Activation of phospholipase A2 of human spermatozoa by proteases

The effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A₂ activity (PA₂: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA₂ activity optimally with trypsin concentrations of 1.0–1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA₂ activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA₂ activity at basal levels and a by-product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA₂ activation upon the addition of proteases, thus indicating that the increase in PA₂ activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A₂ that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A₂ could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.     

Activation of phospholipase A2 by ternary model membranes

Formation of liquid-ordered domains in model membranes can be linked to raft formation in cellular membranes. The lipid stoichiometry has a governing influence on domain formation and consequently, biochemical hydrolysis of specific lipids has the potential to remodel domain features. Activation of phospholipase A₂ (PLA₂) by ternary model membranes with three components (DOPC/DPPC/Cholesterol) can potentially change the domain structure by preferential hydrolysis of the phospholipids. Using fluorescence microscopy, this work investigates the changes in domain features that occur upon PLA₂ activation by such ternary membranes. Double-supported membranes are used, which have minimal interactions with the solid support. For membranes prepared in the coexistence region, PLA₂ induces a decrease of the liquid-disordered (L_d) phase and an increase of the liquid-ordered (L_o) phase. A striking observation is that activation by a uniform membrane in the L_d phase leads to nucleation and growth of L_o-like domains. This phenomenon relies on the initial presence of cholesterol and no PLA₂ activation is observed by membranes purely in the L_o phase. The observations can be rationalized by mapping partially hydrolyzed islands onto trajectories in the phase diagram. It is proposed that DPPC is protected from hydrolysis through interactions with cholesterol, and possibly the formation of condensed complexes. This leads to specific trajectories which can account for the observed trends. The results demonstrate that PLA₂ activation by ternary membrane islands may change the global lipid composition and remodel domain features while preserving the overall membrane integrity.     

Activation of phospholipase A2 by Hsp70 in vitro

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer β-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg(2+).     

Activation of phospholipase D in normodense human eosinophils

Normodense human eosinophils have been labeled in 1-0-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubating isolated cells with alkyl-[32P]lysoPC. Stimulation of these 32P-labeled cells with C5a, A23187 or PMA in the presence of 0.5% ethanol resulted in time- and dose-dependent formation of alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because cellular ATP does not contain 32P, alkyl-[32P]PA must have been formed by the hydrolytic action of phospholipase D (PLD) and not by the combined actions of phospholipase C and DG kinase. Regardless of the stimulating agent, alkyl-[32P]PEt formation paralleled that of alkyl-[32P]PA, suggesting that alkyl-PEt was the result of a PLD-catalyzed transphosphatidylation reaction between alkyl-PC and ethanol. These data provide the first definitive proof of receptor- and nonreceptor-mediated activation of PLD in normodense eosinophils derived from human blood.     

Activation of phospholipase Cepsilon by free fatty acids and cross talk with phospholipase D and phospholipase A2

This paper uses phospholipase Cepsilon as a model to demonstrate that lipids can act as ligands to bind to specific motifs and regulate protein activity via allosteric effects. Phospholipids such as phosphatidic acid and free fatty acids such as arachidonate are potent activators of PLCepsilon, increasing the rate of PI hydrolysis by 8-fold and 50-fold, respectively. The mechanism appears to be a reduction of K(m), as the substrate dependence curve is shifted to the left and K(m) is reduced 10-fold. The regulation of PLCepsilon by lipids appears to be physiologic, as reconstitution or cotransfection of either cPLA(2) or PLD with PLCepsilon leads to activation of phosphodiesterase activity. Additionally, TSA-201 cells transfected with PLCepsilon and fed arachidonic acid complexed with BSA had increased (4-5-fold) hydrolysis of polyphosphoinositides. This study demonstrates the ability of lipids to act as potent and direct mediators of protein function and identifies cross talk between different classes of phospholipase (PLD and PLA(2) with PLC) mediated via lipid products.     

Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor

Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA(2)s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA(2) (bromophenacyl bromide-inactivated human sPLA(2) and the H48Q mutant of the porcine sPLA(2)) were as effective as the catalytically active sPLA(2)s in inducing cytokine production. HLM expressed the M-type receptor for sPLA(2)s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA(2) activity as well as binding to the M-type receptor, suppressed sPLA(2)-induced cytokine production. Incubation of HLM with the sPLA(2)s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA(2)s. In conclusion, two distinct sPLA(2)s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.     

Activation of N-acylethanolamine-releasing phospholipase D by polyamines

N-acylethanolamines including anandamide (an endogenous ligand of cannabinoid receptors) are biosynthesized from N-acyl-phosphatidylethanolamine (PE) by a phosphodiesterase of the phospholipase D type. The enzyme partially purified from the particulate fraction of rat heart hydrolyzed N-palmitoyl-PE to N-palmitoylethanolamine with a specific activity of 50 nmol/min per mg protein at 37 degrees C in the presence of 10 mM CaCl2. We found that the enzyme was highly activated in dose-dependent manner by polyamines like spermine, spermidine, and putrescine. Spermine was the most potent with an EC50 value around 0.1 mM, and increased the specific enzyme activity 27 fold up to 53 nmol/min per mg protein. However, a synergistic effect of spermine and the known activator (Ca2+ or Triton X-100) was not observed. The spermine-stimulated enzyme was also active with N-arachidonoyl-PE (a precursor of anandamide). Thus, polyamines may function as endogenous activators to control the biosynthesis of anandamide and other N-acylethanolamines.     

Activation of phospholipase A2 by freshly added lysophospholipids

Reaction progress curves for the hydrolysis of dimyristoylphosphatidylcholine by pig pancreatic phospholipase A₂ exhibits a latency phase. Addition of 1-palmitoyllysophosphatidylcholine to the preformed vesicles reduces the latency phase and enhances the binding of phospholipase A₂ to the vesicles. In contrast, the binary codispersions prepared from diacylphospholipids premixed with lysophosphatidylcholine do not exhibit such enhanced susceptibility to the phospholipase. This effect appears to be due to organizational defects created by asymmetrical incorporation of lysophospholipid molecules into the outer monolayer of the vesicles, and the action of phospholipase is not observed when the additive is equilibrated in both the monolayers of the vesicles.     

Activation of cytosolic phospholipase A2 and 15-lipoxygenase by oxidized low-density lipoproteins in cultured human lung fibroblasts

In cell cultures of human lung fibroblasts, we found that oxidized LDL (oxLDL), after 24-h treatment, stimulated arachidonic acid release. A putative role for phospholipases A(2) and MAPK activities in this process was postulated. Consequently, we studied the contribution of either Ca(2+)-dependent, cytosolic phospholipase A(2) (cPLA(2)) or Ca(2+)-independent phospholipase A(2) (iPLA(2)), and the role of the MAP kinase family in oxLDL toxicity to fibroblastic cells in vitro. Activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH(2)-terminal kinase (JNK) was also assessed with Western blotting. Compared with cellular samples untreated or treated with native LDL, treatment with oxLDL (50-100 microM hydroperoxides) for 24 h significantly increased the levels of either cPLA(2) protein expression or constitutively phosphorylated cPLA(2) protein; in addition we observed enzyme translocation to membranes. iPLA(2) activity was not stimulated by oxLDL. Arachidonic acid release appeared to be associated with phosphorylation of ERK1/2 which was significantly enhanced in a dose-dependent manner whereas no activation of p38 and JNKs was found, indicating that these MAPKs are not involved in mediating the maximal oxLDL response. Western blotting on subcellular fractions and confocal microscopy analyses confirmed an increase in 15-lipoxygenase (15-LO) protein expression and translocation upon activation. A significant increase of cyclooxygenase-2 expression into membrane fraction was also found. Collectively, the data presented link the stimulation of ERK-cPLA(2)-15-LO pathway by oxLDL to the prooxidant mechanism of the lipoprotein complex. It may initially stimulate the fibroblast reaction against the oxidation challenge as well as metabolic repair, such as during lung inflammation and pulmonary fibrosis.     

Activation of phospholipase D by PKC and GTPgammaS in human neuroblastoma cells overexpressing MARCKS

Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCalpha). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPgammaS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPgammaS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPgammaS, stimulation of PLD mediated by GTPgammaS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPgammaS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.     

Activation of phospholipase D by osmotic cell swelling

In response to osmotic cell swelling, Intestine 407 cells react with a rapid and transient activation of phospholipase D (PLD). To investigate the role of PLD during the regulatory volume decrease, cells were treated with 1-butanol resulting in a depletion of PLD substrates. Activation of volume-regulated anion channels, but not the cell swelling-induced release of taurine, was largely inhibited in the presence of low concentrations of 1-butanol. In addition, hypotonicity-induced exocytosis, ATP release and subsequent endocytosis were found to be largely abrogated. The results support a model of cell volume regulation in which PLD plays an essential role in the cell swelling-induced vesicle cycling and in the activation of volume-sensitive anion channels.     

Activation of phospholipase D1 by ADP-ribosylated RhoA

Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-gamma-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-gamma-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho-PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.     

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells

The ability of endothelin to promote phospholipid hydrolysis has been studied in myo-[2-3H]-inositol-, [3H]-arachidonic acid- or methyl-[3H]choline chloride-prelabelled cultured vascular smooth muscle cells (VSMC) from rat and bovine thoracic aortae and human omental vessels. The biochemical responses to endothelin were comparable between the different VSMC isolates. Endothelin promoted the accumulation of glycerolphospho[3H]inositol and concomitant loss of [3H]-inositol label from phosphatidylinositol. Exposure of [3H]choline-labelled VSMC to endothelin resulted in a loss of radioactivity from phosphatidylcholine that was inversely parallelled by an increase in water-soluble [3H]-choline metabolites. In [3H]-arachidonic acid ([3H]-AA)-labelled VSMC, endothelin induced extracellular release of [3H]-AA which derived from both phosphatidylcholine and phosphatidylinositol. Half-maximally effective concentrations of endothelin for all these responses were approximately 2-7 nM and did not vary between VSMC types. Endothelin-induced release of [3H]-AA into VSMC medium-overlay was inhibited by quinacrine and nordihydroguaiaretic acid but not by neomycin or indomethacin. The data herein implicate activation of phospholipase A2 by endothelin with subsequent metabolism of arachidonic acid via the lipoxygenase pathway.     

Activation of phospholipase A2 by cannabinoids. Lack of correlation with CNS effects

Cannabinoids delta 1-tetrahydrocannabinol, cannabinol, cannabidiol and cannabigerol have been shown to affect directly the activity of phospholipase A2 in a cell-free assay. The compounds produced a biphasic activation of the enzyme, with EC50 values in the range 6.0-20.0 X 10(-6) M and IC50 values in the range 50.0-150.0 X 10(-6) M. These results correlated well with the relative potencies reported for the stimulation of prostaglandin release from human synovial cells in vitro, confirming that activation of phospholipase A2 is the predominant action of cannabinoids on arachidonate metabolism in tissue culture. However, since delta 1-tetrahydrocannabinol is unique among these compounds in possessing cataleptic activity, it is unlikely that phospholipase A2 is the major receptor mediating the psychotropic effects of cannabis.     

Activation of phospholipase A2 by adriamycin in vitro. Role of drug-lipid interactions

To probe adriamycin-phospholipid interactions, the effects of this cytotoxin on the hydrolysis of a pyrene-labeled acidic alkyl-acyl phospholipid analog 1-octa-cosanyl-2-(6-pyren-1-yl)hexanoyl-sn-glycero-3-phos p hatidylmethanol (C28-O-PHPM) by porcine pancreatic phospholipase A2 (PLA2) were studied. In the absence of added Ca2+ adriamycin caused a 3-4-fold activation of hydrolysis of this pyrenelipid whereas an inhibition of action of PLA2 on the corresponding phosphatidylcholine derivative C28-O-PHPC was observed. Under similar conditions adriamycin also enhanced the rate of hydrolysis of the pyrene-labeled diacyl lipid 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylgly cer ol and inhibited the hydrolysis of PLA2 on the phosphatidylcholine derivative. Increasing calcium concentrations abolished the activating and most of the inhibitory effects of adriamycin with the above phospholipid substrates. Quenching of pyrene excimer fluorescence by adriamycin revealed efficient binding of the drug to acidic lipids. Addition of 1 mM calcium reduced fluorescence quenching by adriamycin maximally by approximately 90%. In comparison, quenching by adriamycin of pyrene-labeled phosphatidylcholine was much weaker and calcium had only an insignificant effect. Monolayer experiments at an air/water interface showed a rapid and surface pressure-dependent penetration of the drug into a film of C28-O-PHPM. Increase in surface pressure was reversed by 80% by the inclusion of 1 mM Ca2+ into the subphase. Penetration of adriamycin into a monolayer of C28-O-PHPC was much weaker. In agreement with earlier studies two types of binding of adriamycin to C28-O-PHPM are proposed.     

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.