Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

An acidic phospholipase A₂ with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A₂ (PLA₂s) can be found. Basic PLA₂s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA₂s tend to have a lower toxicity. A novel PLA₂, here named PnPLA₂, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA₂ is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA₂ had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA₂s from viperid venoms. PnPLA₂ displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA₂ showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA₂, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C₂C₁₂. This is the first report on a bactericidal protein of Porthidium nasutum venom.     

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A₂ (PLA₂) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-β-cyclodextrin. Activation of calcium-independent PLA₂ (iPLA₂) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p < 0.01), which was blocked by PLA₂ inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (< 10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA₂ to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA₂ may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA₂ may provide a therapeutic target for viral infections.     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

A [Lys49]phospholipase A2 from Protobothrops flavoviridis Venom Induces Caspase-Independent Apoptotic Cell Death Accompanied by Rapid Plasma-Membrane Rupture in Human Leukemia Cells

Protobothrops flavoviridis venom contains plural phospholipase A₂ (PLA₂) isozymes. A [Lys⁴⁹]PLA₂ called BPII induced cell death in human leukemia cells. PLA2, an [Asp⁴⁹]PLA₂ that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA₂ lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.     

Release of GPI-Anchored Zn2+-Glycerophosphocholine Cholinephosphodiesterase as an Amphiphilic Form from Bovine Brain Membranes by Bee Venom Phospholipase A2

Enzymatic release of Zn²⁺-glycerophosphocholine (GPC)cholinephosphodiesterase, as an amphiphilic form, from bovine brain membranes was examined. Of various membrane hydrolases, bee PLA₂ was the most effective in the release of the GPC cholinephosphodiesterase (amphiphilic form, 63–70%) from membrane. Compared to pancreatic PLA₂, bee PLA₂ was more efficient in the release of GPC cholinephosphodiesterase. In pH-dependent release of GPl-anchored phosphodiesterase, there was a similar pH-release profile between PLA₂-mediated release and spontaneous one, implying the involvement of membrane disruption in the PLA₂ action. The PLA₂-mediated release showed a limited time-dependence (until 45 min) and a limited dose dependence (up to 3 units / ml), characteristic of a receptor-type binding. An ionic binding of PLA₂ to membrane may be alluded from the interfering effect of anionic phospholipids on the PLA₂ action. In support of an interaction between PLA₂ and membrane glycoproteins, the PLA₂ action was found to be blocked by lectins, wheat germ agglutinin or concanavalin A. In combination with detergent, the PLA₂-mediated release was found to be enhanced synergistically by saponin, a cholesterol-complexing agent. Meanwhile, an additive interaction between PLA₂ and lysolecithin suggests that PLA₂ action is independent of lysolecithin. It is suggested that the binding of PLA₂ to specific sites of membranes, probably rich in GPI-anchored glycoproteins, may be related to the facilitated release of GPI-anchored proteins as amphiphilic form.     

Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A₂ hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA₂ shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA₂, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d₃₁-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA₂ molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA₂.     

Activation of Arachidonate Release and Cytosolic Phospholipase A2 via Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in Macrophages Stimulated by Bacteria or Zymosan

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A₂ (cPLA₂). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA₂ in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA₂ activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA₂ activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA₂ activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA₂ and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes

The activities of enzymes involved in lipid metabolism—phospholipase A₂ (PLA₂) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)—were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA₂ activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA₂ activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA₂ activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA₂ activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA₂ activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.     

Purification and inflammatory edema induced by two PLA2 (Anch TX-I and Anch TX-II) from sea anemone Anthothoe chilensis (Actiniaria: Sagartiidae)

The Anch TX-I and II PLA₂ were purified from Anthothoe chilensis (Lesson, 1830) from the extract of the anemone after only two chromatographic step using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on μ-Bondapak C18 column. Both PLA₂ showed a molecular mass of ~ 14 kDa determined by MALDI-TOF mass spectrometry and showed a high catalytic activity (data not showed). Although homologous with mammalian or snake venom group I PLA₂s, Anch TX-I and II is sufficiently structurally different for the question of its placement into the existing PLA₂ classification scheme to arise. In addition, Anch TX-I and II, despite possessing many common structural features, also differ in some important structural properties. The amino acid sequence of both PLA₂ (Anch TX-I and III) showed high amino acid sequence identity with PLA₂Rhopilema nomadica and Bunodosoma caissarum Cnidaria and PLA₂ of group III protein isolated from the Mexican lizard Heloderma horridum horridum and Heloderma suspectum. In addition, Anch TX-I and Anch TX-II showed high amino acid sequence identity with PLA₂ from group III also showed significant overall homology to bee Apis dorsata, Bombus terrestris and Bombus pennsylvanicus and PLA₂. We also investigated the in vivo edematogenic activity of Anch TX-I and Anch TX-II in a model of paw and skin edema in rats and observed that both are able to induce dose-dependent edema.     

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold in crease in [³H]-AA release, a 7-fold increase in PGE₂ production and a 2-fold increase in PLA₂ activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1. A combination of the two cytokines had an additive effect on [³H]-AA release and PLA₂ activity while PGE₂ production was similar to that detected using interleukin-1 alone. [³H]-AA, was released in substantial amounts when sodium fluoride was used as a stimulus but PGE₂ was not. These data show that tumor necrosis factor and interleukin-1 can both activate synovial cell PLA₂ and induce generation of PGE₂, but act in an additive rather than a synergistic fashion. Furthermore, the data show that PGE₂ production is not always concordant with [³H]-AA release, suggesting that appropriate enzyme(s) must be activated.     

Purification and Amino Acid Sequence of MP-III 4R D49 Phospholipase A2 from Bothrops pirajai Snake Venom, a Toxin with Moderate PLA2 and Anticoagulant Activities and High Myotoxic Activity

MP-III 4R PLA₂ was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA₂ from Bothrops pirajai was determined by amino acid sequencing of reduced and carboxymethylated MP-III 4R and the isolated peptides from clostripain and protease V8 digestion. MP-III 4R is a D49 PLA₂ with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA₂ and anticoagulant activity. This PLA₂ does not have a high degree of homology with other bothropic PLA₂-like myotoxins (~75%) and nonbothropic myotoxins (~60%). MP-III 4R is a new PLA₂, which was isolated using exclusively analytical and preparative HPLC methods. Based on the N-terminal sequence and biological activities, MP-III 4R was identified as similar to piratoxin-III (PrTX-III), which was isolated by conventional chromatography based on molecular exclusion ion exchange chromatography. Clinical manifestations indicate that at the site of toxin injection, there may be pain of variable intensity, because animals continue to lick the limb. No clinical sign indicating general toxicity was noticed. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R, with a high frequency (70%) of affected muscle fibers.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

Biological and Structural Characterization of a New PLA<Subscript>2</Subscript> from the Crotalus durissus collilineatus Venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A₂ (PLA₂) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80 with crotalic PLA₂s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50 if compared to other sources of PLA₂s such as from the Bothrops snake venom. Also, this PLA₂ induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and q-BPB showed a similar reduction. The q-BPB did not reduce significantly the myotoxic activity induced by the PLA₂, but the anhydrous acetic acid treatment and the pre-incu-bation of PLA₂ with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA₂ with q-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or b-wing region may be involved in the binding of this PLA₂ to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.