on the inclusion and exclusion criteria: Group I - 25 subjects with severe generalized CP with CAL ≥ 5 mm in more than 30% of sites. Group II -25 subjects with moderate generalized CD with clinical CAL 3 mm-4 mm in more than 30% of sites. Group III- 25 systemically and periodontally healthy volunteers who served as control. Clinical parameters such as Plaque Index (PI), BOP, Probing Depth (PD) and CAL, lipid profile such as Total Cholesterol (TC), Triglyceride (TG), High density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL) as well as Lp-PLA2 were assessed. One-way ANOVA and Tukey's Post-hoc test were used for data analysis. Age was higher in Group I and II when compared to Group III. Group II showed a significant correlation between PI and LDL. In Group III, lower PI was significantly correlated with high HDL and low LDL and BOP was positively correlated with TG and HDL. A significant association of Lp-PLA2 was found to be higher with increase in the TG and VLDL level in Group I and Group II when compared to Group III. The study concluded that Lp-PLA2, TG and VLDL already being a predictor biomarker for atherosclerotic disease can be an inflammatory marker for periodontitis.

Asthenozoospermia and membrane remodeling enzymes: a new role for phospholipase A2.

Phosholipase A2 (PLA2 ) activity in the seminal plasma and in sperm heads is closely related to sperm motility and male fertility. Therefore, the purpose of this study was to investigate the possible involvement of different isoforms of phospholipase in asthenozoospermia. To accomplish this, cPLA2 , phospho-cPLA2 , iPLA2 , and sPLA2 were evaluated by immunofluorescence and immunoblot analyses in spermatozoa obtained from 22 normozoospermic men and 28 asthenozoospermic patients. We found significant differences in cPLA2 and its phosphorylated/activated form, iPLA2 , and sPLA2 content and distribution in normal and asthenozoospermic patients. cPLA2 was localized in heads, midpieces, and tails of all spermatozoa as constitutive enzyme, less expressed in the tail of spermatozoa with low progressive motility. While active phospho-cPLA2 distribution was homogeneous throughout the cell body of control-donor spermatozoa, lower levels were detected in the tails of asthenozoospermic patients, as opposed to its strong presence in heads. Low immunofluorescence signal for iPLA2 was found in astenozoospermic patients, whereas sPLA2 was significantly lower in the heads of asthenozoospermic patients. Spermatozoa with low progressive motility showed differences both in terms of total specific activity and of intracellular distribution. cPLA2 , iPLA2 , and sPLA2 specific activities correlated positively and in a significantly manner with sperm progressive motility both in normozoospermic men and asthenozoospermic patients. In conclusion, PLA2 s are expressed in different areas of human spermatozoa. Spermatozoa with low motility showed differences in total specific activity and enzyme distributions. We speculated that PLA2 expression and/or different distribution could be potential biomarkers of asthenozoospermia, one of the major causes of male factor infertility.

Evaluation of lipoprotein-associated phosholipase A2 and plaque burden/composition in young adults.

The total burden of subclinical coronary atherosclerosis is significant in young adults. Serum lipoprotein-associated phospholipase A2 (Lp-PLA2) is an established predictor of morbidity and mortality because of cardiovascular disease. The aim of the present investigation was to evaluate the relationship between subclinical coronary atherosclerosis and serum Lp-PLA2 concentrations in a population of young adults. A total of 261 individuals younger than 45 years of age who had undergone coronary computed tomography angiography were evaluated. The study group included 101 patients in whom coronary computed tomography angiography detected subclinical coronary atherosclerosis; the control group included 160 sex-matched and age-matched healthy control patients. Serum Lp-PLA2 levels were increased significantly in the study group patients compared with the control patients (15.42±11.88 vs. 8.06±4.32 ng/ml, P<0.001). Furthermore, a positive correlation was identified between the Lp-PLA2 levels and the total number of plaques and diseased arteries (r=0.495, P<0.001, and r=0.621, P<0.001, respectively). The presence of mixed plaque composition was also correlated with the Lp-PLA2 levels (r=0.657, P<0.001). Multivariate regression analysis identified four independently significant predictors of subclinical coronary atherosclerosis: high-sensitivity C-reactive protein levels, tobacco use, uric acid levels, and serum Lp-PLA2 levels. The presence of subclinical coronary atherosclerosis is associated independently with Lp-PLA2, and it has potential utility as a novel indicator of cardiovascular disease risk in the young adult population.

Molecular mechanisms of short-term habituation in the leech Hirudo medicinalis

Although habituation is ubiquitous in the animal kingdom, its underlying mechanisms remain poorly understood. In this study, we began to explore the molecular cascades underlying short-term habituation in the leech Hirudo medicinalis. In H. medicinalis, a training paradigm, consisting of low-frequency repetitive electrical stimulation of the skin, produces a gradual increase in the latency to swim that spontaneously recovers within 20–30min. As first step in determining the molecular pathways in short-term habituation, we examined the role of Ca2+. Both Ca2+ influx through voltage-gated channels and Ca2+ release from intracellular stores were found to contribute to short-term habituation. The analysis of the downstream targets of elevated cytosolic Ca2+ revealed that the activation of the phosholipase A2 was required for the induction of short-term habituation. Finally, we reported that the recruitment of arachidonic acid metabolites, generated by the 5-lipoxygenase pathway, was also necessary for the induction of swim induction habituation. These results provide the framework for a comprehensive characterization of the molecular underpinnings of habituation. This outcome will allow us to compare the mechanisms of habituation with those underlying other forms of nonassociative learning in the leech, such as sensitization and dishabituation, and, more in general, with those governing habituation in different vertebrate and invertebrate model systems.

PIBF: The Double Edged Sword. Pregnancy and Tumor

The role of progesterone-dependent immunomodulation in the maintenance of normal pregnancy. In vitro and in vivo data on the effect that progesterone and its mediator progesterone-induced blocking factor (PIBF) exert on the immune functions of pregnant women are reviewed, together with clinical findings. Activated pregnancy lymphocytes express progesterone receptors, which enable progesterone to induce a protein called PIBF. PIBF increases Th2 type cytokine production by signaling via a novel type of IL-4 receptor and activating the Jak/STAT pathway. PIBF inhibits phosholipase A2, thus reduces prostaglandin synthesis. PIBF inhibits perforin release in human decidual lymphocytes and reduces the deleterious effect of high NK activity on murine pregnancy. PIBF production is a characteristic feature of normal human pregnancy, and its concentration is reduced in threatened pregnancies. PIBF mRNA and protein are expressed in a variety of malignant tumors. Inhibition of PIBF synthesis increases survival rates of leukemic mice. Progesterone-induced blocking factor is produced by pregnancy lymphocytes and also by malignant tumors. The PIBF-induced Th2-dominant immune response is favorable during pregnancy but might facilitate tumor growth by suppressing local antitumor immune responses.

Elevated Lp-PLA 2 Levels Add Prognostic Information to the Metabolic Syndrome on Incidence of Cardiovascular Events Among Middle-Aged Nondiabetic Subjects

To explore potential interrelationships between lipoprotein-associated phospholipase A2 (Lp-PLA2), the metabolic syndrome (MetS), and incident cardiovascular disease (CVD). MetS was defined by the National Cholesterol Education Program Adult treatment Panel III criteria in 4480 nondiabetic Malmö Diet and Cancer Study subjects without history of CVD. Incidence of first CVD event (stroke [130 cases] or myocardial infarction [131]) was monitored over 10 years of follow-up. Lp-PLA2 activity and mass were significantly higher in subjects with MetS. Lp-PLA2 activity compared with Lp-PLA2 mass was more strongly correlated to individual components and increased more linearly with number of MetS components. Elevated Lp-PLA2 activity (top compared with bottom tertile), but not elevated Lp-PLA2 mass, increased risk for incident CVD (relative risk, RR: 1.54, 95% CI 1.07 to 2.24), as did MetS (1.42, 1.06 to 1.90) after taking possible confounders into account. Relative to those without either elevated Lp-PLA2 activity or MetS, combination of MetS and elevated Lp-PLA2 activity increased risk for CVD (1.97, 1.34 to 2.90). Elevated Lp-PLA2 activity without MetS increased risk for CVD (1.40, 1.03 to 1.92) but not MetS without elevated Lp-PLA2 activity (1.46, 0.94 to 2.27). Lp-PLA2 is associated to the MetS. Higher plasma levels of Lp-PLA2 increased risk for incident CVD regardless of MetS. The simultaneous presence of elevated Lp-PLA2 activity and MetS may identify an especially high risk individual.

Cytosolic PLA2 in Zymogen Granule Fusion and Amylase Release: Inhibition of GTP-induced Fusion by Arachidonyl Trifluoromethyl Ketone Points to cPLA2 in G-Protein-mediated Secretory Vesicle Fusion

Previously we reported that the G-protein Galphai3 localized in pancreatic zymogen granule (ZG) membrane participates in vesicular fusion at the cell plasma membrane (PM). In the present study, the presence of cytosolic phosholipase A2 (cPLA2) in rat ZGs was demonstrated and its potential role in G-protein-mediated ZG-PM fusion was investigated. In vitro fusion assays utilizing both enzymatic and fluorimetric techniques demonstrate that ZGs fuse with PM with a greater potency in the presence of GTP. Arachidonyl trifluoromethyl ketone (AACOCF3) at 40 microM reduces GTP-induced ZG-PM fusion by 25-50%. Anti-cPLA2 antibody reduces ZG-PM fusion in a dose-dependent manner and a 50% reduction of the fusion takes place in the range of 0.48-0.64 ratios of cPLA2 antibody to ZG proteins. PLAP, a cPLA2 activator synthetic peptide increases ZG-PM fusion in a limited dose-dependent manner and tends to inhibit at higher concentrations. Exogenous arachidonic acid inhibits GTP-induced ZG-PM fusion in a dose-dependent manner. Furthermore, a non-hydrolysable GTP analogue, Gpp(NH)p, reduces PLAP effect in ZG-PM fusion; and the net effect of Gpp(NH)p and PLAP differs significantly from the net effect of GTP and PLAP on ZG-PM fusion suggesting that cPLA2 is involved in G-protein-mediated secretory vesicle fusion.

THE ROLE OF PANCREATIC PHOSPHOLIPASE A2 IN EXPERIMENTAL PANCREATITIS

Premature intracellular activation of digestive zymogens is regarded as an initial and initiating event for acute pancreatitis. While the activation of proteolytic zymogens was shown in numerous studies, the role of other digestive enzymes is much less clear. Several authors have suggested that Phosholipase A2 is responsible for proteolytic injury of pancreatic acini. However, earlier studies have never investigated the role of the ancreatic secretory phospholipase A2 (PLA2, type 1b) in contrast to the well known and studied leukocyte PLA2, (PLA2, type 2a). Pancreatic secretory-PLA2-deficient mice were generated by targeted disruption of the PLA2 gene (Gastroenterology 2001; 120:1193-1202). Acute pancreatitis was induced by 7 hourly injections of caerulein (50μg/h, i.p.). Control mice received i.p. saline. Activities of amylase, lipase, myeloperoxidase, trypsin and PLA2 were enzymatically measured with specific substrates. Histological evaluation of pancreatic injury was performed after H&E staining, and apoptosis was analysed by 3-OH nick-end labelling. Pancreatic edema was calculated as ratio of pancreatic wet weight to body mass. CCK-induced necrosis in isolated acinar cells was measured by propidium iodide inclusion. PLA2 expression and PLA2 activity was absent in PLA2-KO animals. In wild type animals we detected a high PLA2 basal activity with a 3-fold increase after supramaximal caerulein stimulation. As an endogenous activator of PLA2 we identified Cathepsin L. Supramaximal caerulein stimulation induced early pancreatic injury as indicated by high levels of serum amylase and lipase in wild-type mice but there was no significant difference in comparison to knock-out animals. The pancreatic inflammatory reaction indicated by myeloperoxidase levels and CD45 staining was reduced; in parallel we detected a 7 fold increase in the rate of apoptosis in PLA2 KO animals. Isolated acinar cells from both animal groups showed no difference in CCK-induced necrosis or trypsin activity. Our data clearly indicate that activation of the secretory pancreatic pro-PLA2 plays a role in mediating the systemic inflammatory response, but has little or no impact on the initiation or propagation of the digestive enzyme activation cascade in pancreatic acini eventually leading to cell necrosis. The deletion of PLA2 leads to a shift of cell death from predominantly necrosis to apoptosis.

Requirement for Annexin A1 in Plasma Membrane Repair

Ca2+ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca2+, binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca2+ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca2+ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.

Cl−secretion in ATP‐treated renal epithelial C7–MDCK cells is mediated by activation of P2Y1 receptors, phospholipase A2 and protein kinase A

This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.

Role for Calcium Signaling and Arachidonic Acid Metabolites in the Activity-Dependent Increase of AHP Amplitude in Leech T Sensory Neurons

Previous studies have revealed a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) neurons of the leech Hirudo medicinalis. The firing of T cells is characterized by an AHP, which is mainly due to the activity of the Na+/K+ ATPase. Low-frequency repetitive stimulation of T neurons leads to a robust increment of the AHP amplitude, which is correlated with a synaptic depression between T neuron and follower cells. In the present study, we explored the molecular cascades underlying the AHP increase. We tested the hypothesis that this activity-dependent phenomenon was triggered by calcium influx during neural activity by applying blockers of voltage-dependent Ca2+ channels. We report that AHP increase requires calcium influx that, in turn, induces release of calcium from intracellular stores so sustaining the enhancement of AHP. An elevation of the intracellular calcium can activate the cytosolic isoforms of the phosholipase A2 (PLA2). Therefore we analyzed the role of PLA2 in the increase of the AHP, and we provide evidence that not only PLA2 but also the recruitment of arachidonic acid metabolites generated by the 5-lipoxygenase pathway are necessary for the induction of AHP increase. These data indicate that a sophisticated cascade of intracellular signals links the repetitive discharge of T neurons to the activation of molecular pathways, which finally may alter the activity of critical enzymes such as the Na+/K+ ATPase, that sustains the generation of the AHP and its increase during repetitive stimulation. These results also suggest the potential importance of the poorly studied 5-lipoxygenase pathway in forms of neuronal plasticity.

Possible synergistic prostate cancer suppression by anatomically discrete pomegranate fractions

We investigated whether dissimilar biochemical fractions originating in anatomically discrete sections of the pomegranate (Punica granatum) fruit might act synergistically against proliferation, metastatic potential, and phosholipase A2 (PLA2) expression of human prostate cancer cells in vitro . Proliferation of DU 145 human prostate cancer cells was measured following treatment with a range of therapeutically active doses of fermented pomegranate juice polyphenols (W) and sub-therapeutic doses of either pomegranate pericarp (peel) polyphenols (P) or pomegranate seed oil (Oil). Invasion across Matrigel by PC-3 human prostate cancer cells was measured following treatment with combinations of W, P and Oil such that the total gross weight of pomegranate extract was held constant. Expression of PLA2, associated with invasive potential, was measured in the PC-3 cells after treatment with the same dosage combinations as per invasion. Supra-additive, complementary and synergistic effects were proven in all models by the Kruskal-Wallis non-parametric H test at p < 0.001 for the proliferation tests, p < 0.01 for invasion, and p < 0.05 for PLA2 expression. Proliferation effects were additionally evaluated with CompuSyn software median effect analysis and showed a concentration index CI < 1, confirming synergy. The results suggest vertical as well as the usual horizontal strategies for discovering pharmacological actives in plants.

Toxicity of glutathione depletion in mesencephalic cultures: a role for arachidonic acid and its lipoxygenase metabolites.

The contribution of arachidonic acid (AA) release and metabolism to the toxicity that results from glutathione (GSH) depletion was studied in rat mesencephalic cultures treated with the GSH synthesis inhibitor l-buthionine sulfoximine. Our data show that GSH depletion is accompanied by increased release of AA, which is phosholipase A2 (PLA2) dependent. Exogenous AA is toxic to GSH-depleted cells. This toxicity is prevented by inhibition of lipoxygenase activity, suggesting participation of toxic byproducts of AA metabolism. Hydroxyperoxyeicosatetraenoic acid (HPETE), one of the primary products of AA metabolism by lipoxygenase is also toxic to GSH-depleted cells, whereas hydroeicosatetraenoic acid (HETE) is not. Cell death caused by GSH depletion is prevented by: (i) replenishment of GSH levels with GSH-ethyl ester; (ii) inhibition of PLA2 activity; (iii) inhibition of lipoxygenase activity; and (iv), treatment with ascorbic acid. These data suggest that the following events likely contribute to cell death when GSH levels become depleted. Loss of GSH results in increased release of AA, which is PLA2 dependent. Metabolism of arachidonic acid via the lipoxygenase pathway results in generation of oxygen free radicals possibly produced during conversion of HPETE to HETE, which contribute to cellular damage and death. Our study suggests that limiting AA release and metabolism may provide benefit in conditions with an existing depletion of GSH, such as Parkinson's disease.

Resistance to Endotoxic Shock in Phospholipase A2 Receptor-deficient Mice

Mammals possess various types of secretory phospholipase A2, which differ in the primary structure and tissue distribution. The phosholipase A2 receptor (PLA2R) recognizes group IB phospholipase A2 (PLA2-IB) and mediates the PLA2-IB-induced biological responses in non-digestive organs, including eicosanoid production and contraction of airway smooth muscles. In this study, we generated PLA2R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA2-IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA2R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA2-IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-alpha and interleukin-1beta elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA2R in the progression of endotoxic shock.

Changes in the levels of glycerophosphoinositols during differentiation of hepatic and neuronal cells.

Glycerophosphoinositols are metabolites formed by a phosholipase A2 and a lysolipase specifically acting on membrane phosphoinositol lipids. High levels of these compounds characterize epithelial cells and fibroblasts transformed by ras and other cellular oncogenes. Here we have analyzed the glycerophosphoinositol levels in cells that are considered models of cell differentiation. Using rat hepatocytes at different stages of liver development we have shown that the glycerophosphoinositol basal levels of fetal cells were up to fourfold higher than in adult hepatocytes. No changes in glycerophosphoinositol were observed in regenerating rat liver, a model of differentiated cells proliferating in a synchronous manner, where only glycerophosphoinositol 4-phosphate increased by 80%. Similarly to fetal hepatocytes, a modest but significant increase (30%) in the levels of glycerophosphoinositols was observed in undifferentiated NG-108-15 cells as compared to the same cells induced to differentiate by cAMP. In a different neuronal cell line, PC12 cells, increased glycerophosphoinositol levels characterized the differentiated cells. Based on these observations we suggest that high glycerophosphoinositol levels characterize cellular phenomena associated with the activation of ras/mitogen-activated protein kinase pathways.

Calmodulin-antagonism inhibits human keratinocyte proliferation

Calcium is a major regulator on intracellular metabolism. It is now established that its effects are mediated by calmodulin (CAM), a specific receptor protein [10]. The calcium-calmodulin complex influences enzymes such as adenylate cyclase, phosphodiesterase, protein kinase, and phosholipase A2 [10]. Calmodulin-dependent processes have been implicated as being important regulatory components of cell proliferation or cell-cycle progression, especially at the G1 S-boundary [1]. As keratinocytes have been shown to contain calmodulin in significant amounts [5, 12], the protein is accepted as a regulatory component of keratinocyte proliferation and differentiation [9]. Recent works have studied the role of calmodulin in the hyperproliferation of epidermal disorders, e.g., psoriasis; calmodulin levels are grossly elevated in both lesional [3, 8, 11, 14, 15] and nonlesional epidermis [4, 8, 11, 14] in psoriasis. Clearance of psoriatic plaques is accompanied by a significant overall reduction in epidermal calmodulin, irrespective of the treatment regime used [13]. Therefore we tested the effects of fendiline (Fen), a drug with potent calmodulin-antagonistic activities [6] in a system of cultured human keratinocytes; keratinocytes were removed from human skin by trypsinization (0.5% trypsin, Sigma, in PBS, 24 h at 4~ Cells were washed and placed into microtiter plates (2 x 105 cells/ml). Culture medium was RPMI 1640 (Seromed) completed with 1% nonessential amino acids, 1% L-glutamine, hydrocortisone 0.4 gg/ml, 10% fetal calf serum, 1% penicillin/streptomycin 10 E/10,000 pg/ml. The cells were incubated for 48 h (37~ 5% CO2) either in the presence or in the absence of fendiline 10 8 1 0 -5 M. In some series the tumor promotor PMA (4-beta-phorbol, 12-beta-myristate 13-alphaacetate, Sigma) was added. After a further incubation of 24 h with 3H-thymidine (Amersham, 0.5 I.tgCi/ sample) the cells were removed and filtered. Solubilized radioactivity was counted in a beta-scintillation counter.

Mechanisms of enhancement of cholesteryl ester transfer protein activity by lipolysis.

Lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from plasma high density lipoproteins (HDL) to very low density lipoproteins (VLDL). In time course studies the stimulation of cholesteryl ester transfer by bovine milk lipase was correlated with accumulation of fatty acids in VLDL remnants. As the amount of fatty acid-poor albumin in the incubations was increased, there was decreased accumulation of fatty acids in VLDL remnants and a parallel decrease in the stimulation of cholesteryl ester transfer by lipolysis. Addition of sodium oleate to VLDL and albumin resulted in stimulation of the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. The stimulation of transfer of cholesteryl esters into previously lipolyzed VLDL was abolished by lowering the pH from 7.5 to 6.0, consistent with a role of lipoprotein ionized fatty acids. CETP-mediated cholesteryl ester transfer from HDL to VLDL was also augmented by phosholipase A2 and by a bacterial lipase which lacked phospholipase activity. When VLDL and HDL were re-isolated after a lipolysis experiment, both lipoproteins stimulated CETP activity. Postlipolysis VLDL and HDL bound much more CETP than native VLDL or HDL. Lipolysis of apoprotein-free phospholipid/triglyceride emulsions also resulted in enhanced binding of CETP to the emulsion particles. Incubation conditions which abolished the enhanced cholesteryl ester transfer into VLDL remnants reduced binding of CETP to remnants, emulsions, and HDL. In conclusion, the enhanced CETP-mediated transfer of cholesteryl esters from HDL to VLDL during lipolysis is related to the accumulation of products of lipolysis, especially fatty acids, in the lipoproteins. Lipids accumulating in VLDL remnants and HDL as a result of lipolysis may augment binding of CETP to these lipoproteins, leading to more efficient transfer of cholesteryl esters from HDL to VLDL.

Mechanism of action of bradykinin-induced release of prostaglandin E.

1. The mode of action of bradykinin-induced release/biosynthesis of prostaglandin E (PGE) was investigated using the method of the isolated perfused rabbit ear. 2. Phentolamine (1 μg/ml) hardly reduced the total amount of PGE released by bradykinin. Only in the first fraction (3 min) following bradykinin injection was PGE release significantly inhibited. 3. Papaverine (5–10 μg/ml) did not affect the bradykinin-induced PGE release but totally abolished the vasoconstriction evoked by the peptide. 4. Mepacrine (10 μg/ml) greatly inhibited the bradykinin-stimulated PGE release but did not reduce the conversion of arachidonic acid by cyclo-oxygenase into PGs when used in this concentration. 5. Phospholipase A2 — injected intra-arterially into the perfused rabbit ear — strongly stimulated the release of PGE. As with bradykinin, the phospholipase A2-induced PGE release was also significantly inhibited by meparcrine (10 μg/ml). 6. Bradykinin or arachidonic acid, even in high doses (20 or 100 μg respectively), were unable to promote the release of rabbit aorta contracting substance (RCS) from the rabbit ear. 7. PGs were not metabolized when injected intraarterially through the rabbit ear. 8. The results suggest that PGE release induced by bradykinin is hardly mediated via liberating catecholamines or the constricting vessel wall. It is concluded that bradykinin stimulates PGE release by selectively activating a phospholipase A2 without affecting cyclo-oxygenase and subsequent enzymes. The phosholipase A2 catalyzes the splitting of PG precursors from membrane phospholipids, before they are converted by the active PG synthetase system into PGs.