Natural killer cells: endothelial interactions, migration, and target cell recognition

Our previous studies with mice showed that chronic ethanol (EtOH) administration affected the incorporation of unsaturated free fatty acids (FFA) into four major brain phospholipids (PL). In the current study, we investigated the effects of ganglioside GM1 pretreatment on EtOH-induced changes in the incorporation of various FFA into cerebral PL in mice. Consistent with our earlier findings, the results suggest that chronic EtOH exposure alters the incorporation of unsaturated fatty acids into phosphatidylinositol (PI), phosphatidylserine, and phosphatidylcholine (PC), but not into phosphatidylethanolamine (PE). No significant differences were observed with stearic acid. The ganglioside GM1 treatment led to increased incorporation of linoleic acid (LA) into PE and PC and appeared to enhance the EtOH-produced effects especially for docosahexaenoic acid (DHA) and to a lesser extent for oleic acid, LA, and arachidonic acid, when compared to the untreated control group. However, when comparison was made with the EtOH-alone group, significant differences were observed only with DHA incorporation and mainly into PE and PI. Thus acyltransferases may play an important role in membrane adaptation to the injurious effects of EtOH and GM1 appears to enhance selective incorporation of FFA into membrane PL; a process that may represent a repair mechanism.     

Inhibitory effects of proadrenomedullin N-terminal 20 peptide on antidiuresis and norepinephrine overflow induced by stimulation of renal nerves in anesthetized dogs

In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [3H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [3H]AnNH and [3H]N-ArPE (p < 0.05) (1.43-fold for [3H]AnNH and 1.65-fold for [3H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24 h) inhibited initially the formation of [3H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [3H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [3H]AnNH. The formation of [3H]AnNH and [3H]N-ArPE by EtOH was enhanced by the Ca2+ ionophore A23187 or by the depolarizing agent veratridine and the K+ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [3H]AnNH and [3H]N-ArPE was inhibited by exogenous AnNH, whereas only [3H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and G(i/o) protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.     

The relationship between compositional phase separation and vesicle morphology: implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A2 (PLA2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Amino acid residue 149 of lecithin:cholesterol acyltransferase determines phospholipase A2 and transacylase fatty acyl specificity

Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleoyl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmitoyl-2-arachidonoyl PC (PAPC). Six recombinant human LCAT cDNA clones were constructed with unique clusters of rat sequence substitutions in the human background spanning the region encoding amino acids 121-296. Media from transfected COS cells expressing each of the constructs were assayed for LCAT cholesterol esterification (CE) or phospholipase A2 (PLA2) activity using substrate particles containing POPC or PAPC. The PAPC/POPC CE activity ratio of the cluster 1 construct (amino acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clones produced CE activity ratios <0.3, unchanged from human LCAT. The cluster 6 clone (Y292H/W294F) had an intermediate ratio (0.6). Similar results were observed for LCAT PLA2 activity. In additional studies, position 149 of human LCAT was changed to the rat sequence (hE149A) and compared to a triple mutation containing the remainder of the cluster 1 changes (G151R/E154D/R158Q). CE and PLA2 activity ratio for the hE149A construct was >1.7, similar to rat LCAT, whereas the triple mutation construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fatty acyl specificity of human LCAT to that of rat LCAT, with an increase in activity toward PAPC. This is the first example of a point mutation in an enzyme with PLA2 activity that results in an increase in activity toward arachidonic acid.     

Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation

Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.     

Elevation of the phospholipase A2 activity in peritoneal fluid cells from women with endometriosis

OBJECTIVE: To assess the prostaglandin (PG) production on peritoneal fluid (PF) cells, phospholipase A2 (PLA2) activity of those cells in women with endometriosis was measured and compared with that of women without endometriosis. DESIGN: Prospective clinical controlled study. PATIENTS: Women who underwent laparoscopy and were found either to have endometriosis (n = 15) or not (n = 9) were included in this study. Mononuclear cells obtained from the patients at laparoscopy were immediately separated by a Ficoll-Paque technique, lysed by nitrogen cavitation, and stored at -80 degrees C. INTERVENTIONS: Phospholipase A2 activity was measured by Dole assay using 1-palmitoyl-2-[1-14C] palmitoyl phosphatidyl choline and assessed on a protein basis and a cell number basis. RESULTS: There were at least four measurable kinds of PLA2 activity detected in the cells: two calcium-dependent pH optima 7.0 and 9.0 activities and two calcium-independent pH optima 7.5 and 8.5 activities. A calcium-dependent and pH optima 9.0 activity was the highest, and it was significantly higher in women with endometriosis when compared with those who did not have endometriosis. CONCLUSION: These results indicate that the increase in the PGs in PF with endometriosis may be produced by PF cells in which PLA2 activity is elevated.     

Influence of nicotine and cotinine on retinal phospholipase A2 and its significance to macular function

The macula is a constituent of the sensory retina that is necessary for sharp contrast and color vision. A significant relationship has been found between tobacco smoking and age-related macular degeneration. Opsin, rhodopsin and phospholipase A2 (PLA2) are located in excitable membranes of retina which are enriched with polyunsaturated fatty acids (PUFA). A question may arise as to whether nicotine and its major metabolite cotinine influence PLA2 so that arachidonic acid (AA) and proinflammatory prostaglandins (PG) are produced in the retina. Therefore, the effects of nicotine and cotinine on the retinal PLA2 were studied. PLA2 activity of rat retinal sonicates was assayed using 1-palmitoyl-2[1-14C]arachidonyl-Phosphatidylethanolamine (PE, 2.2 nmol) as a substrate in Tris buffer (10 mM, pH 7.4) at 37 degrees C with and without nicotine or cotinine in the assay medium. These studies gave the following results: (1) Rat retinal PLA2 activity was 4.2+/-0.8 pmol PE hydrolyzed/100 ng protein/hr. (2) Nicotine in low concentrations (1-150 nM) activated PLA2 (EC50 5 nM). (3) Cotinine also activated PLA2 (EC50 300 nM). (4) Only high concentrations of nicotine (> 1.0 microM) and cotinine (> 25 microM) exhibit inhibition of PLA2. (5) All three known PLA2 inhibitors, mepacrine, 4-bromophenacyl bromide and bromoacetylcholine bromide (IC50: 0.5mM, 88 microM, 30 mM, respectively) inhibited retinal PLA2 activity. These observations suggest that polyunsaturated fatty acids are cleaved, and arachidonic acid, the precursor for prostaglandins and related pro-inflammatory mediators, is liberated by nicotine and cotinine. Oxidative stress (reduced levels of antioxidants), vascular insufficiency, as well as activation of PLA2 by nicotine and cotinine may contribute for retinal degeneration in smokers during aging.     

Use of Percoll gradient centrifugation for the isolation of diatoms from Wadden Sea sediments; diatom yields, species recoveries and population diversity

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.     

Phospholipid interactions affect substrate hydrolysis by bovine brain phospholipase A1

The specificity of substrate hydrolysis by bovine brain phospholipase A1 (PLA1) was examined. In the presence of Mg2+, using pH values of 7 to 9, the purified enzyme deacylated 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine yielding 2-[1-14C]arachidonoyl-lysophosphatidylethanolamine at a rate of 70 mumol/min per mg. In the absence of Mg2+, however, the reaction rate slowed at pH values above 7.25. In contrast, brain PLA1 slowly (3.8 mumol/min per mg) hydrolyzed 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine (PAPC) unless phosphatidylserine (PS) was included. Maximal PAPC hydrolyzing activity required a PAPC/PS molar ratio of 2.5:1, Mg2+, and a pH value of 8.5-9.5. Replacing PS with phosphatidylethanolamine (PE) or phosphatidic acid (PA), but not phosphatidylinositol (PI), produced a similar effect. Moreover, hydrolysis of either arachidonoyl-substituted or dipalmitoyl-substituted PC at pH 7.5 was enhanced by increasing the mol fraction of PE. Brain PLA1 also hydrolyzed 1-stearoyl-[1-14C]arachidonoyl-PI with high velocity, but only if the substrate was dispersed in PE vesicles. In contrast, the velocity of PS, 1-palmitoyl-lyso-PC or diacylglycerol hydrolysis was low and unaffected by PE. In summary, PLA1 hydrolyzed PE with high velocity and specificity, whereas a high rate of PC or PI hydrolysis was observed only if PS, PE, or PA was present. In addition, PLA1 activity was greatly influenced by pH and Mg2+, implying that the substrate conformation is important to the catalytic efficiency of PLA1. Finally, the high rate of PE, PC or PI hydrolysis suggests PLA1 significantly contributes to the turnover of these phospholipids in the brain.     

Free-roaming and feral cats--their impact on wildlife and human beings

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in which beta-galactosidase (beta-gal) was entrapped. Exposing the POPC liposomes entrapping beta-gal inside to heat treatment (40-50 degrees C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of beta-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping beta-gal were heated at 45 degrees C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45 degrees C, 30 min) also supported the translocation of beta-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of beta-gal was maximized at the temperature of 40-50 degrees C. The mechanisms of beta-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and beta-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of beta-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of beta-gal were well corresponding to those from model calculation.     

Interfacial activation, lysophospholipase and transacylase activity of group VI Ca2+-independent phospholipase A2

The Group VI 80-kDa Ca2+-independent phospholipase A2 (iPLA2) has been purified from murine P388D1 macrophages and Chinese hamster ovary (CHO) cells. The amino acid sequence of the iPLA2 has been determined and shown to contain a lipase consensus sequence and eight ankyrin repeats, which makes it distinct from Group I-V PLA2s. This enzyme appears to play a key role in mediating basal phospholipid remodeling. We now report that the Group VI iPLA2 displays interfacial activation toward short chain phospholipids, 1-octanoyl-2-heptanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, and 1,2-dihexanoyl-sn-glycero-3-phosphocholine micelles. ATP protects the iPLA2 from a loss in activity as a result of prolonged incubation during the assay. Hence higher enzyme activity is observed in the presence than in the absence of ATP. Similar protection was obtained with glycerol. In addition, the iPLA2 exhibits multiple activities which are strongly dependent on substrate presentation. The lysophospholipase activity of this enzyme was diminished by Triton X-100 and stimulated by glycerol. With the combination of 50 microM Triton X-100 and 50% glycerol, the enzyme's lysophospholipase activity achieved equivalent activity to its PLA2 activity. The iPLA2 displayed both lysophospholipid/transacylase and phospholipid/transacylase activity, supporting the conclusion that the mechanism of action of iPLA2 proceeds through an acyl-enzyme intermediate as proposed for the Group IV cPLA2.     

Arachidonic acid up-regulates and prostaglandin E2 down-regulates the expression of pancreatic-type phospholipase A2 and prostaglandin-endoperoxide synthase 2 in uterine stromal cells

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two-series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic-type phospholipase A2 (PLA2I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line UIII, which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1, which is expressed at a high level in UIII cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up-regulated by increasing arachidonate concentrations (1-10 microM). The maximal response was obtained at 24 h, reaching a 2.3-fold and 2.6-fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate-induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose-dependent manner, the maximal response being reached at 1 microM. We conclude that arachidonic acid up-regulates the expression of PLA2 I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down-regulating enzyme expression.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 mu M; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 mu M) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE1 receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE1 receptor number (Bmax) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPgammaS (100 mu M) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of GSalpha and S49 cyc- reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE1 receptors and GS in situ by application of a C-terminal anti-GSalpha antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available GSalpha within the PGE1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 mu M)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

Antigen-induced generation of lyso-phospholipids in human airways

The goal of the current study was to examine the formation of phospholipids, 1-radyl-2-lysosn-glycero-phospholipids (lyso-PL) and 2-acetylated phospholipids (such as PAF) as well as mechanisms responsible for generating these phospholipids in bronchoalveolar lavage fluid (BAI.F) from allergic subjects challenged with antigen. Bronchoalveolar lavage was performed in normal and allergic subjects before, 5-30 min, 6 h, and 20 h after segmental antigen challenge via a wedged bronchoscope. Levels of 1-hexadecyl-2-lyso-phospholipids and 1-hexadecyl-2-acetyl-phospholipids were initially determined by negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS). Antigen dramatically elevated quantities of 1-hexadecyl-2-lyso-phospholipids in allergic subjects 20 h after challenge when compared to non-allergic controls. In contrast, there was not a significant increase in levels of 1-hexadecyl-2-acetyl-phospholipids after antigen challenge. Closer examination of 1-radyl-2-lyso-sn-glycero-3-phosphocholine (GPC) revealed that 1-palmitoyl-2-lyso-GPC, 1-myristoyl-2-lyso-GPC and 1-hexadecyl-2-lyso-GPC were three major molecular species produced after antigen challenge. 1-palmitoyl-2-lyso-GPC increased sevenfold to levels of 222 +/- 75 ng/ml of BALF 20 h after antigen challenge. The elevated levels of lyso-PL correlated with levels of albumin used to assess plasma exudation induced by allergen challenge. In contrast, the time course of prostaglandin D2 (PGD2) or 9 alpha, 11 beta PGF2 (11 beta PGF2) formation did not correlate with lyso-PL generation. To examine the mechanism leading to lyso-phospholipid formation in antigen-challenged allergic subjects, secretory phospholipase A2 (PI.A2) and acetyl hydrolase activities were measured. There was a significant increase in PLA2 activity found in BALF of allergic subjects challenged with antigen when compared to saline controls. This activity was neutralized by an antibody directed against low molecular mass, (14 kD) human synovial PLA2 and dithiothreitol. Acetyl hydrolase activity also markedly increased in BALF obtained after antigen challenge. This study indicates that high levels of lyso-PLs are present in airways of allergic subjects challenged with antigen and provides evidence for two distinct mechanisms that could induce lyso-PL formation. Future studies will be necessary to determine the ramifications of these high levels of lyso-phospholipids on airway function.     

GM2 promotes ciliary neurotrophic factor-dependent rescue of immortalized motor neuron-like cell (NSC-34)

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblastoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, beta 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.     

Properties of phospholipase A1/transacylase in the white muscle of bonito Euthynnus pelamis (Linnaeus)

The properties of phospholipase A1 (PLA1) obtained from the white muscle of bonito, Euthynnus pelamis (Linnaeus), were examined. The PLA1 activity had a pH optimum from 6.5 to 7.0 for phosphatidylcholine (PC), and calcium ion was not required. The optimum temperature was from 20 to 30 degrees C. When a fatty alcohol was used as an acceptor, a wax ester was produced by transferring a fatty acid at the sn-1 position of the donor's PC. The maximum production of lysophosphatidylcholine was shifted by 0.5 pH units to the acidic side and the pH optimum of wax ester synthesis was from 6.0 to 6.5. The synthesis was independent of calcium ion and Coenzyme A. The transacylation was also observed when 1-lyso-2-acyl-sn-glycero-3-phosphocholine was used as an acceptor. Fatty acid at the sn-1 position of the donor PC was transferred to the unoccupied hydroxy group of the acceptor at the sn-1 position. When 2,3-dipalmitoyl-sn-glycero-1-phosphocholine was used as the acyl donor, a similar amount of palmitic acid was transferred as in the case of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. However, 1-acyl-2-lyso-sn -glycero-3-phosphocholine, a positional isomer, was a poor acceptor. These results indicate that the transacylation by the PLA1 from bonito muscle is not stereospecific, but is position-specific both for the acyl donor and acceptor.     

Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.     

Evidence for functional involvement of asparagine 67 in substrate recognition by snake venom phospholipases A2

Site-directed mutagenesis studies of recombinant Trimeresurus flavoviridis venom gland phospholipase A2 (PLA2) showed that the Asn residue at position 67 takes part in recognition of the substrate 2-arachidonoyl sn-glycero-3-phosphocholine in both monomeric and micellar states. The amount of arachidonate released from phosphatidylcholine mixed micelles was reduced to 30% for N67D and N67K mutants, and to 70% for N67G mutant, but remained unchanged for N67S mutant. In contrast, for monomeric substrate, the activity was decreased to 40% for N67D and N67G and to 60% for N67K but remained unchanged for N67S. These results suggest that the properties of the side chain of residue 67 exert a significant influence on recognition of 2-arachidonoyl sn-glycero-3-phosphocholine.