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PubMed1996

A controlled biochemical and immunohistochemical study of human synovial-type (group II) phospholipase A2 and inflammatory cells in macroscopically normal, degenerated, and herniated human lumbar disc tissues.

STUDY DESIGN: Group II phospholipase A2 enzyme activity was studied biochemically and immunohistochemically in tissue samples from disc prolapses, degenerated discs, and macroscopically normal discs. In parallel, phospholipase A2 and inflammatory cells were studied by indirect immunocytochemistry. OBJECTIVES: To compare phospholipase A2 activity in normal discs and abnormal discs by an identical assay for phospholipases A2, and to compare the occurrence of inflammatory cells with phospholipase A2 activity and immunoreactivity. SUMMARY OF BACKGROUND DATA: It has been suggested that a high phospholipase A2 enzyme activity in herniated disc tissue could be significant in abnormal states such as sciatica and discogenic pain. No comparison between healthy disc tissue and samples of abnormal discs (degenerated or herniated) has been carried out. In particular, an identical assay for phospholipase A2 for such tissue samples, supported by immunohistochemical staining data, has never been applied in parallel to normal and abnormal disc tissue, and neither have such results been compared with the demonstration of inflammatory cells. METHODS: Group II phospholipase A2 enzyme activity was determined, in parallel, using an identical assay for tissue samples from 11 macroscopically normal discs, 33 disc herniations, and six discs showing degeneration by discography. For determination of phospholipase A2 enzyme activity, a radioassay using 1-palmitoyl-2-(1-14C)linoleoyl-L-3-phosphatidylethanolamine as the phospholipid substrate was used. Total tissue DNA as an estimate of total tissue cell number was measured in parallel with phospholipase A2 activity. All tissue samples also were studied by indirect immunocytochemistry, locating phospholipase A2 and T and B lymphocytes. RESULTS: Neither degenerated nor herniated disc tissue samples demonstrated a higher phospholipase A2 activity than control disc tissue samples. Average phospholipase A2 activity was actually higher in the control samples than in herniated disc samples (Mann-Whitney test, P < 0.001), possibly a result of a higher total DNA (P < 0.005). The observed level of phospholipase A2 activity was lower than that of inflammatory human synovial fluid. Neither was there marked immunoreactivity for phospholipase A2, which was observed in chondrocytes in areas of cartilage and occasional disc cells, supporting the biochemical results. Lymphocytes were more numerous only in herniated disc samples (15%), and their presence showed little overlap with phospholipase A2 immunoreactivity. CONCLUSIONS: Synovial-type (Group II) phospholipase A2 enzyme activity is not particularly high in disc tissue and does not appear to be higher in herniated or degenerated discs than control disc tissue. Immunoreactivity to phospholipase A2 is seen only occasionally and is strong only when cartilage tissue is present. Neither are inflammatory lymphocytes commonly observed.

Adult↗

PubMed1998

Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnant rat myometrial tissue.

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidinium thiocyanate-phenol-chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde-1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase-polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase-polymerase chain reaction studies have confirmed expression of the phospholipase C-beta1a, phospholipase C-beta3, phospholipase C-gamma1, phospholipase C-beta2, and phospholipase C-delta1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-delta1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-beta1 a isoform was expressed at the highest level, the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-gamma2 isoforms at an intermediate level, and the phospholipase C-delta1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle.

Animals↗

PubMed2003

Regulation of EGF-induced phospholipase C-gamma1 translocation and activation by its SH2 and PH domains.

Translocation of phospholipase C-gamma1 is essential for its function in response to growth factors. However, in spite of recent progress, the phospholipase C-gamma1 translocation pattern and the molecular mechanism of the translocation are far from fully understood. Contradictory results were reported as to which domain, PH or SH2, controls the epidermal growth factor-induced translocation of phospholipase C-gamma1. In this communication, we studied epidermal growth factor-induced translocation of phospholipase C-gamma1 by using comprehensive approaches including biochemistry, indirect fluorescence and live fluorescence imaging. We provided original evidence demonstrating that: (i) endogenous phospholipase C-gamma1, similar to YFP-tagged phospholipase C-gamma1, translocated to endosomes following its initial translocation from cytosol to the plasma membrane in response to epidermal growth factor; (ii) phospholipase C-gamma1 remained phosphorylated in endosomes, but phospholipase C-gamma1 activity is not required for its translocation, which suggests a signaling role for phospholipase C-gamma1 in endosomes; (iii) the PH domain was not required for the initial translocation of phospholipase C-gamma1 from cytosol to the plasma membrane, but it stabilizes phospholipase C-gamma1 in the membrane at a later time; (iv) the function of the phospholipase C-gamma1 PH domain in stabilizing phospholipase C-gamma1 membrane association is very important in maintaining the activity of phospholipase C-gamma1; and (v) the role of the PH domain in phospholipase C-gamma1 membrane association and activation is dependent on PI3K activity. We conclude that the phospholipase C-gamma1 SH2 and PH domains coordinate to determine epidermal growth factor-induced translocation and activation of phospholipase C-gamma1.

Animals↗

PubMed1996

Serum phospholipase A2 in patients after splenectomy.

Phospholipase A2 values increase in serum in various inflammatory states, infections, and postoperatively in surgical patients. Several organs, including the liver and spleen have been suggested as sources of circulating phospholipase A2. The purpose of the present work was to examine the possible role of the spleen as a source of elevated serum concentrations of phospholipase A2 after surgery. Pre- and postoperative serum samples of patients undergoing splenectomy were studied for group I phospholipase A2, group II phospholipase A2, and C-reactive protein mass concentrations and catalytic activity concentration of phospholipase A2. The catalytic activity concentration of phospholipase A2 and the mass concentrations of group II phospholipase A2 and C-reactive protein increased postoperatively (8.08 +/- 1.40 U/l vs. 3.96 +/- 0.89 U/l (mean +/- SEM) for phospholipase A2 catalytic concentration (p < 0.03), and 154.8 +/- 32.1 micrograms/l vs. 47.5 +/- 14.7 micrograms/l (mean +/- SEM) for group II phospholipase A2 mass concentration (p < 0.02, n = 7). The mass concentration of group I phospholipase A2 remained unchanged. The catalytic concentration of phospholipase A2 correlated well with the mass concentration of group II phospholipase A2 (p < 0.001, r = 0.846, n = 43). The concentration of C-reactive protein correlated well with the mass concentration of group II phospholipase A2 (p < 0.001, r = 0.566, n = 43) in serum. The results indicate that group II phospholipase A2 is released into the circulation after splenectomy, and the spleen seems not to be the source of circulating group II phospholipase A2.

Adult↗

PubMed2000

Elevations of group II phospholipase A2 concentrations in serum and amniotic fluid in association with preterm labor.

OBJECTIVE: The purpose of this study was to determine whether the elevation of secretory group II phospholipase A(2) concentration in the serum and amniotic fluid in preterm labor is associated with intrauterine inflammation. STUDY DESIGN: Serum and amniotic fluid were collected from women with preterm delivery (<37 weeks' gestation; n = 38) and term delivery (n = 20). Phospholipase activity was measured with a highly sensitive system that was based on reverse-phase high-performance liquid chromatographic separation of 9-anthracenylmethyl derivatives of fatty acids released by phospholipase A(2). The concentrations of immunoreactive isozymes (group I or II) of secretory phospholipase A(2) were assayed with a radioimmunoassay kit with a monoclonal antibody against human pancreatic phospholipase A(2) and splenic IIA phospholipase A(2). Localization of immunoreactive group II phospholipase A(2) in the amniotic membrane was determined by immunostaining visualized with the Vectastain ABC (Vector Laboratories, Inc, Burlingame, Calif) method. RESULTS: Enzymatic activities of phospholipase A(2) in the serum and amniotic fluid specimens obtained from patients in preterm labor with chorioamnionitis were significantly higher than those in specimens from patients in term labor. Significant elevations of phospholipase A(2) activities were observed in patients with preterm labor without histologically evident chorioamnionitis. The activity of phospholipase A(2) was clearly correlated with the concentration of the immunoreactive group II phospholipase A(2). Group II phospholipase A(2) was localized in amniotic cells obtained from patients with a pathologically determined diagnosis of chorioamnionitis. The predictive value for chorioamnionitis of the group II phospholipase A(2) concentration was relatively higher than the predictive values of the concentrations of C-reactive protein and interleukins 6 and 8. CONCLUSION: Significant elevations of group II phospholipase A(2) concentrations were detected in the serum and amniotic fluid of women with preterm labor. Group II phospholipase A(2) concentration may be a useful indicator for preterm labor, and phospholipid metabolism is certainly activated both in preterm labor and in apparent inflammatory diseases.

Amniotic Fluid↗

PubMed2006

Inhibitors of brain phospholipase A2 activity: their neuropharmacological effects and therapeutic importance for the treatment of neurologic disorders.

The phospholipase A(2) family includes secretory phospholipase A(2), cytosolic phospholipase A(2), plasmalogen-selective phospholipase A(2), and calcium-independent phospholipase A(2). It is generally thought that the release of arachidonic acid by cytosolic phospholipase A(2) is the rate-limiting step in the generation of eicosanoids and platelet activating factor. These lipid mediators play critical roles in the initiation and modulation of inflammation and oxidative stress. Neurological disorders, such as ischemia, spinal cord injury, Alzheimer's disease, multiple sclerosis, prion diseases, and epilepsy are characterized by inflammatory reactions, oxidative stress, altered phospholipid metabolism, accumulation of lipid peroxides, and increased phospholipase A(2) activity. Increased activities of phospholipases A(2) and generation of lipid mediators may be involved in oxidative stress and neuroinflammation associated with the above neurological disorders. Several phospholipase A(2) inhibitors have been recently discovered and used for the treatment of ischemia and other neurological diseases in cell culture and animal models. At this time very little is known about in vivo neurochemical effects, mechanism of action, or toxicity of phospholipase A(2) inhibitors in human or animal models of neurological disorders. In kainic acid-mediated neurotoxicity, the activities of phospholipase A(2) isoforms and their immunoreactivities are markedly increased and phospholipase A(2) inhibitors, quinacrine and chloroquine, arachidonyl trifluoromethyl ketone, bromoenol lactone, cytidine 5-diphosphoamines, and vitamin E, not only inhibit phospholipase A(2) activity and immunoreactivity but also prevent neurodegeneration, suggesting that phospholipase A(2) is involved in the neurodegenerative process. This also suggests that phospholipase A(2) inhibitors can be used as neuroprotectants and anti-inflammatory agents against neurodegenerative processes in neurodegenerative diseases.

Animals↗

PubMed2004

Retinoic acid-mediated phospholipase A2 signaling in the nucleus.

Retinoic acid modulates a wide variety of biological processes including proliferation, differentiation, and apoptosis. It interacts with specific receptors in the nucleus, the retinoic acid receptors (RARs). The molecular mechanism by which retinoic acid mediates cellular differentiation and growth suppression in neural cells remains unknown. However, retinoic acid-induced release of arachidonic acid and its metabolites may play an important role in cell proliferation, differentiation, and apoptosis. In brain tissue, arachidonic acid is mainly released by the action of phospholipase A2 (PLA2) and phospholipase C (PLC)/diacylglycerol lipase pathways. We have used the model of differentiation in LA-N-1 cells induced by retinoic acid. The treatment of LA-N-1 cells with retinoic acid produces an increase in phospholipase A2 activity in the nuclear fraction. The pan retinoic acid receptor antagonist, BMS493, can prevent this increase in phospholipase A2 activity. This suggests that retinoic acid-induced stimulation of phospholipase A2 activity is a retinoic acid receptor-mediated process. LA-N-1 cell nuclei also have phospholipase C and phospholipase D (PLD) activities that are stimulated by retinoic acid. Selective phospholipase C and phospholipase D inhibitors block the stimulation of phospholipase C and phospholipase D activities. Thus, both direct and indirect mechanisms of arachidonic acid release exist in LA-N-1 cell nuclei. Arachidonic acid and its metabolites markedly affect the neurite outgrowth and neurotransmitter release in cells of neuronal and glial origin. We propose that retinoic acid receptors coupled with phospholipases A2, C and D in the nuclear membrane play an important role in the redistribution of arachidonic acid in neuronal and non-nuclear neuronal membranes during differentiation and growth suppression. Abnormal retinoid metabolism may be involved in the downstream transcriptional regulation of phospholipase A2-mediated signal transduction in schizophrenia and Alzheimer disease (AD). The development of new retinoid analogs with diminished toxicity that can cross the blood-brain barrier without harm and can normalize phospholipase A2-mediated signaling will be important in developing pharmacological interventions for these neurological disorders.

Alzheimer Disease↗

PubMed2000

Investigation of the relocation of cytosolic phospholipase A2 and annexin V in activated platelets.

Cytosolic phospholipase A(2) is a Ca(2+)-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A(2). Annexin V is a Ca(2+)-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A(2). Here, we have studied the association of cytosolic phospholipase A(2) and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A(2) and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A(2) to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A(2) or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A(2) remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A(2) with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A(2) can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A(2) copurify annexin V and cytosolic phospholipase A(2) from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A(2) and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A(2) and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A(2) into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.

Annexin A5↗

PubMed1997

Phospholipase A2 activity in herniated lumbar discs. Clinical correlations and inhibition by piroxicam.

STUDY DESIGN: Prospective study of phospholipase A2 activity in the serum and intervertebral discs of patients undergoing surgery for sciatica due to disc herniation. OBJECTIVES: To determine correlations between herniated disc phospholipase A2 and clinical, radiographic, and anatomic signs of common sciatica; to evaluate serum phospholipase A2 activity as a marker of disc phospholipase A2; and to investigate the in vivo effect of piroxicam on disc phospholipase A2. SUMMARY OF BACKGROUND DATA: Several studies suggest disc inflammation as a mechanism of sciatica due to disc herniation, and phospholipase A2 emerges as a key enzyme of cartilage and disc tissues. METHODS: Phospholipase A2 activity was determined, using the degradation of a specific substrate, in the serum and discs of 31 patients (14 treated with acetaminophen and 17 treated with piroxicam) undergoing surgery for sciatica due to lumbar disc herniation. Visual analog scale for pain, Dallas Pain Questionnaire, Lasègue's sign, radiographic stage of degeneration of the herniated disc, volume of disc herniation shown by computed tomography, and surgical findings were recorded. RESULTS: Disc phospholipase A2 activity was independent of the patient's age or sex, the radiologic stage of disc degeneration, and the volume of the herniation, and showed no significant correlation with Lasègue's sign or pain measured on a visual analog scale. The correlation between disc phospholipase A2 and the Dallas category of items measuring the impact of pain on daily activities approached the level of significance (P = 0.07). Disc phospholipase A2 activity was significantly higher in cases of sequestrated discs than in other herniations. Disc phospholipase A2 was significantly correlated with serum phospholipase A2, and was significantly lower in patients treated with piroxicam than in those treated with acetaminophen. CONCLUSIONS: Disc phospholipase A2 is thought to participate in the physiopathology of sciatica and to bemodulated by nonsteroidal anti-inflammatory drug therapy. Serum phospholipase A2 is suggested as a biologic marker of disc inflammation in patients with sciatica.

Acetaminophen↗

PubMed1984

Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices.

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.

Acetophenones↗

PubMed2006

Direct modulation of phospholipase D activity by Gbetagamma.

Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.

Binding Sites↗

PubMed2001

Fear conditioning-induced alterations of phospholipase C-beta1a protein level and enzyme activity in rat hippocampal formation and medial frontal cortex.

We investigated the effects of one-trial fear conditioning on phospholipase C-beta1a catalytic activity and protein level in hippocampal formation and medial frontal cortex of untreated control rats and rats prenatally exposed to ethanol. One hour following fear conditioning of untreated control rats, phospholipase C-beta1a protein level was increased in the hippocampal cytosolic fraction and decreased in the hippocampal membrane and cortical cytosolic and cortical membrane fractions. Twenty-four hours after fear conditioning, phospholipase C-beta1a protein level was reduced in the hippocampal cytosolic fraction and elevated in the cortical nuclear fraction; in addition, 24 h after conditioning, phospholipase C-beta1a activity in the cortical cytosolic fraction was increased. Rats that were exposed prenatally to ethanol displayed attenuated contextual fear conditioning, whereas conditioning to the acoustic-conditioned stimulus was not different from controls. In behavioral control (unconditioned) rats, fetal ethanol exposure was associated with reduced phospholipase C-beta1a enzyme activity in the hippocampal nuclear, cortical cytosolic, and cortical membrane fractions and increased phospholipase C-beta1a protein level in the hippocampal membrane and cortical cytosolic fractions. In certain cases, prenatal ethanol exposure modified the relationship between fear conditioning and changes in phospholipase C-beta1a protein level and/or activity. The majority of these effects occurred 1 h, rather than 24 h, after fear conditioning. Multivariate analysis of variance revealed interactions between fear conditioning, subcellular fraction, and prenatal ethanol exposure for measures of phospholipase C-beta1a protein level in hippocampal formation and phospholipase C-beta1a enzyme activity in medial frontal cortex. In the majority of cases, fear conditioning-induced changes in hippocampal phospholipase C-beta1a protein level were augmented in rats prenatally exposed to ethanol. In contrast, fear conditioning-induced changes in cortical phospholipase C-beta1a activity were, often, in opposite directions in prenatal ethanol-exposed compared to diet control rats. We speculate that alterations in subcellular phospholipase C-beta1a catalytic activity and protein level contribute to contextual fear conditioning and that learning deficits observed in rats exposed prenatally to ethanol result, in part, from dysfunctions in phospholipase C-beta1a signal transduction.

Acoustic Stimulation↗

PubMed2001

Phospholipase A2 isoforms in acute pancreatitis.

OBJECTIVE: To assess phospholipase A2 isoforms during human and experimental acute necrotizing pancreatitis. Phospholipase A2 isoforms (group I, II, and IV) were examined in acute pancreatitis tissues in humans and rats to determine whether the exocrine pancreas itself is a source of these mediators. SUMMARY BACKGROUND DATA: Phospholipase A2 has important regulatory functions, especially in inflammation. METHODS: Using Northern blot analysis and immunohistochemistry, the expression and localization of phospholipase A2 isoforms were analyzed in pancreatic tissue obtained from 21 patients with acute necrotizing pancreatitis and in pancreatic tissues of rats with acute edematous and necrotizing pancreatitis. Rat samples were examined daily for 1 week. RESULTS: In human acute pancreatitis, phospholipase A2-I mRNA expression was 8.9-fold decreased. By contrast, phospholipase A2-II (7.8-fold) and phospholipase A2-IV (8.1-fold) mRNA levels were increased. By in situ hybridization, phospholipase A2-IV was found to be expressed in remaining acinar and ductal cells adjacent to the necrotic areas. Immunostaining revealed moderate to intense phospholipase A2-II immunoreactivity in remaining acinar and ductal cells next to the necrosis. In rat pancreatitis, phospholipase A2-II mRNA levels in the pancreas were unchanged in the early phase (8 hours) but markedly increased after 24 hours, with a fluctuating pattern until day 7. CONCLUSIONS: Enhanced expression of phospholipase A2-II and A2-IV isoenzymes in human and experimental acute pancreatitis suggests that these enzymes play a role in modulating the inflammatory reaction in the pancreas. Because phospholipase A2-II and A2-IV mRNA was strongly present in remaining viable pancreatic acinar and ductal cells, the pancreas itself seems to be at least partly a source and a regulator of phospholipase A2-II- and A2-IV-dependent inflammatory reactions in acute pancreatitis.

Adolescent↗

PubMed1996

Phospholipases A2 in the serum of patients after coronary artery bypass surgery.

OBJECTIVE: To study the catalytic activity of phospholipase A2 and the concentrations of group I and group II phospholipase A2 in the sera of patients during and after coronary artery bypass surgery. DESIGN: Prospective study. SETTING: University hospital and research laboratory. PATIENTS: Fourteen consecutive patients operated on for ischemic heart disease. INTERVENTIONS: The catalytic activity of phospholipase A2 and the serum concentrations of group I and group II phospholipases A2 were measured before, during, and after the operation until the seventh postoperative day. A total of 196 blood samples were taken from 14 patients. MEASUREMENTS AND MAIN RESULTS: The catalytic activity of phospholipase A2 and the concentration of group II phospholipase A2 increased in the sera of patients after coronary artery bypass surgery. The concentration of Group I phospholipase A2 did not change. The catalytic activity of phospholipase A2 correlated significantly with group II but not with group I phospholipase A2 values. CONCLUSIONS: The increase in the catalytic activity of phospholipase A2 in serum after coronary artery bypass surgery is due to group II phospholipase A2. The present results suggest that group II phospholipase A2 is responsible for the physiologic and pathophysiologic effects of catalytically active phospholipase A2 after coronary artery bypass surgery.

Adult↗

PubMed1988

Activation of phospholipases A1 and A2 in heart, liver, and blood during endotoxin shock.

The effects of endotoxin shock on the activities of phospholipases A1 and A2 were studied in canine heart sarcolemma, liver plasma membrane, and blood serum. Results obtained from control dogs indicate that phospholipases A1 and A2 were equally active in cardiac sarcolemma. In liver plasma membrane the activity of phospholipase A1 was twice that of phospholipase A2, while in blood serum phospholipase A1 activity was only half that of phospholipase A2. Results obtained 4 hr after endotoxin administration (0.5 mg/kg, iv) demonstrate that phospholipase A1 activities were activated by 117% in cardiac sarcolemma, 188% in liver plasma membrane, and 150% in blood serum, while phospholipases A2 activities were stimulated by 71% in cardiac sarcolemma, 175% in liver plasma membrane, and 31% in blood serum. The in vitro incubation of all three enzyme preparations from control dogs with endotoxin (5-15 micrograms/0.5 ml) stimulated both phospholipase A1 and A2 activities and the stimulation was concentration dependent. These data suggest that endotoxin may have a direct stimulatory effect on phospholipases A1 and A2 activities in the cell membranes of heart and liver, and also in blood serum. Since phospholipases A play an important role in the regulation of membrane structure and function, the observed activation of phospholipases A1 and A2 may have a physiological significance in the pathogenesis of cellular dysfunction in endotoxin shock.

Animals↗

PubMed1998

Phospholipase C isozymes in the human brain and their changes in Alzheimer's disease.

Phosphoinositide-specific phospholipase C is a key enzyme in signal transduction. We have previously demonstrated that an isozyme of phospholipase C, phospholipase C-delta1, accumulates aberrantly in the brains of patients with Alzheimer's disease. In the present study, we examined the property of phospholipase C isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimer's disease brains. The chromatofocusing profile of human brain phospholipase C activity on a Mono P HR column demonstrated that phospholipase C-gamma1, exhibiting an isoelectric point value of 5.2, and phospholipase C-delta1, exhibiting isoelectric point values of 5.2 and 4.6, are partly overlapped in their elution. In contrast, the elution profiles of control and Alzheimer's disease brain phospholipase C on Superdex 200 pg column gel filtration chromatography indicated that phospholipase C-gamma1 and phospholipase C-delta1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in the human brain. Using this gel filtration chromatography it was revealed that the phospholipase C-gamma1 activity was significantly decreased and the phospholipase C-delta1 activity was significantly increased in Alzheimer's disease brains compared with controls. These results suggest that the phospholipase C isozymes are differentially involved in Alzheimer's disease.

Aged↗

PubMed2001

5-Aminosalicylate stimulates phospholipase D activity in macrophages.

5-Aminosalicylate, which is considered to be the active moiety of sulfasalazine, is one of the most widely used agents for treatment of inflammatory bowel disease. However, its mechanism of action is unclear. In this report, we provide evidence that the phospholipase D pathway is a target for this drug in macrophages. Activation of phospholipase D leads to the generation of important second messengers such as phosphatidic acid, lysophosphatidic acid and diacylglycerol, all of which can regulate cellular responses involved in inflammation. Murine peritoneal macrophages were labeled with [(3)H]myristate, incubated with various drugs, agonists, or inhibitors, and phospholipase D activity was assayed. 5-Aminosalicylate or sulfasalazine stimulated phospholipase D in a time- and concentration-dependent manner. Chelation of extracellular Ca(2+) inhibited phospholipase D activation by either of these drugs whereas pretreatment of macrophages with the tyrosine kinase inhibitor genistein had no effect. Downregulation of protein kinase C by prolonged incubation with phorbol ester completely blocked the activation of phospholipase D. Pertussis toxin decreased the activation of phospholipase D. The levels of inositol 1,4,5-trisphosphate increased by 260% after treatment of macrophages with 5-aminosalicylate. A phosphoinositide-specific phospholipase C inhibitor U73122 blocked phospholipase D activation completely. Interestingly, long-term preincubation of the macrophages with a relatively low concentration of 5-aminosalicylate that did not stimulate phospholipase D activity by itself, potentiated the effect of phorbol ester-induced activation of phospholipase D. Taken together, these results show that 5-aminosalicylate activates phospholipase D via a pathway involving inositol 1,4,5-trisphosphate generation, calcium fluxes, and Gi/Go. Although the mechanisms by which phospholipase D activation by 5-aminosalicylate or sulfasalazine might attenuate inflammatory responses in the intestine remain to be defined, these results highlight a novel potential mechanism of action for these drugs.

Animals↗

PubMed1998

Extracellular phospholipases as universal virulence factor in pathogenic fungi.

Microbial pathogens use a number of genetic strategies to invade the host and cause infection. These common themes are found throughout microbial virulence factors. Secretion of enzymes, such as phospholipase, has been proposed as one of these themes which is used by bacteria, parasite, and pathogenic fungi. The role of extracellular phospholipase as a potential virulence factor in pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus has gained credence recently. In this address data implicating phospholipase as a virulence factor in Cryptococcus neoformans and Aspergillus fumigatus will be presented. This will be followed by a more detailed description of our molecular and biochemical approaches we used to more definitively delineate the role of phospholipase in the virulence of C. albicans. First, we purified the phospholipase B protein, the dominant phospholipase secreted by C. albicans, obtained the amino acid sequence of its N-terminus and an internal peptide fragment, and used this information to clone the gene encoding the protein using a PCR-based approach. Nucleotide sequence analysis revealed an ORF of 1818 bp that predicted for a pre-protein of 605 amino acid residues. The deduced amino acid sequences of the cloned gene (PLB 1) showed 42.3%, 45%, and 47.8% overall sequence identity, with the reported sequences of phospholipase B cloned from Penicillium notatum, Saccharomyces cerevisiae, and Saccharomyces rosei, respectively. Second, using targeted gene disruption, URA blaster, we created C. albicans null mutants which failed to secrete phospholipase B. Third, we tested the ability of these isogenic strain pairs to cause lethality using a murine model of hematogenously disseminated candidiasis. Our data demonstrate that the parent phospholipase-producing strain caused more fatality in mice, while the null phospholipase-deficient strain was avirulent. Importantly, the parent and null mutants had similar growth and germination rates. These data prove that phospholipase B is essential for candidal virulence, and pave the way for studies directed at determining the mechanism/s through which phospholipase modulate candidal virulence. Understanding phospholipase as a common theme in fungal pathogenicity is critical for developing new antifungal strategies based on anti-virulence.

Animals↗