Anti-masculinizing action of estradiol and cyproterone acetate: regulation of a protein fraction with phospholipase-A2 stimulatory and masculinizing activities

Recently we have identified a protein fraction (55-63 K) from male and testosterone-exposed female mouse genital tract, which stimulates phospholipase A2 (PLA2) and induces masculine differentiation in an undifferentiated mouse genital explant, suggesting a role of this protein in the action of testosterone. In the current study we have further investigated the role of this protein by determining whether anti-masculinizing agents, namely, estradiol and cyproterone acetate, have any effect on the production of this protein. The results described here indicate that a protein fraction containing PLA2 stimulatory activity was present in both control male and estradiol- or cyproterone acetate-exposed male fetal genital tract. However the specific activity of the PLA2-stimulatory protein was significantly higher in the control males than in the experimental males. We did not find any major difference in the behavior of this protein fraction in various chromatographic steps except that in CM-sepharose column; the PLA2-stimulatory activity from the male preparation was eluted in two overlapping peaks with 0.3 and 0.25 M NaCl and that from the treated males was eluted only with 0.25 M NaCl. The SDS-gel analysis of this protein fraction revealed a doublet band (55 and 63 K) in control samples and primarily a 63 K band in experimental samples. The protein fraction from all these sources showed a significant difference in their biological activity. The control male preparation induced Wolffian duct whereas the estradiol sample was completely ineffective and the cyproterone acetate sample was partially effective in inducing Wolffian duct. Thus, it appears that the protein fraction has a role in the masculinizing action of testosterone.     

Attenuating the defeminization of the neonatal rat brain: Mechanisms of action of cyproterone acetate, 1,4,6-androstatriene-3,17,-dione and a synthetic progestin, R5020

Although defeminization of the rat brain appears to depend significantly on the conversion of testosterone (T) to estradiol (E₂), the antiandrogenic steroid cyproterone acetate (CA) is able to attenuate defeminization. In order to study the mechanism of action of CA on brain sexual differentiation, newborn male rats were given subcutaneous injections of this steroid on postnatal Days 2–6. When castrated on Day 70 and given estrogen and progesterone, these CA-treated males displayed elevated lordosis quotients (LQ) compared to controls. CA-treated neonatal males were also examined at the end of the drug treatment to ascertain the mechanism of drug action: (1) Serum T levels were normal; (2) Brain cell nuclear estrogen receptor occupation, estimated by an exchange assay, was reduced by ≈ 30% in the brains of the CA-treated males, although the ability of exogenous E₂ to occupy these brain estrogen receptors was not reduced. Other work has demonstrated a weak competitive effect of cyproterone on aromatization, and thus cyproterone acetate may have interfered with the conversion of T to E₂ CA also has progestogenic activity, and 5-mm capsules of a potent synthetic progestin, R5020, given to newborn male rats on Days 2–6, are shown to elevate the LQ after postnatal Day 70 to the same extent as CA. However, R5020 did not reduce estrogen receptor occupation in the neonatal male rat brain and was without effect on serum T levels in the neonatal male. Because of the implied role of T-derived estrogens in defeminization, an experiment was conducted showing that the defeminizing action of estradiol benzoate given to 3-day-old female rat pups is attenuated by the antiestrogen, CI628, and not by the potent inhibitor of aromatization, 1,4,6-androstatriene-3,17-dione (ATD). This result complements previous experiments showing that both ATD and CI628 attenuate the defeminization produced by T. Taken together, the results lend further support to a pivotal role for aromatization and for estrogen-receptor interactions in the defeminizing effects of T. The actions of progestins such as CA and R5020 in attenuating defeminization are discussed in relation to the recent demonstration of progestin receptors in the neonatal rat brain. It is concluded that CA may act by a combination of actions, both by inhibiting aromatization and by acting as a progestin.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30–80 days gestation) contains a factor which inhibits phospholipase A₂ (PLA₂) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA₂ assay system. Inhibitory activity was associated with a 35–45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA₂-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA₂ activity was of a non-competitive nature (K_i = 4.1 ± 0.7μg/ml, ca 118 nmol/1). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

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.     

Phospholipase A2-like activity of human bocavirus VP1 unique region

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A₂ (PLA₂) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA₂ enzymatic activity and how these critical residues contribute to its PLA₂ activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca²⁺-dependent secreted PLA₂-like (sPLA₂) activity, which was inhibited by sPLA₂-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA₂ activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA₂-like enzymatic activity, and these residues are crucial for its sPLA₂-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.     

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract

Summary In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity.Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Biochemical characteristics of immune-associated phospholipase A<Subscript>2</Subscript> and its inhibition by an entomopathogenic bacterium, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A₂ (PLA₂) activity. Recently, an immune-associated PLA₂ gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA₂ gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA₂ (TcPLA₂) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA₂ (sPLA₂) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA₂ inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA₂). The X. nematophila culture broth contained PLA₂ inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA₂ inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA₂-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA₂ gene expression by RNA interference.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

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.     

Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na⁺ dependent transporter TauT and taurine release are increased and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA₂ activity, exposure to exogenous reactive oxygen species as well as inhibition of protein tyrosine phosphotases (PTP) potentiate the swelling-induced taurine loss. Inhibition of PI3K and PTEN reduces and potentiates swelling-induced taurine release, respectively. Inhibition of CK2 has no effect on PLA₂ activity and ROS production by NADPH oxidase, whereas it lifts the effect of PTEN and PTP inhibition. It is suggested that CK2 regulates the taurine release downstream to known swelling-induced signal transducers including PLA₂, NADPH oxidase and PI3K.     

Phospholipase A2 activation in cultured mouse hepatocytes exposed to tumor necrosis factor-α

High concentrations of tumor necrosis factor α (TNFα) are cytotoxic to cultured hepatocytes. Impairment of energy metabolism and generation of an intracellular oxidant stress are important events in the pathogenesis of this toxicity (6). In the present study, we have examined the role of phospholipase A₂ activation in TNFα-in-duced toxicity in mouse hepatocytes, since it has been reported to play a key role in TNFα cytolytic activity in other cell types. Recombinant murine TNFα (0.1 μg/mL) caused a dose-dependent increase in PLA₂ activity in cultured mouse hepatocytes. The increase in PLA₂ activity was observed after only 0.5 hour of exposure (152 ± 10% of control), and continued to increased over the first 4 hours of exposure (292 ± 32%). However, TNFα-induced GSSG efflux and ATP depletion did not occur until after 2 hours of exposure. Furthermore, a small level of cytotoxicity was observed after a 24 hour incubation period. Putative PLA₂ inhibitors, chlorpromazine (CPZ) and 4-bromophenacyl bromide (BPB), both prevented the TNFα-induced increase in PLA₂ activity. They also reduced ATP depletion, GSSG efflux, and cytotoxicity. The PLA₂ inhibitor, manoalide (a natural marine product), completely prevented PLA₂ activation and cytotoxicity induced by TNFα. Pretreatment of hepatocytes with cycloheximide, to inhibit protein synthesis, increased TNFα-induced cytotoxicity. Cycloheximide pretreatment also potentiated PLA₂ activation, ATP depletion, and GSSG efflux. CPZ and BPB both reduced the extent of PLA₂ activation, ATP depletion, GSSG formation, and cytotoxicity in the cycloheximide pretreated cells exposed to TNFα. Taken together, these results demonstrate that TNFα activates PLA₂ which occurs prior to other deleterious events in hepatocytes, and that inhibition of PLA₂ activity reduces cell injury by TNFα. This suggests that PLA₂ activation may lead to impairment of energy metabolism, an oxidant stress, and cytotoxicity in cells exposed to TNFα. Additionally, protein synthesis inhibition potentiates TNFα induction of PLA₂ and toxicity, suggesting that there is a protein-synthesis-dependent protective mechanism in hepatocytes which ameliorates the effects induced by PLA₂. These findings provide strong evidence that PLA₂ activation plays an important role in the pathogenesis of toxicity induced by TNFα in cultured mouse hepatocytes.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.     

Interaction of parvovirus B19 with human erythrocytes alters virus structure and cell membrane integrity

The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A₂ (PLA₂) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA₂ motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA₂ motif as well as a proportional increase of the PLA₂ activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA₂ motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA₂ activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA₂ constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells.     

Arachidonic acid release from NIH 3T3 cells by group-I phospholipase A2: Involvement of a receptor-mediated mechanism

Group I pancreatic phospholipase A₂ (PLA₂ I) is primarily a digestive enzyme. Recently, however, in addition to its catalytic activity a receptor-mediated function has been described for this enzyme. PLA₂ I binding to its receptor induces cellular chemokinesis, proliferation, and smooth muscle contraction. This enzyme also induces the production of prostaglandin E₂ in certain cells and may have a proinflammatory role. However, despite its ability to hydrolyze phospholipids in in vitro assays, PLA₂-I does not efficiently catalyze release of AA from intact cells. Here, we demonstrate that while short-term exposure of NIH 3T3 cells to PLA₂-I is ineffective, exposure of 6 h or longer significantly increases the basal release of AA. Dose-response curve of PLA₂-I-induced AA release was saturable with an EC₅₀ of 14.01 ± 1.36 nM (n = 3). [³H]-AA was preferentially released over [³H]-oleic acid by PLA₂-I, inactivated with 4-bromophenacyl bromide, was fully capable of mediating AA release. These data suggest that a non-catalytic, receptor-mediated mechanism is involved in PLA₂-I-induced AA release in NIH-3T3 cells. This relase of AA is not dependent on protein kinase C or Ca²⁺ concentration. Comparison of the effect of PLA₂-I with those of ATP and platelet-derived growth factor indicates that each of these agonists regulates AA release via independent pathways. Neither the basal enzymatic activity of the 85-kDa cytosolic PLA₂ nor the protein level of this enzyme was affected by treatment of cells with PLA₂-I. However, the increase in basal enzymatic activity of 85 kDa PLA₂ due to protein kinase C activation was further enhanced by pretreatment of cells with PLA₂-I. We conclude that: (1) short-term exposure of cells to PLA₂ I does not cause measurable AA release; (2) release of AA from intact cells by this enzyme requires long-term exposure; (3) AA release is not mediated by a direct catalytic effect of PLA₂ I; and (4) AA release by PLA₂ I is accomplished via a receptor-mediated process. Taken together, these results raise the possibility that PLA₂ I, in addition to its digestive function, may also contribute to aggravate preexisting inflammatory processes and/or to initiate new ones when chronic exposure of cells to this enzyme occurs. © 1995 Wiley-Liss Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.