SEARCH · PubMed Health

Results for “PHOSPHOLIPASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 739 records · Page 41Linked to original sources

PubMed2005

mTOR-dependent suppression of protein phosphatase 2A is critical for phospholipase D survival signals in human breast cancer cells.

A critical aspect of tumor progression is the generation of survival signals that overcome default apoptotic programs. Recent studies have revealed that elevated phospholipase D activity generates survival signals in breast and perhaps other human cancers. We report here that the elevated phospholipase D activity in the human breast cancer cell line MDA-MB-231 suppresses the activity of the putative tumor suppressor protein phosphatase 2A in a mammalian target of rapamycin (mTOR)-dependent manner. Increasing the phospholipase D activity in MCF7 cells also suppressed protein phosphatase 2A activity. Elevated phospholipase D activity suppressed association of protein phosphatase 2A with both ribosomal subunit S6-kinase and eukaryotic initiation factor 4E-binding protein 1. Suppression of protein phosphatase 2A by SV40 small t-antigen has been reported to be critical for the transformation of human cells with SV40 early region genes. Consistent with a critical role for protein phosphatase 2A in phospholipase D survival signals, either SV40 small t-antigen or pharmacological suppression of protein phosphatase 2A restored survival signals lost by the suppression of either phospholipase D or mTOR. Blocking phospholipase D signals also led to reduced phosphorylation of the pro-apoptotic protein BAD at the protein phosphatase 2A dephosphorylation site at Ser-112. The ability of phospholipase D to suppress protein phosphatase 2A identifies a critical target of an emerging phospholipase D/mTOR survival pathway in the transformation of human cells.

Adaptor Proteins, Signal Transducing↗

PubMed1987

Partial characterization of phospholipase C activity in normal, psoriatic uninvolved, and lesional epidermis.

Arachidonic acid (AA), the precursor of prostaglandins and leukotrienes, can be directly liberated from membrane phospholipids by phospholipase A2 or indirectly by phospholipase C. One or both of these enzymes may be responsible for the increased content of AA found in psoriatic lesional epidermis. Keratome biopsies were obtained from normal and psoriatic individuals. After homogenization and sonication, a 10,000 g supernatant was used as the enzyme source. The activities of both phospholipase A2 and C were assayed in each sample using phosphatidylcholine and phosphatidylinositol, respectively, as substrates. Phospholipase A2 activity was found to be significantly higher than normal in both uninvolved and lesional psoriatic epidermis. In contrast, phospholipase C activity was significantly higher than normal in only the psoriatic plaque on the basis of wet weight (p less than 0.001), protein (p = 0.01), and DNA (p = 0.004) content. Phospholipase C activity in pmol diacylglycerol formed/min/microgram DNA was: normal 4.96 +/- 0.80, n = 13; uninvolved 7.29 +/- 1.06, n = 18; plaque 14.44 +/- 2.50, n = 18. Analysis (pH profile, calcium requirement, substrate specificity, and saturation kinetics) of pooled epidermal extracts showed no inherent differences in phospholipase C from normal and psoriatic epidermis, suggesting either a higher concentration or the presence of an activated form of the enzyme in psoriatic plaque. Since phospholipase C activity, in contrast to phospholipase A2 activity, is elevated only in lesional epidermis, it is possible that this enzyme contributes to AA accumulation observed in this tissue.

Calcium↗

PubMed2000

Potential role of phospholipases in virulence and fungal pathogenesis.

Microbial pathogens use a number of genetic strategies to invade the host and cause infection. These common themes are found throughout microbial systems. Secretion of enzymes, such as phospholipase, has been proposed as one of these themes that are used by bacteria, parasites, 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 review, data implicating phospholipase as a virulence factor in C. albicans, Candida glabrata, C. neoformans, and A. fumigatus are presented. A detailed description of the molecular and biochemical approaches used to more definitively delineate the role of phospholipase in the virulence of C. albicans is also covered. These approaches resulted in cloning of three genes encoding candidal phospholipases (caPLP1, caPLB2, and PLD). By using targeted gene disruption, C. albicans null mutants that failed to secrete phospholipase B, encoded by caPLB1, were constructed. When these isogenic strain pairs were tested in two clinically relevant murine models of candidiasis, deletion of caPLB1 was shown to lead to attenuation of candidal virulence. Importantly, immunogold electron microscopy studies showed that C. albicans secretes this enzyme during the infectious process. These data indicate that phospholipase B is essential for candidal virulence. Although the mechanism(s) through which phospholipase modulates fungal virulence is still under investigations, early data suggest that direct host cell damage and lysis are the main mechanisms contributing to fungal virulence. Since the importance of phospholipases in fungal virulence is already known, the next challenge will be to utilize these lytic enzymes as therapeutic and diagnostic targets.

Amino Acid Sequence↗

PubMed1967

Lethal toxin of Bacillus cereus. I. Relationships and nature of toxin, hemolysin, and phospholipase.

Bacillus cereus phospholipase was characterized as a phospholipase C by the analysis of lecithin degradation products by thin-layer and paper chromatography. Methanol in the growth menstruum inhibited completely the synthesis of phospholipase C, whereas the synthesis of lethal toxin and hemolysin were only partially inhibited. Dialysis of preformed B. cereus products against ethyl alcohol and methanol did not inactivate hemolytic, phospholipase C, or lethal activity. The hemolytic and lethal activities of culture filtrates were completely abolished by trypsin, but phospholipase C activity was resistant to inactivation. Lethal and phospholipase C properties of culture filtrates were resistant to inactivation at 45 C, whereas the hemolytic activity was completely destroyed. Lethal, hemolytic, and phospholipase C activities appeared simultaneously in a complex growth menstruum, but the kinetics of synthesis were different in all cases. Resolution of B. cereus filtrates on columns of Sephadex showed that the phospholipase C, hemolysin, and lethal toxin are distinct proteins. Evidence is also presented which suggests a correlation between the synthesis of B. cereus toxin and the period of transition from vegetative growth to sporulation. The activity of each B. cereus product was cation-independent, as opposed to cation-dependency of the phospholipase C and lethal activities of Clostridium perfringens alpha-toxin. Immunological cross-reactivity between the B. cereus products and C. perfringens alpha-toxin was not apparent; indeed, they were shown to be antigenically distinct.

Alcohols↗

PubMed2004

Phospholipase D.

Phospholipase D catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup. Phospholipase D activities have been detected in simple to complex organisms from viruses and bacteria to yeast, plants, and mammals. Although enzymes with broader selectivity are found in some of the lower organisms, the plant, yeast, and mammalian enzymes are selective for phosphatidylcholine. The two mammalian phospholipase D isoforms are regulated by protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families. Mammalian and yeast phospholipases D are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. This review discusses the identification, characterization, structure, and regulation of phospholipase D. Genetic and pharmacological approaches implicate phospholipase D in a diverse range of cellular processes that include receptor signaling, control of intracellular membrane transport, and reorganization of the actin cytoskeleton. Most ideas about phospholipase D function consider that the phosphatidic acid product is an intracellular lipid messenger. Candidate targets for phospholipase-D-generated phosphatidic acid include phosphatidylinositol 4-phosphate 5-kinases and the raf protein kinase. Phosphatidic acid can also be converted to two other lipid mediators, diacylglycerol and lyso phosphatidic acid. Coordinated activation of these phospholipase-D-dependent pathways likely accounts for the pleitropic roles for these enzymes in many aspects of cell regulation.

Animals↗

PubMed1991

Phospholipase A activity in seminal plasma of men attending an infertility clinic.

Seminal plasma from 40 men attending an infertility clinic was analyzed for phospholipase A activity and compared with serum from 20 healthy blood donors. The imprecision of the method was acceptable and the overall coefficient of variation was 8.1%. A huge discrepancy existed between seminal plasma and blood serum with regard to phospholipase A activity, the former fluid displaying on average a 180-fold higher activity than the latter. Preincubation of seminal plasma for 1 h at 37 degrees C resulted in an even higher phospholipase A activity while the corresponding activity in serum remained unchanged by such a treatment. This suggested the existence of a zymogen form of phospholipase A in seminal plasma. A significant correlation existed between sperm concentration and phospholipase A activity in seminal plasma (r = 0.46; p less than 0.01) and preincubated seminal plasma samples from normozoospermic men displayed a significantly higher mean value than those of azoospermic men. Phospholipase A activity also correlated significantly to zinc concentration in seminal plasma (r = 0.67; p less than 0.001). 4-Bromophenacyl bromide was inhibitory to a certain extent, the inhibition being most evident in the samples with high phospholipase A activity. Dibucaine and quinacrine, known phospholipase inhibitors in other systems, had no inhibitory effects. Only 58% of the seminal plasma samples contained measurable amounts of triglycerides. No significant correlation existed between triglyceride concentration and phospholipase A activity, r = 0.11.

Acetophenones↗

PubMed1992

Flow cytometric kinetic measurements of neutrophil phospholipase A activation.

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.

Boron Compounds↗

PubMed1992

Inhibition of agonist-induced activation of phospholipase C following poxvirus infection.

Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.

Adenosine Triphosphate↗

PubMed1991

Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein.

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.

Aluminum↗

PubMed1991

Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes.

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.

Angiotensin II↗

PubMed1990

Purification from bovine liver membranes of a guanine nucleotide-dependent activator of phosphoinositide-specific phospholipase C. Immunologic identification as a novel G-protein alpha subunit.

Cholate-solubilized extracts from bovine liver plasma membranes preincubated with the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) displayed enhanced phosphoinositide-specific phospholipase C activity compared with extracts from membranes incubated without nucleotide or with ATP or GDP analog. Resolution of the GTP gamma S-elicited activator of phospholipase C was achieved using heparin-Sepharose which bound the phospholipase C activity. Recombination of non-adsorbed extract with salt-eluted phospholipase C activity resulted in a stimulation of enzyme activity. The GTP gamma S-dependent activator was purified, on the basis of its ability to activate partially purified phospholipase C, by sequential chromatography on Q-Sepharose, Sephacryl S-300, octyl-Sepharose, and Mono Q. The presence of G-protein beta subunits and the alpha subunits of Gi1, Gi2, and Gi3 was detected, by immunoblot analysis, in Mono Q-purified phospholipase C activator preparations. Resolution of the activator from these alpha subunits was achieved by incubation with pertussis toxin in the presence of millimolar NAD+ followed by rechromatography on Mono Q. The phospholipase C activator, thus resolved from ADP-ribosylated alpha i subunits, possessed an approximate Mr of 42 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copurified with a substoichiometric amount of beta subunit. Immunoblot analysis of fractions from the final Mono Q column revealed cross-reactivity of the 42-kDa phospholipase C activator with antipeptide antibodies raised against residues 160-169 of alpha i1 and a region of sequence common to all known G-protein alpha subunits. The 42-kDa activator was not recognized by other alpha subunit-specific or common antibodies. These findings identify the purified phospholipase C activator as a novel G-protein alpha subunit. This may represent the active subunit of the pertussis toxin-insensitive G-protein mediating receptor-stimulated phosphoinositide breakdown in mammalian liver.

Adenosine Diphosphate Ribose↗

PubMed1990

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the purified enzyme reconstituted with turkey erythrocyte ghosts.

The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstitution of the enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.

Adenosine Diphosphate↗

PubMed1975

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.

Anilino Naphthalenesulfonates↗

PubMed1989

Coupling of exogenous receptors to phospholipase C in Xenopus oocytes through pertussis toxin-sensitive and -insensitive pathways. Cross-talk through heterotrimeric G-proteins.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) can be categorized into molecularly divergent groups by their differential sensitivity to pertussis toxin. Receptors specifically use either pertussis toxin-sensitive or-insensitive G-proteins to couple to specific effectors. Receptor stimulation of phospholipase C, however, is pertussis toxin sensitive in some systems and pertussis toxin insensitive in others. We studied the coupling of receptors to phospholipase C by expressing receptors from both systems into a single cell, the Xenopus oocyte. [Arg8]Vassopressin (AVP) receptors from liver and cholecystokinin-8(sulfated) (CCK) receptors from brain were expressed in oocytes by intracellular injection of RNA. Both receptors stimulated a Ca2+-dependent Cl- current which can also be evoked by intracellular injection of inositol 1,4,5-tris-phosphate. Hence, receptor stimulation of phospholipase C was measured as the evoked Ca2+-dependent Cl- current. The liver AVP receptor, which is known to stimulate phospholipase C in a pertussis toxin-insensitive manner (Lynch, C. J., Prpic, V., Blackmore, P. F., and Exton, J. H. (1986) Mol. Pharmacol. 29, 196-203), was found to stimulate phospholipase C through a pertussis toxin-sensitive pathway in the Xenopus oocyte. The CCK receptor from brain stimulated phospholipase C through a pertussis toxin-insensitive pathway. Both AVP and CCK stimulation of phospholipase C were attenuated by the intracellular injection of excess G-protein beta gamma subunits. Neither pertussis toxin treatment nor intracellular injection of beta gamma subunits affected any steps subsequent to inositol 1,4,5-tris-phosphate production. From these data we conclude that both the pertussis toxin-sensitive and -insensitive pathways for receptor coupling to phospholipase C are transduced by heterotrimeric G-proteins. We also find that there is a lack of coupling fidelity of receptors to G-proteins in stimulation of phospholipase C which can be influenced by the membrane environment.

Animals↗

PubMed1989

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets.

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1995

Annexin I concentration, phospholipase activity and thromboxane synthesis in irradiated rat lung.

Annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins (PLBP), has been suggested as a regulator of prostaglandin metabolism as a result of its inhibitory effect on phospholipase A2. Synthesis of prostaglandin is increased in irradiated tissue, but the mechanism underlying this increase has not been delineated. It is conceivable that a decrease in the level of annexin I resulting in increased phospholipase activity may be responsible for the enhanced synthesis of prostaglandin. Accordingly, we measured the level of a lung 36 kDa PLBP, which possesses characteristics of annexin I, as well as the activity of phospholipase and the synthesis of thromboxane A2 (TXA2) in irradiated rat lung. The right lung of rats was irradiated with 0, 15 or 30 Gy of X rays and the animals were sacrificed after 3 months. Phospholipid binding protein was assayed by its ability to transfer unilamellar liposomes to multilamellar liposomes and by immunoblotting against anti-36 kDa rabbit PLBP antisera. Production of TXA2 by minced lung tissue was determined by radioimmunoassay of its stable metabolite TXB2. Phospholipase activity was assayed by hydrolysis of [14C]dioleoylphosphatidylcholine. Our results showed that PLBP activity in the lungs irradiated with 30 Gy was lower than that in the lungs irradiated with 0 and 15 Gy (8.82 +/- 0.47 compared to 9.73 +/- 0.49 and 9.95 +/- 0.78 nmol phospholipid transferred/mg protein, respectively). Western blotting demonstrated a near total depletion of annexin I in the lungs irradiated with 30 Gy. Phospholipase activity was also lower in the lungs irradiated with 30 Gy compared to that in the lungs irradiated with 0 Gy (0.23 +/- 0.01 vs 0.32 +/- 0.01 nmol phosphatidylcholine liberated/mg protein/min, P < 0.001). Reduced phospholipase activity was observed not only in the cytosolic or soluble fraction of lung homogenate, but also in precipitates obtained after 21,000g and 100,000g centrifugation. Despite this decline in phospholipase activity, there was a 2.8-fold increase in the synthesis of thromboxane (367 +/- 65 compared to 1076 +/- 143 pg TXB2/mg tissue/10 min for lungs irradiated with 0 and 30 Gy, respectively). These results are not consistent with the hypothesis that increased synthesis of thromboxane A2 in irradiated rat lung is a direct result of elevated phospholipase activity. In fact, phospholipase activity is decreased in the irradiated lung, despite a decline in the concentration of annexin I, its putative inhibitor.

Animals↗

PubMed1993

Comparative studies on fatty acid composition and phospholipases A2 and C activities in rabbit and bovine iris-ciliary body.

It is well established that production of prostaglandins by ocular tissues is dependent upon the species. The rabbit iris-ciliary body produces greater amounts of prostaglandins than that of the bovine. To throw more light on the biochemical basis underlying these differences we have compared the fatty acid composition and phospholipases A2 and C activities in rabbit and bovine irides. When the concentration of arachidonic acid is expressed as % of total fatty acids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, separated from rabbit iris phospholipids, contained 56, 38 and 18% more arachidonic acid, respectively, than those of the bovine. The total lipid phosphorus in rabbit and bovine iris-ciliary body were found to be 13.74 and 9.34 mumol g-1 wet tissue, respectively. Subcellular fractions, prepared from rabbit iris, contained 30-230% more phospholipase C activity than those of the bovine, and about 5-41 times higher phospholipase A2 activity than those of the bovine. In the rabbit iris microsomal fraction, phospholipase C activity is 33 times higher than that of phospholipase A2. However, the data presented suggest that phospholipase A2, rather than phospholipase C, is the enzyme which is more involved in arachidonic acid release for eicosanoid biosynthesis. These findings suggest that the high contents of arachidonic acid and phospholipases A2 and C in the rabbit iris could contribute to its unique capacity to synthesize and release prostaglandins in the anterior segment as compared to that of other mammalian species.

Animals↗

PubMed1983

The influence of phospholipase A2 and glutathione peroxidase on the elimination of membrane lipid peroxides.

The relationship between release of membrane lipid peroxidation products and phospholipase action was examined. Rat liver microsomes and phosphatidylcholine liposome-phospholipase A2 preparations were subjected to iron ascorbate-induced lipid peroxidation. Peroxidation products were characterized by measurement of malondialdehyde and lipid peroxides. Experiments were designed to demonstrate phospholipase dependent removal of peroxidation products origination in the membrane. Increased lysophosphatidylcholine formation was evident following lipid peroxidation in phospholipase A2-containing liposomes which was inhibited by p-bromophenacyl bromide and mepacrine. Lipoxygenase-dependent oxygen consumption, as well as peroxide transfer from microsomes to the incubation medium, was largely dependent on phospholipase and could be diminished by phospholipase inhibitors. Furthermore, lipid hydroperoxides formed by subjecting phosphatidylcholine liposomes to iron ascorbate-induced peroxidation, or those present in aged liposomes, were effectively reduced by glutathione peroxidase when phospholipase A2 was present in the assay. Low level glutathione peroxidase activity was observed in the absence of phospholipase A2.

Animals↗