Formation Of Lysophospholipids Research Articles

Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.

Inhibition of phosphatidic acid production and lysophospholipid formation in collagen-stimulated human platelets by staurosporine, a protein kinase inhibitor

To investigate a possible regulatory role of protein kinase C (PKC) on collagen-induced phospholipase activity, human platelets were prelabelled with either [3H] arachidonic acid or [14C]stearic acid and stimulated with collagen (2 micrograms/ml) in the presence or absence of the protein kinase inhibitor, staurosporine (1 microM). The collagen-induced release of [3H]arachidonic acid and formation of [14C]stearoyl-labelled lysophospholipids was inhibited by prior incubation with staurosporine, as was the formation of 3H-labelled thromboxane B2, thereby suggesting inhibition of the collagen-induced phospholipase A2 activity. The degradation of phosphatidylinositol (PI) and elevation of phosphatidic acid (PA) in platelets prelabelled with either radiotracer were also completely blocked by staurosporine pretreatment, indicating a suppression of collagen-stimulated phospholipase C activity. Suppressed phospholipase C activity may have been due to diminished thromboxane A2 formation since treatment with the dual cyclo-oxygenase/lipoxygenase inhibitor, BW755C, also resulted in an inhibition of the collagen-stimulated loss of 14C-labelled PI and rise in PA by 75-80%. Our results suggest that protein kinase, possible PKC, may be involved in the regulation of these phospholipases in collagen-stimulated human platelets.

.beta.-Bungarotoxin-mediated liposome fusion: spectroscopic characterization by fluorescence and ESR

The capacity of the snake venom neurotoxin beta-bungarotoxin to induce fusion of small unilamellar liposomes was demonstrated. The fusion process was studied spectroscopically using three different methods: (i) by resonance energy transfer, using fluorescent lipid analogues; (ii) by the Tb/DPA assay; (iii) by electron spin resonance, using spin-labeled phospholipids. For the latter technique, a new method of analysis based on Fourier-transform component separation was developed. The fusogenic activity was found to be strongly correlated with the known phospholipase A2 activity of the toxin: both functions were shown to have a specific requirement of Ca2+ at almost stoichiometrical concentrations, much below the threshold values found for unspecific divalent cation induced vesicle fusion. Similarly, the presence of phosphatidic acid in the target membrane was essential for both fusogenic and enzymatic activities. The results suggest a molecular mechanism of fusion involving protein binding to negatively charged groups on the membrane surface, followed by local formation of lysophospholipids and as a consequence hereof the creation of point defects in the lipid structure. On the basis of these findings, a model is put forward to explain the specific mode of action of beta-bugarotoxin in vivo.

Evidence for tight coupling of phospholipase activation and Ca 2+ influx during acrosome reaction of golden hamster spermatozoa

1. 1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity, Ca 2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. 2. Upon exposure of the spermatozoa to 1.7 mM Ca 2+, a net uptake by the cells occurred in two distinguishable phases. 3. 3. Depletion of extracellular Ca 2+ by ethylene glycol bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) at a time that an initial Ca 2+ uptake was observed to reach almost steady-state, prevented the secondary Ca 2+ uptake and acrosome reaction. 4. 4. The time course of an initial Ca 2+ uptake seemed to precede that of the acrosome reaction. 5. 5. Incubation of the spermatozoa with Ca 2+ in the presence of [ 3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca 2+. 6. 6. Phospholipase A 2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca 2+ uptake and phosphatidic acid production. 7. 7. It is concluded that first Ca 2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A 2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.

Molecular mechanisms of platelet activation.

Molecular mechanisms of platelet activation.

Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C.

Many types of peptide hormone and neurotransmitter receptors mediate hydrolysis of phosphoinositides (PI) and arachidonic acid and arachidonic acid metabolite (AA) release, but the relation between these responses is not clearly defined. We have characterized bradykinin (BK)-mediated AA release and PI hydrolysis in clonal Madin-Darby canine kidney cells (MDCK-D1). Both responses occurred over a similar dose range in response to the B1 and B2 receptor agonist, BK, but not in response to the B1 receptor-selective agonist des-Arg-BK. To test whether AA release occurs via a mechanism which is sequential to and dependent upon PI hydrolysis, we used the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C. TPA treatment blocked BK-mediated PI hydrolysis in MDCK-D1 cells, while at the same time and at similar concentrations enhancing BK-mediated AA release. Thus, TPA treatment dissociated BK-mediated AA release from PI hydrolysis. In addition, treatment of MDCK-D1 cells with neomycin blocked BK-mediated hydrolysis of phosphatidylinositol bisphosphate without reducing BK-mediated AA release. BK treatment increased formation of lysophospholipids with a time course in accord with BK-mediated AA release, indicating that at least part of the BK-mediated AA release was likely derived from activation of phospholipase A2. BK-mediated lysophospholipid production was enhanced by pretreatment with TPA, suggesting that the mechanism of AA release before and after treatment with TPA was the same. BK-mediated AA release and lysophospholipid production was dependent on the presence of extracellular calcium, while the enhanced responses to BK in the presence of TPA were not dependent on the presence of extracellular calcium. TPA treatment also enhanced AA release and lysophospholipid production in response to the calcium ionophore A23187. From these data we propose that BK, acting at B2 receptors, promotes AA release in MDCK cells via a mechanism which is 1) independent of polyphosphoinositide hydrolysis by phospholipase C, 2) dependent upon influx of extracellular calcium and activation of phospholipase A2, and 3) enhanced by activation of protein kinase C.

The role of lipids in Plasmodium falciparum invasion of erythrocytes: a coordinated biochemical and microscopic analysis.

The role of lipids in Plasmodium falciparum invasion of erythrocytes was investigated by biochemical and fluorescent microscopic analysis. Metabolic incorporation of radioactive oleate or palmitate and fractionation of radiolabeled phospholipids by thin-layer chromatography revealed no difference in the major phospholipid classes of schizonts and early ring forms after merozoite invasion. Fluorescent anthroyloxy derivatives of oleate and palmitate were also metabolically incorporated into parasite phospholipids. By microscopic analysis, the fluorescent phospholipids were seen localized in the plasma membrane and, within the merozoite, concentrated near the apical end. During invasion fluorescent phospholipid appeared to be injected from the apical end of the merozoite into the host membrane, both within and outside the parasite-host membrane junctions. After invasion fluorescent lipid was only found in the parasite plasma membrane and/or parasitophorous vacuole membrane. Parallel experiments with a fluorescent cholesterol derivative, incorporated into parasite membranes by exchange, revealed neither heterogeneous distribution of label within the parasite nor evidence for cholesterol transfer from merozoite to host cell membrane. Results suggest that during invasion no major covalent alteration of parasite lipids, such as lysophospholipid formation, occurs. However, invasion and formation of the parasitophorous vacuolar membrane apparently involves insertion of parasite phospholipids into the host membrane.

Preterm labor and bacterial intra-amniotic infection: arachidonic acid liberation by the action of phospholipase A2.

There is a strong association between preterm labor and infection, presumably through an increase in prostaglandin (PG) formation. The studies presented in this report were undertaken to evaluate whether bacterial products stimulate endogenous phospholipid hydrolysis and arachidonic acid liberation by the action of phospholipase A2, a rate-limiting step for PG synthesis. When human endometrial cells prelabelled with [3H]arachidonic acid to an isotopically steady state were exposed to a medium conditioned with Bacteroides fragilis, arachidonic acid liberation was stimulated, accompanied with lysophospholipid formation. Similar stimulatory effect on phospholipid degradation was also observed in the experiments with the bacterial extract. These results demonstrate that the organism produces phospholipase A2 acting on endogenous phospholipids and/or factor(s) activating phospholipase A2. Phospholipase A2 is suggested as a mechanism for the onset of labor associated with intra-amniotic infection and arachidonic acid liberation.

No evidence for lysophospholipid formation during peroxidation of phospholipids by NADPH-cytochrome P-450 reductase and iron ions.

Liposomes comprised of liver microsomal phospholipids and radioactive phosphatidylcholine or phosphatidylethanolamine as tracers were incubated with isolated liver microsomal NADPH-cytochrome P-450 reductase, NADPH and ADP-EDTA-chelated iron ions, a system which stimulates peroxidation of unsaturated fatty acids of phospholipids. Phospholipids and their reaction products were extracted and chromatographed on HPLC. Phosphatidylcholine and phosphatidylethanolamine considerably decreased after 30 min incubation, depending on the enzyme and NADPH as measured by UV absorbance and radioactivity. However, neither a lysophospholipid peak nor a lysophospholipid-like peak were detectable. We suggest that lysophospholipid formation during microsomal lipid peroxidation is exclusively due to phospholipase A2 and not due to peroxidative breakdown of the unsaturated fatty acid in the beta-position of glycerol.

Involvement of phospholipase A2 in norepinephrine release from synaptosomes isolated from rat cerebral cortex

Phospholipase A(2) added directly to superfused [(3)H]norepinephrine-labeled synaptosomes could cause the release of neurotransmitter molecules. Chloroquine and quinacrine, which block the action of phospholipase A(2), inhibited either the phospholipase A(2)-stimulated or the high potassium-stimulated release of [(3)H]norepinephrine from synaptosomes. Only quinacrine blocked the high potassium-stimulated influx of Ca(2+). It appears that during stimulation of synaptosomes, Ca(2+) influx leads to the activation of phospholipase A(2), which in turn, hydrolyzes membrane phospholipids in situ. The formation of lysophospholipids may alter the microenvironment and the physicochemical properties of membranes, resulting in the release of neurotransmitter through exocytosis.

活性化血小板の膜脂質代謝とサイクリックヌクレオチド ｃＡＭＰとｃＧＭＰの比較

Platelet activation is associated with the active metabolism of inositol phospholipids. The effects of cAMP and cGMP on thrombin-stimulated human platelet responses and membrane phospholipid metabolism were investigated. Thrombin (0.1U/ml)-induced aggregation and serotonin release were prevented by pretreatment with dibutyryl cyclic AMP (dbcAMP) in a dose-dependent manner. 1, 2-Diacylglycerol production as a result of phospholipase C activation and formation of lysophospholipids were also suppressed by addition of dbcAMP. Two opposite hypotheses (stimulation or inhibition) are still discussed about the role of cGMP in platelet function. 8-Bromo cyclic GMP (8bcGMP) partially inhibited aggregation and serotonin secretion stimulated by thrombin. Phospholipase C activity, monitored by phosphatidylinositol breakdown and phosphatidic acid formation, and arachidonic acid liberation were also diminished by 8bcGMP. Although the inhibitory effect of 8bcGMP on thrombin-stimulated platelet is less than that of the same dose of dbcAMP, 8bcGMP suppresses platelet responses. A plausible explanation for its inhibitory effect is that 8bcGMP may inhibit the receptor-linked breakdown of inositol phospholipids and arachidonate liberation.

Comparison of the hydrolysis of phosphatidylethanolamine and phosphatidyl ( N-acyl) ethanolamine in Dictyostelium discoideum amoebae

In Dictyostelium discoideum, ( N-acyl)ethanolamine glycerophospholipids disappear as the amoebae aggregate, whereas the amount of ethanolamine glycerophospholipids remains relatively constant, suggesting that each type of ethanolamine-containing phospholipid might have a separate metabolic pathway. To study their metabolism, phosphatidylethanolamine and phosphatidyI( N-acyl)ethanolamine containing either [ 14C]ethanolamine or a 14C-labeled sn-2 fatty acyl group were incubated with D. discoideum homogenates, and the conversion of the substrates into radioactive products was monitored. At pH values 3.8 and 4.5, phosphatidyl( N-acyl)ethanolamine was hydrolyzed by a phospholipase A 1 to form the sn-2 acyl form of the lipid. Only minor hydrolysis occurred at pH values of 5.2 or higher. ( N-acyl)Ethanolamine was also released by a phospholipase D type activity at 0.1 the rate of the lysophospholipid formation. Phosphatidyl ( N-acyl)ethanolamine was not hydrolyzed to form phosphatidylethanolamine or water soluble components. At pH 7.2 and at the low pH range of 3.8–4.5, phosphatidylethanolamine was hydrolyzed to lysophosphatidylethanolamine, which was then further degraded to water-soluble components. At pH 7.2, a phospholipase A 2 initially hydrolyzed the phosphatidylethanolamine, whereas at the low pH range a phospholipase A 1 was the most active enzyme. Although both types of ethanolamine-containing phospholipid were hydrolyzed by a phospholipase A 1 at the low pH range, phosphatidylethanolamine hydrolysis was more sensitive to inhibition by Trition X-100, and phosphatidylethanolamine was hydrolyzed to water-soluble components, whereas phosphatidyl( N-acyl)ethanolamine was not. At pH 7.2, phosphatidylethanolamine was hydrolyzed, but phosphatidyl( N-acyl)ethanolamine was not hydrolyzed at all. These results indicate that there are separate routes of degradation for the two types of ethanolamine-containing phospholipid in D. discoideum.

Inhibition of prostacyclin synthesis in endothelial cells by methylisobutylxanthine is not mediated through elevated cAMP level

Methylisobutylxanthine (MIX) raised cAMP levels and inhibited prostacyclin synthesis and arachidonic acid release in endothelial cells from both pig aorta and human umbilical vein. These effects were reversible and dose dependent on MIX concentrations. Dibutyryl cAMP (3 mM) alone did not inhibit prostacyclin synthesis or arachidonic acid release. When added with MIX, dibutyryl cAMP did not enhance the inhibition elicited by MIX. MIX inhibited the formation of lysophospholipids, 1,2-diacylglycerol and phosphatidic acid in bradykinin-stimulated pig endothelial cells, suggesting that the inhibition of prostacyclin synthesis resulted from an apparent inhibition of both phospholipase A 2 and phospholipase C. Other phosphodiesterase inhibitors, theophylline and mopidamole, also raised cAMP levels and inhibited arachidonic acid release. However, there was no correlation between cAMP levels and these inhibitions. Forskolin, an adenylate cyclase activator, elevated intracellular cAMP levels with no apparent inhibition on prostacyclin synthesis. We conclude that the inhibitory effect of MIX on phospholipase A 2 and phospholipase C is probably through mechanisms other than the elevation of the cAMP level.

Role of Ca2+ in phosphatidylinositol response and arachidonic acid release in formylated tripeptide- or Ca2+ ionophore A23187-stimulated guinea pig neutrophils.

The role of Ca2+ in phospholipid metabolism and arachidonic acid release was studied in guinea pig neutrophils. The chemotactic peptide formylmethionyl-leucyl-phenyl-alanine (fMLP) activated [32P]Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) without any effects on the labeling of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). This activation was observed in Ca2+-free medium. Even in the neutrophils severely deprived of Ca2+ with EGTA and Ca2+ ionophore A23187, the stimulated labeling was not inhibited. When [3H]arachidonic acid-labeled neutrophils were stimulated by fMLP, a loss of [3H]arachidonic acid moiety in PI and the resultant increase in [3H]arachidonyl-diacylglycerol (DG), -PA, and free [3H]arachidonic acid was marked within 3 min. With further incubation, a loss of [3H]arachidonic acid in PC and PE became significant. These results suggest the activation of phospholipase C preceded the activation of phospholipase A2. In Ca2+-free medium, the decrease in [3H]arachidonyl-PI and the increase in [3H]arachidonyl-PA were only partially inhibited, although the release of [3H]arachidonic acid and a loss of [3H]arachidonyl-PC and -PE was completely blocked. These results show that PI-specific phospholipase C was not as sensitive to Ca2+ deprivation as arachidonic acid cleaving enzymes, phospholipase A2, and diacylglycerol lipase. Ca2+ ionophore A23187, which is known as an inducer of secretion, also stimulated [32P]Pi incorporation into PI and PA, although the incorporation into other phospholipids, such as PC and PE, was inhibited. This stimulated incorporation seemed to be caused by the activation of de novo synthesis of these lipids, because the incorporation of [3H]glycerol into PA and PI was also markedly stimulated by Ca2+ ionophore. But the chemotactic peptide did not increase the incorporation of [3H]glycerol into any glycerolipids including PI and PA. Thus, it is clear that fMLP mainly activates the pathway, PI leads to DG leads to PA, whereas Ca2+ ionophore activates the de novo synthesis of acidic phospholipids. When [3H]arachidonic acid-labeled neutrophils were treated with Ca2+ ionophore, the enhanced release of arachidonic acid and the accumulation of [3H]arachidonyl-DG, -PA with a concomitant decrease in [3H]arachidonyl-PC, -PE, and -PI were observed. Furthermore, the Ca2+ ionophore stimulated the formation of lysophospholipids, such as LPC, LPE, LPI, and LPA nonspecifically. These data suggest that Ca2+ ionophore releases arachidonic acid, unlike fMLP, directly from PC, PE, and PI, mainly by phospholipase A2. When neutrophils were stimulated by fMLP, the formation of LPC and LPE was observed by incubation for more than 3 min. Because a loss of arachidonic acid from PI occurred rapidly in response to fMLP, it seems likely the activation of PI-specific phospholipase C occurred first and was followed by the activation of phospholipase A2 when neutrophils are activated by fMLP...

Characterization of substances that restore impaired cell division of UV-irradiated E. coli B.

Substances which restore impaired cell division in UV-irradiated E. coli B were surveyed among various bacteria. The active substance was found only in several genera of Gram-negative bacteria, i.e., Escherichia, Enterobacter, Salmonella and some species of Pseudomonas. The activity in the dialyzed cell extract of E. coli B/r was observed in the presence of β-NAD and was enhanced by Mg2 + and Mn2+. The active substance was very labile, but the activity was protected by 1 mM dithiothreitol in the process of purification. The activity of a fraction recovered through DEAE-cellulose column chromatography was stimulated by the presence of membrane fraction. Upon treatment with lipid-degrading enzymes and proteases, the division-stimulating activity was lost or reduced. It appears that the inactivation by lipase and phospholipase A2 was due to the formation of lysophospholipids and that a proteinous substance participated in the recovery of impaired cell division of UV-irradiated E. coli B.

Anti-inflammatory and platelet anti-aggregant activity of phospholipase-A2 inhibitors.

Mepacrine, papaverine, p-bromophenacyl bromide and 2,3-dibromo(4'-cyclohexyl-3'-chloro)-phenyl-4-oxo-butyric acid (CB 874) inhibit the hydrolysis of phospholipids induced by thrombin in dog platelets. They also exhibit anti-inflammatory and anti-aggregant properties. These biological activities may be explained by a direct or indirect inhibitory action on phospholipase A2. Phospholipase A2 inhibitors may block not only the release of arachidonic acid and its subsequent conversion into prostaglandins but also the formation of lysophospholipids involved in inflammation and/or platelet aggregation.

Lysophospholipids in Pig Pancreatic Zymogen Granules in Relation to Exocytosis

A hypothesis to explain the stimulatory role of cyclic AMP (adenosine 3':5'-monophosphate) in pancreatic enzyme secretion. has been tested. In this hypothesis cyclic AMP would activate a phospholipase activity, which would lead to a locally increased lysophospholipid formation, resulting in a fusion between the zymogen granule membrane and the apical plasma membrane. Cyclic AMP added to isolated pig pancreatic zymogen granules leads to an increased lysis of these granules, but the slowness of this effect makes its physiological significance dubious. In pancreatic homogenates or zymogen granules no stimulating effect of cyclic AMP on lipase of phospholipase activity could be demonstrated. Isolated zymogen granules have a high lysophospholipid content (27% of total phospholipids), consisting of the 1-acyl and 2-acyl forms of lysophosphatidylcholine and lysophosphatidylethanolamine. Experiments with radioactive phosphatidylcholine indicate that the lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure. It is concluded that these experiments do not lend support to the above hypothesis for the mechanism of action of cyclic AMP in pancreatic enzyme secretion