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PubMed2006

Galpha13 mediates activation of the cytosolic phospholipase A2alpha through fine regulation of ERK phosphorylation.

Heterotrimeric GTP-binding (G) proteins transduce hormone-induced signals to their effector enzymes, which include several phospholipases. In particular, the G(o)/G(i) and G(q) protein families have been shown to couple signaling to phospholipase A(2) (PLA(2)), phospholipase C, and phospholipase D, while the G(12)/G(13) family has been linked to the activation of small GTPases of the Rho family, and hence, to phospholipase D activation. Here, we demonstrate that in CHO cells, the G(12)/G(13) family is also able to activate cPLA(2)alpha, through the activation of RhoA and, subsequently, ERK1/2. Hormone-induced arachidonic acid release increased as a consequence of Galpha(13) overexpression, and was inhibited through inhibition of Galpha(13) signaling. The Galpha(13)-mediated cPLA(2)alpha activation was inhibited by pharmacological blockade of ERK1/2 with either U0126 or PD98059, and by RhoA inactivation with C3 toxin or a dominant-negative RhoA (N19RhoA), and was stimulated by the serine-threonine phosphatase inhibitor calyculin A. Our data thus identify a pathway of cPLA(2)alpha regulation that is initiated by thrombin and purinergic receptor activation, and that signals through Galpha(13), RhoA and ERK1/2, with the involvement of a calyculin-sensitive phosphatase.

Animals↗
PubMed2005

Phospholipase A2 activation by melittin enhances spontaneous glutamatergic excitatory transmission in rat substantia gelatinosa neurons.

In order to know a role of phospholipase A2 in modulating nociceptive transmission, the effect of a secreted phospholipase A2 activator melittin on spontaneous glutamatergic excitatory transmission was investigated in substantia gelatinosa neurons of an adult rat spinal cord slice by using the whole-cell patch-clamp technique. Bath-applied melittin at concentrations higher than 0.5 microM increased both the amplitude and the frequency of spontaneous excitatory postsynaptic current in a manner independent of tetrodotoxin; the latter effect of which was examined in detail. In 80% of the neurons examined (n = 64), melittin superfused for 3 min gradually increased spontaneous excitatory postsynaptic current frequency (by 65+/-6% at 1 microM; n = 51) in a dose-dependent manner (effective concentration for half-maximal effect = 1.1 microM). This effect subsided within 3 min after washout. The spontaneous excitatory postsynaptic current frequency increase produced by melittin was reduced by the phospholipase A2 inhibitor 4-bromophenacryl bromide (10 microM) while being unaffected by the cyclooxygenase inhibitor indomethacin (100 microM) and the lipoxygenase inhibitor nordihydroguaiaretic acid (100 microM). A similar increase in spontaneous excitatory postsynaptic current frequency was produced by exogenous arachidonic acid (50 microM); this effect was also unaffected by the cyclooxygenase or lipoxygenase inhibitor. Melittin failed to increase spontaneous excitatory postsynaptic current frequency in a nominally Ca2+-free or La3+-containing Krebs solution. We conclude that melittin increases the spontaneous release of L-glutamate to substantia gelatinosa neurons by activating secreted phospholipase A2 and increasing Ca2+ influx through voltage-gated Ca2+ channels in nerve terminals, probably with an involvement of arachidonic acid but not its metabolites produced by cyclooxygenase and lipoxygenase. Considering that the substantia gelatinosa plays an important role in regulating nociceptive transmission, it is suggested that this transmission may be positively modulated by secreted phospholipase A2 activation in the substantia gelatinosa.

Acetophenones↗
PubMed2006

Phospholipase C epsilon: linking second messengers and small GTPases.

Although several observations over the years suggested a link between the Ras superfamily of GTPases and second messengers generated by the isoforms of phospholipase C, such links had not been substantiated at the molecular level until recently. In particular, identification of a novel phospholipase C isoform, PLC epsilon, which also incorporates domains for guanine nucleotide exchange and Ras binding, have prompted an interest in the interplay between small GTPases and phospholipase C and possible significance of these interconnectivities. Research that followed suggests that activation of each of the major classes of phospholipase C by small GTPases could have a different mechanism and different function, and also that phospholipase C enzymes in turn control Ras GTPases through regulatory proteins that respond directly to second messengers.

Animals↗
PubMed1991

Cytokine- and forskolin-induced synthesis of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by dexamethasone.

We have previously described that treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin stimulates the synthesis and secretion of prostaglandin E2 and group II phospholipase A2. We now report that pretreatment of the mesangial cells with dexamethasone dose-dependently suppresses the cytokines- and forskolin-induced synthesis of prostaglandin E2 as well as the induced synthesis and secretion of group II phospholipase A2. These observations implicate that the inhibition of the cellular or secreted phospholipase A2 activity by dexamethasone in rat mesangial cells is not due to induced synthesis of phospholipase A2 inhibitory proteins but caused by direct inhibition of phospholipase A2 protein expression.

Animals↗
PubMed1986

Activation of NADPH-oxidase by arachidonic acid involves phospholipase A2 in intact human neutrophils but not in the cell-free system.

The mechanism involved in the stimulation of NADPH-oxidase by arachidonic acid (AA) in intact human neutrophils was studied and compared with that involved in a cell-free system. [3H]-AA was released from pre-labeled cells upon AA stimulation, and phospholipase A2 inhibitors reduced in parallel the release of [3H]-AA and superoxide. Cyclooxygenase, lipoxygenase or protein kinase inhibitors failed to affect either response. In a cell-free system, no release of [3H]-AA was observed after AA addition, whereas NADPH-oxidase was activated; the generation of superoxide was not inhibited by phospholipase inhibitors and was not initiated by adding phospholipase A2 to the preparation. Thus AA stimulates NADPH-oxidase through a phospholipase A2 mediated pathway in intact cells, but activates the oxidase independent of phospholipase A2 in a broken cell system, suggesting distinctive mechanisms of activation for each system.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Hyposmotically induced amino acid release from the rat cerebral cortex: role of phospholipases and protein kinases.

In an evaluation of the contribution of swelling-induced amino acid release, through the regulatory volume decrease (RVD) process, to cerebral ischemic injury, studies of the role of phospholipases and protein kinases in the response to hyposmotic stress were undertaken using an in vivo rat cortical cup model. Hyposmotic stress induced significant releases of aspartate, glutamate, glycine, phosphoethanolamine, taurine and GABA from the rat cerebral cortex. Taurine release was most affected, exhibiting a greater than 9-fold increase during the hyposmotic stimulus. The phospholipase A2 (PLA2) inhibitors 4-bromophenacyl bromide (1 microM) and 7,7-dimethyleicosadienoic acid (5 microM) had no significant effects on hyposmotically induced amino acid release. AACOCF3 (50 microM), an inhibitor of cytosolic PLA2 decreased taurine release to 84% of DMSO controls. The release of the other amino acids was not affected. The phospholipase C inhibitor U73122 (5 microM) had no significant effects on amino acid release. The protein kinase C (PKC) inhibitor chelerythrine (5 microM) significantly reduced hyposmotically induced taurine release to 72% of saline controls but had no significant effects on the other amino acids. Stimulation of PKC with phorbol 12-myristate, 13-acetate (10 microM) did not significantly change taurine, glutamate, glycine or phosphethanolamine release. The releases of aspartate and GABA were enhanced 2 to 3 fold. Phorbol 12,13-didecanoate (10 microM), another potent stimulator of PKC, significantly increased taurine release to 122% of DMSO controls. The releases of aspartate, glutamate and glycine were enhanced 2.5 to 3.5 fold. Similarly, stimulation of protein kinase A with forskolin (100 microM) significantly increased taurine, aspartate, and glycine release 1.5- to 2-fold compared to DMSO controls. In summary, phospholipases may play a minor role in volume regulation. These studies also support the hypothesis that protein kinases play a modulatory role in the RVD response. The results show that although RVD may play a role, additional mechanisms, including phospholipase activation, must be involved in the ischemia-evoked release of excitotoxic amino acids.

Acetophenones↗
PubMed1999

Luteal phospholipase A2 activity increases during functional and structural luteolysis in pregnant rats.

We determined cytosolic phospholipase A2 activity of the corpus luteum during luteolysis in pregnant and post-partum rats. Phospholipase A2 activity and its metabolite prostaglandin F2alpha in the corpus luteum remarkably increased just before parturition and further rose transiently during post-partum structural luteolysis. The absence of a pups' suckling stimulus delayed corpus luteum involution, being associated with an altered fluctuation in phospholipase A2 activity and depressed prostaglandin F2alpha levels. Exogenous prolactin had a reversal effect. Pharmacological and immunochemical characterization suggests multiple isoforms of phospholipase A2 in a pregnant corpus luteum. These results show the increased phospholipase A2 activity and its possible implication in luteolysis in pregnant rats.

Animals↗
PubMed2002

Observation of additional calcium ion in the crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A2.

Phospholipase A(2) catalyses hydrolysis of the ester bond at the C2 position of 3-sn-phosphoglycerides. Here we report the 1.9A resolution crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A(2). The structure was solved by molecular replacement method using the orthorhombic form of the recombinant phospholipase A(2). The final protein model contains all the 123 amino acid residues, two calcium ions, 125 water molecules and one 2-methyl-2-4-pentanediol molecule. The model has been refined to a crystallographic R-factor of 19.6% (R(free) of 25.9%) for all data between 14.2A and 1.9A. The residues 62-66, which are in a surface loop, are always disordered in the structures of bovine pancreatic phospholipase A(2) and its mutants. It is interesting to note that the residues 62-66 in the present structure is ordered and the conformation varies substantially from those in the previously published structures of this enzyme. An unexpected and interesting observation in the present structure is that, in addition to the functionally important calcium ion in the active site, one more calcium ion is found near the N terminus. Detailed structural analyses suggest that binding of the second calcium ion could be responsible for the conformational change and the ordering of the surface loop. Furthermore, the results suggest a structural reciprocity between the k(cat)(*) allosteric site and surface loop at the i-face, which represents a newly identified structural property of secreted phospholipase A(2).

Allosteric Site↗
PubMed1990

Phospholipase A2 in venom extracts from honey bees (Apis mellifera L.) of different ages.

We measured phospholipase A2 activity in the venom of worker honey bees (Apis mellifera L.) of known ages using chemical (titrimetric) and radioallergosorbent methods. The two techniques give similar results. Low levels of phospholipase A2 are present in the venom system at the time of eclosion. Phospholipase A2 activity in the venom increases steadily through the 10 days after eclosion. Maximal phospholipase A2 levels (about 40 micrograms phospholipase A2/venom sac) are maintained through the rest of the life of a worker bee in summer.

Aging↗
PubMed1998

Mitochondrial swelling and oxygen consumption during respiratory state 4 induced by phospholipase A2 isoforms isolated from the South American rattlesnake (Crotalus durissus terrificus) venom.

The non-covalent interaction between two molecular entities namely, phospholipase A2 and crotapotin, results in the main toxin, crotoxin, present in the venom of the South American rattlesnake Crotalus durissus terrificus. High performance liquid chromatography has enabled us the isolation of three phospholipase A2 isoforms (F1, F2 and F3), characterized through denaturing and non-denaturing polyacrylamide gel electrophoresis and also through the N-terminal amino acid sequence analysis. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined through mitochondrial swelling and O2 consumption during respiratory state 4. F1 showed a dose-dependent stimulation of O2 consumption while F2 and F3 caused stimulation only at low doses and inhibition at high amounts. These effects were completely suppressed by the presence of 0.1% bovine serum albumin or 0.5 mM EGTA in the incubation medium. Taking the mitochondrial swelling as an activity parameter, all of them presented the same behaviour at different intensities, leading to permeabilization of the mitochondrial membrane. In this case, addition of EGTA prevented it whereas bovine serum albumin was ineffective, indicating that the lipid microenvironment was affected. These results suggest that free fatty acids are directly responsible for the observed effects induced by phospholipase A2 isoforms on oxygen consumption experiments. The protection conferred by cyclosporin-A on swelling induced by the isoforms, when present in low concentrations, may suggest that cyclosporin-A binds to a mitochondrial membrane site protecting the membrane against the phospholipase A2 attack.

Amino Acid Sequence↗
PubMed1997

Phospholipase A2 in human placental serum.

Intervillous blood was collected from term placentae at delivery, and sera were tested for phospholipase A2 under various experimental conditions. Enzyme activity was found to develop upon extended storage in the cold or at 37 degrees C. The enzyme is reversibly inhibited by dithiothreitol, requires Ca++ ions for activity, and tolerates various detergents. The apparent molecular weight is 42 kDa. In all these parameters the serum enzyme behaves similar to the 42 kDa phospholipase A2 which we recently purified to homogeneity from thoroughly washed placental tissue. Serum phospholipase A2 appears to be generated by proteolytic processing from a slightly larger inactive precursor which was detected immunochemically. Most likely this protein originates from fetal cells and may be released by membrane damage. We conclude that both placental serum and tissue harbour a novel type of phospholipase A2 which is distinct from cytosolic and secretory phospholipases A2. Preference for arachidonate containing substrate suggests a role in eicosanoid production within gestational tissues.

Blood↗
PubMed1997

Serum phospholipase A2 in dengue.

Group II phospholipase A2 is an enzyme involved in the pathologies of various inflammatory diseases, including infections. We measured the concentration of group II phospholipase A2 in sera of 49 patients suffering from dengue, virus serotype 3, during a recent epidemic in The Cook Islands. Group II phospholipase A2 concentrations were elevated above the normal level in 90% of the patients. There was a significant negative correlation between group II phospholipase A2 levels and platelet counts. It was concluded that dengue virus infection causes a generalized inflammatory reaction and acute phase response. Determination of serum group II phospholipase A2 level gives useful information for assessing the severity of viral infections, including dengue.

Adolescent↗
PubMed1996

A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig.

BACKGROUND: The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. The sequence of GPLRP2 is 63 % identical to that of human pancreatic lipase (HPL), but the so-called lid domain, is much reduced in GPLRP2. A phospholipase A1 from hornet venom (Dolml PLA1) is very similar to HPL and GPLRP2 but is devoid of lipase activity; Dolml PLA1 also contains a reduced lid domain and lacks a region termed the beta9 loop, which is located in the vicinity of the HPL and GPLRP2 active sites. The structure determination of a chimera of GPLRP2 and HPL and domain building of Dolml PLA1 were undertaken to gain a better understanding of the structural parameters responsible for the differences in lipase versus phospholipase activity among these structurally related enzymes. RESULTS: The crystal structure of a chimeric mutant of GPLRP2, consisting of the catalytic domain of GPLRP2 and the C-terminal domain of HPL, has been solved and refined to 2.1 A resolution. This enzyme belongs to the alpha/beta hydrolase fold family and shows high structural homology with classical pancreatic lipases. The active site is closely related to those of serine esterases, except for an unusual geometry of the catalytic triad. Due to the reduced size of the lid domain, the catalytic serine is fully accessible to solvent. Part of the beta9 loop, which stabilizes the lid domain in the closed conformation of the classical HPL, is totally exposed to the solvent and is not visible in the electron-density map. CONCLUSIONS: The structures of the related enzymes, GPLRP2 and HPL and the model of Dolml PLA1, provide insights into the role played by the loops located above the active site in controlling substrate selectivity towards triglycerides or phospholipids. In GPLRP2, the lid domain is reduced in size compared to HPL, and hydrophilic residues are exposed to solvent. GPLRP2 is thus able to accommodate the polar head of phospholipids. The beta9 loop is still present in GPLRP2, making it possible for this enzyme to still accommodate triglycerides. In Dolml PLA1, the beta9 loop is absent, and this enzyme is unable to process triglycerides retaining only the phospholipase A1 activity.

Amino Acid Sequence↗
PubMed2000

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change.

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.

Acrylamide↗
PubMed1993

Conformational changes in oxidized phospholipids and their preferential hydrolysis by phospholipase A2: a monolayer study.

Cleavage of oxidized fatty acids by phospholipase A2 has been implicated as the first step in the repair mechanism for oxidative damage to membrane phospholipids. However, the mechanism by which this enzyme preferentially hydrolyzes oxidized fatty acyl chains is poorly understood. Using a lipid monolayer technique, we found that the molecular surface areas of 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)-phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) were increased by as much as 50% relative to the parent nonoxidized 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC). These experimental data directly indicate a drastically changed molecular conformation of oxidized phospholipids in which the hydroperoxy or hydroxy group in the sn-2 fatty acid is close to the lipid-water interface. Phospholipases A2 from porcine pancreas and from bee venom were shown to break down PLPC-OOH and PLPC-OH monolayers much faster than PLPC monolayers. In all cases, the presence of serum albumin in the subphase enhanced monolayer breakdown by extracting hydrolysis products from the monolayer, but monolayer breakdown was always much faster for oxidized than for nonoxidized PLPC. This did not appear to be due to change in the extent of monolayer penetration by phospholipase A2, since enzyme-monolayer interaction studies revealed essentially identical penetration behavior of bee venom phospholipase A2 with PLPC, PLPC-OOH, and PLPC-OH monolayers. We propose that the altered molecular conformation of oxidized phospholipids facilitates access to the sn-2 ester bond, thereby ensuring their preferential hydrolysis in the presence of a phospholipase A2.

Hydrolysis↗
PubMed1984

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D.

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.

Isotope Labeling↗
PubMed2001

Phospholipases stimulate secretion in RBL mast cells.

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.

Animals↗
PubMed1993

Structural determinants of haloenol lactone-mediated suicide inhibition of canine myocardial calcium-independent phospholipase A2.

Haloenol lactones are potent mechanism-based inhibitors of a novel class of calcium-independent phospholipases A2 which have been implicated as the enzymic mediators of membrane dysfunction during myocardial ischemia (Hazen, S. L.; et al. J. Biol. Chem. 1991, 266, 7227-7232). Herein we demonstrate that the ring size, hydrophobic group, and cryptic electrophile in the haloenol lactone moiety are important and modifiable determinants of the inhibitory potency of haloenol lactone-mediated inhibition of calcium-independent phospholipase A2. Direct comparisons between haloenol lactone-mediated inhibition of calcium-independent phospholipase A2 and the absence of inhibition with calcium-dependent phospholipase A2 further underscore the marked differences in the catalytic strategy employed by these two classes of intracellular phospholipases A2.

Animals↗