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PubMed1996

Synthesis of group II phospholipase A2 and lysozyme in lacrimal glands.

PURPOSE: The aim of this study was to demonstrate the synthesis and cellular distribution of group II phospholipase A2 and lysozyme in the main and accessory lacrimal glands. METHODS: The authors studied samples of normal main lacrimal glands of seven autopsied subjects and accessory lacrimal glands of eight patients who underwent ptosis surgery. The specimens were immunostained with a rabbit antiserum against group II phospholipase A2 and a monoclonal antibody against lysozyme. Expression of group II phospholipase A2 gene was shown using Northern hybridization and in situ hybridization. RESULTS: Lysozyme was present in the secretory granules of most acini, whereas group II phospholipase A2 was seen in a minority of acinar cells, primarily in the central parts of lobules in the main and accessory lacrimal glands. Synthesis of group II phospholipase A2 in the glandular cells was confirmed by Northern hybridization and by in situ hybridization. CONCLUSIONS: There are two specialized cell types in the main and accessory lacrimal glands, one synthesizing group II phospholipase A2 and the other synthesizing lysozyme. These enzymes are important nonspecific antibacterial factors in tears.

Aged↗

PubMed1997

[Phospholipase A2--acute phase protein after liver surgery?].

INTRODUCTION: The exact source of phospholipase A2 is unknown. In this context the liver is discussed because the secretion of phospholipase A2 could be initiated during an acute phase response. PATIENTS AND METHODS: Three groups of patients were prospectively established: A liver resection (n = 12); B control (n = 22; oesophageal resection, gastrectomy, rectum resection); C sepsis (n = 5). Blood was collected preoperatively, each day postoperatively for seven days and at the day of discharge. Biochemical procedures: Phospholipase A2, PMN-elastase, C-reactive protein, GPT, GOT, GLDH, cholinesterase. RESULTS: Postoperatively phospholipase A2 is unchanged in the liver resection and control group, whereas it increases significantly in septic patients. Only following liver resection GPT, GOT and GLDH increase. C-reactive protein is increased in all groups. DISCUSSION: Unchanged phospholipase A2 after liver surgery despite an acute phase response indicated by C-reactive protein does not support the view of phospholipase A2 being of hepatic origin.

Acute-Phase Proteins↗

PubMed1997

Cell swelling activates phospholipase A2 in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells, loaded with 3H-labeled arachidonic acid and 14C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of 3H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of 14C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A2 is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in 14C-labeled stearic acid release nor in the synthesis of phosphatidyl 14C-butanol in the presence of 14C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of 14C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A1, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of 3H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP beta S or with AACOCF3, an inhibitor of the 85 kDa, cytosolic phospholipase A2. Based on these results we propose that cell swelling activates a phospholipase A2--perhaps the cytosolic 85 kDa type--by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.

Arachidonic Acid↗

PubMedDate not supplied

Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis.

Phagocytosis, the process by which leukocytes recognize and destroy invading pathogens, is essential for host defense. The binding of foreign organisms to phagocytic leukocytes initiates a complex signaling cascade which ultimately results in the entrapment and destruction of the pathogen. The signal transduction pathway mediating phagocytosis is the subject of intense investigation and is known to include protein tyrosine kinases, GTP-binding proteins, protein kinase C (PKC), actin polymerization and membrane movement. A rapidly expanding body of evidence suggests that phospholipases play an integral role in phagocytosis by generating essential second messengers. Here we review the data linking activation of phospholipase A2 (PLA2), phospholipase C (PLC) phospholipase D (PLD), and phosphoinositide 3-OH kinase (PI(3)K) to antibody (IgG)-mediated phagocytosis. Evidence is presented that (1) PLA2-derived arachidonic acid (AA) stimulates NADPH oxidase and membrane redistribution during phagocytosis, (2) the inositol-3,4,5-triphosphate (IP3) and diacylglycerol (DAG) products of PLC activate NADPH oxidase and PKC, and (3) sequential activation of PLD and phosphatidic acid phosphohydrolase may provide an alternative pathway for generation of DAG. Additionally, considerable evidence exists that wortmannin, a PI(3)K inhibitor, depresses phagocytosis. This finding is discussed in the context of the extensive effects PI(3)K products have on endocytosis and exocytosis and the potential role of membrane redistribution in phagocytosis. Finally, a model is presented which integrates data obtained from a variety of phagocytic systems and illustrates potential interactions that may exist between phospholipase-derived second messengers and signaling events required for phagocytosis.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2007

Sperm phospholipases and acrosomal exocytosis.

At the time of fertilization, the sperm cell undergoes regulated exocytosis in response to the oocyte-associated agonists progesterone and zona pellucida. An early response generated by agonist-receptor interaction in spermatozoa is the activation of mechanisms leading to Ca2+ influx, this ion being essential for the activation of phospholipases and for the fusion of the plasma membrane with the outer acrosomal membrane. Both a phosphoinositide-specific, and a phosphatidylcholine-specific phospholipase C are involved in the generation of a variety of diacylglycerol molecular species. Phospholipase D, on the other hand, does not seem to play a significant role in the generation of diacylglycerol. Hydrolysis of phospholipids by phospholipase A2 generates free fatty acids and lysophospholipids; these are important either as substrates for the generation of other metabolites (e.g., eicosanoids) or having a direct, essential action in the final stages of membrane fusion. There is still much work to be done in the future in order to characterize phospholipase isozymes and their regulation during acrosomal exocytosis in spermatozoa.

Acrosome↗

PubMed1998

Role of phospholipases during sperm acrosomal exocytosis.

During fertilization the spermatozoon undergoes a process of regulated exocytosis triggered by oocyte-associated agonists. An early response generated by agonist-receptor interaction in spermatozoa is the activation of mechanisms leading to Ca2+ influx, this ion being essential for the activation of phospholipases and for the fusion of the plasma membrane with the outer acrosomal membrane. Both a phosphoinositide-specific, and a phosphatidylcholine-specific phospholipase C are involved in the generation of a variety of diacylglycerol molecular species. Phospholipase D, on the other hand, does not seem to contribute (together with a phosphatidate phosphohydrolase) to a significant generation of diacylglycerol. Phospholipase A2 action serves to release fatty acids and to generate lysophospholipids that, either directly or serving as substrates for the generation of other metabolites, have and essential role in the final stages of membrane fusion. Additional work needs to be carried out in the future to characterize which phospholipase isoenzymes are present in mammalian spermatozoa and mechanisms regulating these enzymes

Acrosome Reaction↗

PubMed1988

Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: evidence for involvement of GTP-binding protein in phospholipase A2 activation.

Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.

Animals↗

PubMed1982

Insulinotropic effects of exogenous phospholipase A2 and C in isolated pancreatic islets.

In isolated pancreatic islets of rats, exogenous phospholipase A2 produced concentration-dependent insulin secretion. p-Bromophenacyl bromide-treated enzyme no longer showed the insulinotropic effect. The insulinotropic effect of phospholipase A2 was inhibited by nordihydroguaiaretic acid (NDGA) and 1-phenyl-3-pyrazolidinone (Phenidone) but not by indomethacin. Exogenous phospholipase C also demonstrated a concentration-related insulinotropic effect. The phospholipase C-induced insulin secretion, however, was inhibited by none of the above inhibitors. These results indicate that a lipoxygenase product(s) is involved in the insulinotropic action of phospholipase A2.

Animals↗

PubMed1989

Lipocortin I and lipocortin II inhibit phosphoinositide- and polyphosphoinositide-specific phospholipase C. The effect results from interaction with the substrates.

Lipocortins I and II, known to inhibit phospholipase A2, have been purified from bovine lung and tested with respect to their ability to affect the enzymatic activities of phosphoinositide- and polyphosphoinositide-specific phospholipase C from human platelets, rat liver cytosol or rat brain membranes. At 0.67 microM, both lipocortins led to complete inhibition of phospholipase C activity with either phosphatidylinositol or phosphatidylinositol 4,5-bisphosphate as substrate. The inhibition could be overcome by increasing the substrate concentration. Ultracentrifugation studies with lipocortin II showed a direct interaction between phosphatidylinositol and the lipocortin, indicating that the lipocortins inhibit phospholipase C not directly but by interacting with the substrate. In experiments with plasma membranes from [3H]inositol-labeled HL-60 cells, lipocortin II did not affect PI-specific phospholipase C activity in the absence or presence of calcium plus or minus GTP-gamma-S.

Animals↗

PubMed1992

Regulation of [3H]nitrendipine binding by phospholipases A2 and C through direct and GTP-sensitive mechanisms.

We compared the effects of Phospholipases A2, C, B and D on [3H]nitrendipine binding to hamster cardiac membranes, in the absence and presence of ATP or GTP. Phospholipase A2, competitively inhibited [3H]nitrendipine binding to hamster cardiac membranes unchanged by ATP or GTP (Ki = 5 ng/ml); as evidenced by complete and reversible displacement of [3H]nitrendipine binding and increase in KD on Scatchard analyses. Phospholipase C also completely inhibited [3H]nitrendipine binding to hamster cardiac membranes (Ki = 5 micrograms/ml) with a decrease in Bmax and no change in KD on Scatchard analyses. The addition of GTP alone inhibited the PLC effect in EGTA-treated membranes. The addition of GTP with either CaCl2 or ATP or both resulted in an equal and opposite enhancement of the PLC effect. Phospholipases B and D had no effect on [3H]nitrendipine binding. These data support: (1) Direct effect of PLA2 on dihydropyridine binding. (2) Indirect regulation of dihydropyridine binding by Phospholipase C through a GTP and ATP-sensitive mechanism.

Adenosine Triphosphate↗

PubMed1987

Prostacyclin synthase and phospholipases in the vascular wall of experimental hypertensive rats.

To reveal the role of enzymes involved in PGI2 synthesis for vascular PGI2 generation in experimental hypertensive models, we defined PGI2 synthase and phospholipases activities in the aortic wall of two different experimental hypertensive rats, e.g. spontaneously hypertensive rats (SHR) and desoxycorticosterone acetate (DOCA)-salt hypertensive rats. In the stage of established hypertension both of the hypertensive models had a significantly large capacity of the vascular wall to produce PGI2, as compared to respective control rats. PGI2 synthase activities in the vascular wall were significantly increased by 27% for SHR and by 80% for DOCA-salt hypertensive rats. Moreover, the enzymatic activities were closely related to the blood pressure values for both of the models. On the other hand, phospholipase C or phospholipase A2 activities were increased or unchanged in SHR, respectively, whereas both of the phospholipases were significantly decreased in DOCA-salt hypertensive rats. Thus, it is indicated that PGI2 synthase is partly responsible for the increased PGI2 generation in the vascular wall of SHR and DOCA-salt hypertensive rats, and that vascular phospholipase C is playing a more important role in providing arachidonate for PGI2 synthesis in SHR.

Animals↗

PubMed1997

Activation of LA-N-2 cell phospholipases by single alanine substitution analogs of amyloid beta peptide (25-35).

A series of single alanine substituted analogs of amyloid beta peptide (25-35) were tested for their ability to activate the phospholipases of cultured LA-N-2 cells. Substitution of alanine for the amino acids 29-34 prevented the activation of phospholipases A2 and D. In addition substitution of alanine at 28 prevented phospholipase D but not phospholipase A2 activation. All the alanine substitutions, except for positions 33 and 35, blunted phospholipase C activations. There were no activations by scrambled amyloid beta peptide.

Alanine↗

PubMed2001

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines.

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.

Animals↗

PubMed1999

Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process.

Aeromonas↗

PubMed1987

Angiotensin II stimulates phospholipases C and A2 in cultured rat mesangial cells.

Angiotensin II stimulates prostaglandin (PG) E2 formation in mesangial cells cultured from rat renal glomeruli. The interactions between angiotensin II and PGE2 are important in modulating glomerular function. We examined the mechanism for stimulation of PGE2 production in mesangial cells using the putative diacylglycerol-lipase inhibitor RHC 80267 and trifluoperazine (TFP), an agent interfering with Ca2+-CaM-mediated processes. Although RHC 80267 inhibited diacylglycerol-lipase activity in mesangial cells, it did not influence PGE2 production in response to either angiotensin II or A23187. In contrast, TFP (50 microM) inhibited basal PGE2 production and stimulation by angiotensin II and A23187. TFP also decreased 14C release in response to angiotensin from cells prelabeled with [14C]arachidonic acid, which was associated with inhibition of 14C loss from phosphatidylinositol. In cells prelabeled with 32P, orthophosphate angiotensin II caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphospate. TFP enhanced 32P labeling of phosphatidylinositides, but did not prevent the loss of phosphatidylinositol 4,5-bisphosphate in response to angiotensin. This was verified in cells prelabeled with myo-[3H]inositol where angiotensin stimulated formation of [3H]inositol trisphosphate. TFP enhanced formation of [3H]inositol trisphosphate both under basal- and angiotensin II-stimulated conditions. Thus TFP did not inhibit phospholipase C activation by angiotensin. Angiotensin II caused marked increases in [32P]lysophospholipids, indicating activation of also phospholipase A2. This process was inhibited by TFP. Taken together, these results are consistent with stimulation of both phospholipase C and A2 by angiotensin, the latter step responsible for the release of arachidonic acid and PGE2 formation. The activation of phospholipase A2, but not that of phospholipase C, is inhibited by TFP, perhaps by interference with calmodulin-dependent steps.

Angiotensin II↗

PubMed1984

Developmental changes in enzyme activities in fetal and neonatal rabbit lung. Cytidylyltransferase, cholinephosphotransferase, phospholipases A1 and A2, beta-galactosidase, and beta-glucuronidase.

Lungs were obtained from rabbit fetuses (on each day from d 24 to d 30 of gestation), neonates and adults, and were fractionated for enzyme assays. The developmental profile of cytidylyltransferase shows a decrease in specific activity from d 25 to d 29 (P less than 0.05) then a sharp rise from d 30 to adult values in d 0 neonates (P less than 0.05). Cholinephosphotransferase specific activity changes little from d 25 to birth, apart from a non-significant peak on d 29. There is a sharp rise from neonatal d 0 to adult values on d 1 (P less than 0.01). The specific activity of microsomal phospholipase A2 declines from d 25 to reach adult values in the neonate (P = 0.05). In contrast, the specific activity of lysosomal phospholipase A2 rises from d 24-28 then falls in the neonate (P less than 0.05). Adult values are higher than those in the fetus and neonate. Three other lysosomal enzyme specific activities rise to d 28 then decline: phospholipase A1, beta-galactosidase, and beta-glucuronidase. The results demonstrate that the level of microsomal phospholipase A2 does not control the extent of remodelling of phosphatidylcholine for surfactant production. Lysosomal phospholipase A2 only increases in parallel with the other lysosomal enzymes, indicating an increase in the number of lysosomes in the lung.

Age Factors↗

PubMedDate not supplied

Subcellular fractionation of bone marrow-derived macrophages: localization of phospholipase A1 and A2 and acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase.

Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA:1-1-acylglycero-3-phosphorylcholine-0-acetyltransferase. After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions. Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-beta-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de- and re-acylation reaction of phospholipids in bone marrow-derived macrophages.

Acyltransferases↗

PubMed1988

Phospholipase activation and arachidonic acid release in isolated intestinal epithelial cells.

A novel method for studying the mobilization of free arachidonic acid (AA) in isolated intestinal epithelial cells is described. The method is based on labeling the cellular phospholipids with 14C-AA and studying the release of this 14C-AA on subsequent phospholipase activation. Cells of high viability were isolated from the small intestine of guinea pigs and incubated with 14C-AA for 2 h; most of the incorporated 14C-AA was then esterified into phosphatidylethanolamine and phosphatidylcholine. When the labeled cells were stimulated with the calcium ionophore A23187 in the presence of external calcium, they released significant amounts of AA. In contrast, the cells released no AA when stimulated with A23187 in the absence of external calcium or in the presence of chlorpromazine or 4-bromophenacyl bromide, both of which are known to inhibit phospholipase A2 activity. On the other hand, the cells released significant AA in response to exogenous phospholipase C from Clostridium perfringens. These findings indicate that AA release in intestinal cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity. They also suggest the further use of 14C-AA-labeled cells for studying agents and mechanisms that may influence the release of AA in the gastrointestinal tract.

Animals↗