Prevention of nitrofurantoin-induced cytotoxicity in isolated hepatocytes by fructose

Nitrofurantoin is a widely utilized urinary antimicrobial drug which has been associated with pulmonary fibrosis, neuropathy, and hepatitis as well as hemolytic anemia in glucose-6-phosphate dehydrogenase-deficient individuals. Incubation of freshly isolated rat hepatocytes with nitrofurantoin caused oxygen activation as a result of futile redox cycling. Glutathione disulfide (GSSG) was formed and rapidly exported from the cell resulting in complete glutathione (GSH) depletion followed by cell death. However, fructose prevented the export of GSSG from the cell and GSH levels recovered rapidly without cytotoxicity occurring. Fructose did not affect nitrofurantoin metabolism but rapidly depleted cellular ATP levels by approximately 80% which remained depressed during the incubation period. Fructose, however, did not protect hepatocytes from nitrofurantoin-induced cytotoxicity if GSH was depleted beforehand. Protection by fructose only occurred at concentrations which caused ATP depletion. These results suggest that fructose prevents nitrofurantoin-induced toxicity by depleting ATP and thereby preventing the ATP-dependent GSSG efflux. GSSG is retained enabling NADPH and glutathione-reductase to reduce the GSSG back to GSH, thereby protecting the cell from nitrofurantoin-induced oxidative stress.     

Phospholipase A2 has a role in proliferation but not in differentiation of HL-60 cells

The role of phospholipase A₂ (PLA₂) and its metabolite arachidonic acid (AA) in the proliferation and differentiation of HL-60 cells was investigated. Addition of either 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) or retinoic acid (RA) to HL-60 cells for 2 h inhibited PMA-stimulated PLA₂ activity measured by [³H]AA release. The inhibitor of PLA₂ activity, p-bromophenacyl bromide (BPB), significantly inhibited the proliferation of HL-60 cells and of fibroblast L929 and Swiss 3T3 cells in a dose-dependent manner. The effect of BPB on proliferation is probably through its inhibitory effect on PLA₂ activity, since the same doses of BPB which inhibited proliferation also inhibited PLA₂ activity determined by [³H]AA release. The importance of PLA₂ activity for cell growth was further supported by the effect of two other PLA₂ inhibitors, AACOCF₃ and scalaradial, which inhibited HL-60 proliferation in a dose-dependent manner. BPB, AACOCF₃ and scalaradial significantly increased the doubling time to 32.4 h, 34.0 h and 31.8 h, respectively, compared with 24.6 h in the control. The inhibitory effect of BPB on HL-60 proliferation was reversed by addition of exogenous free AA to HL-60 cells, indicating the importance of this metabolite for the proliferation process. This reversible effect is specific for AA since it was not achieved by other fatty acids like linolenic acid (LA) or oleic acid (OA). Addition of free AA to HL-60 cells did not induce differentiation, as expected. Although BPB, AACOCF₃, or scalaradial inhibited proliferation, they did not induce differentiation nor affect the differentiation induced by 1,25(OH)₂D₃ or RA. These results implicate that PLA₂ activity has no regulatory role in differentiation of HL-60 cells. The differential effect of PLA₂ inhibitors on proliferation and differentiation of HL-60 cells suggests that these two processes function under different regulatory mechanisms.     

Effect of tumor suppressor PTEN gene on apoptosis and cell cycle of human airway smooth muscle cells

Tumor necrosis factor-alpha (TNFα) plays a crucial role in inflammatory diseases such as rheumatoid arthritis and postmenopausal osteoporosis. Recently, it has been demonstrated that hydrogen gas, known as a novel antioxidant, can exert therapeutic anti-inflammatory effect in many diseases. In this study, we investigated the effect of treatment with hydrogen molecule (H₂) on TNFα-induced cell injury in osteoblast. The osteoblasts isolated from neonatal rat calvariae were cultured. It was found that TNFα suppressed cell viability, induced cell apoptosis, suppressed Runx2 mRNA expression, and inhibited alkaline phosphatase activity, which was reversed by co-incubation with H₂. Incubation with TNFα-enhanced intracellular reactive oxygen species (ROS) formation and malondialdehyde production increased NADPH oxidase activity, impaired mitochondrial function marked by increased mitochondrial ROS formation and decreased mitochondrial membrane potential and ATP synthesis, and suppressed activities of antioxidant enzymes including SOD and catalase, which were restored by co-incubation with H₂. Treatment with H₂ inhibited TNFα-induced activation of NFκB pathway. In addition, treatment with H₂ inhibited TNFα-induced nitric oxide (NO) formation through inhibiting iNOS activity. Treatment with H₂ inhibited TNFα-induced IL-6 and ICAM-1 mRNA expression. In conclusion, treatment with H₂ alleviates TNFα-induced cell injury in osteoblast through abating oxidative stress, preserving mitochondrial function, suppressing inflammation, and enhancing NO bioavailability.     

cDNA cloning and bacterial expression of phospholipase A2 inhibitor PLIα from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus1

The cDNA encoding of a phospholipase A₂ inhibitor (PLIα) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIα. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIα. The PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIα expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIα fusion protein underwent folding to form a trimeric structure like the intact PLIα, and showed inhibitory activity against the group II acidic PLA₂ from A. blomhoffii siniticus venom; although its binding constant (1/K_i) value was 30-fold lower than that of the natural PLIα. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIα against the acidic PLA₂. Furthermore, the carbohydrate chains of the natural PLIα were found to play an important role in the inhibitory activity against the basic PLA₂.     

Mechanisms of Trazodone‐Induced Cytotoxicity and the Protective Effects of Melatonin and/or Taurine toward Freshly Isolated Rat Hepatocytes

It has been reported that the bioactive intermediate metabolites of trazodone might cause hepatotoxicity. This study was designed to investigate the exact mechanism of hepatocellular injury induced by trazodone as well as the protective effects of taurine and/or melatonin against this toxicity. Freshly isolated rat hepatocytes were used. Trazodone was cytotoxic and caused cell death with LC₅₀ of 300 µm within 2 h. Trazodone caused an increase in reactive oxygen species (ROS) formation, malondialdehyde accumulation, depletion of intracellular reduced glutathione (GSH), rise of oxidized glutathione disulfide (GSSG), and a decrease in mitochondrial membrane potential, which confirms the role of oxidative stress in trazodone‐induced cytotoxicity. Preincubation of hepatocytes with taurine prevented ROS formation, lipid peroxidation, depletion of intracellular reduced GSH, and increase of oxidized GSSG. Taurine could also protect mitochondria against trazodone‐induced toxicity. Administration of melatonin reduced the toxic effects of trazodone in isolated rat hepatocytes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:457‐462, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21509     

PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A₂ (PLA₂) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA₂-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca²⁺-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA₂ activity and the release of free arachidonic acid (AA) from 1-acyl 2-¹⁴C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA₂ hyperactivity is Ca²⁺-dependent. There was no considerable alteration in the PLA₂ activity by stimulation of the activity by tyrosine kinase (with vanadate, H₂0₂), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF₄ thus in Tetrahymena PLA₂ activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA₂ activity is in connection with the phospholipid metabolism of Tetrahymena.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102     

Involvement of phospholipase A<Subscript>2</Subscript> in the response of <Emphasis Type="Italic">Solanum</Emphasis> species to an elicitor from <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Changes in activity of phospholipase A₂ (PLA₂), a key enzyme in lipid metabolism and signal network in defence mechanisms, were investigated in Solanum species and Phytophthora infestans interaction. We have compared PLA₂ activity in response to an elicitor, a culture filtrate (CF) derived from P. infestans, in non-host resistant Solanum nigrum var. gigantea, field resistant S. tuberosum cv Bzura and susceptible S. tuberosum clone H-8105. To elucidate the contribution of specific forms of PLA₂ to plant defence mechanism reasonably selective PLA₂ inhibitors, haloenol lactone suicide substrate (HELSS) and p-bromophenacyl bromide (BPB), which discriminate between Ca⁺²-independent PLA₂ (iPLA₂) and Ca⁺²-dependent secretory PLA₂ (sPLA₂), were used. The in vivo and in vitro effects of the inhibitors on PLA₂ activity and on generation of reactive oxygen species (ROS) induced by CF in the studied plants were assayed. We found that PLA₂ activity increased in response to CF treatment, displaying various kinetics and intensity depending on the resistance status of a given genotype. Differences among the genotypes in the effects of each inhibitor on CF-induced PLA₂ activity and on ROS production may reflect the diversity of PLA₂ isoforms in plants. Contrary to BPB, the inhibitory effect of HELSS was observable mainly on CF-induced PLA₂ activity, which suggests that iPLA₂ participates in signal transduction in defence reactions. Various effects of the two inhibitors on PLA₂ activity and ROS production suggest different contribution of sPLA₂ and iPLA₂ to modulation of defence reactions in the interaction between Solanum genotypes and P. infestans.     

Role of nitric oxide and nuclear factor-κB in the CYP2E1 potentiation of tumor necrosis factor α hepatotoxicity in mice

Induction of CYP2E1 by pyrazole (PY) potentiated the hepatotoxicity induced by TNFα in mice. We evaluated the role of nitrosative and oxidative stress and the NF-κB activation pathway in this liver injury. The iNOS inhibitor N-(3-aminomethyl)benzylacetamindine (1400W) or the antioxidant N-acetyl-l-cysteine (NAC) prevented this liver injury. TNFα plus PY treatment triggered radical stress in the liver with increased lipid peroxidation and decreased glutathione and caused mitochondrial damage as reflected by elevated membrane swelling and cytochrome c release. The radical stress and mitochondrial damage were prevented by 1400W and NAC. TNFα plus PY treatment elevated 3-nitrotyrosine adduct formation and induced NOS2 in the liver; 1400W and NAC blocked these changes. A lower extent of liver injury and oxidative stress was found in NOS2^?/? mice treated with TNFα plus PY compared with wild-type controls. Neither 1400W nor NAC modified CYP2E1 activity or protein. Activation of JNK and p38MAPK was weaker in TNFα plus PY-treated NOS2^?/? mice and 1400W and NAC blocked the activation of JNK and p38MAPK in wild-type mice. IKKα/β protein levels were decreased by TNFα plus PY treatment, whereas IκBα and IκBβ protein levels were elevated compared with saline, PY, or TNFα alone. NF-κB DNA binding activity was increased by TNFα alone but lowered by TNFα plus PY. All these changes were blocked by 1400W and NAC. NF-κB activation products such as Bcl-2, Bcl-X_L, cFLIP_S, cFLIP_L, and Mn-SOD were reduced by TNFα plus PY and restored by 1400W or NAC. We conclude that TNFα plus CYP2E1 induces oxidative/nitrosative stress, which plays a role in the activation of JNK or p38MAPK and mitochondrial damage. These effects combine with the blunting of the NF-κB activation pathways and the synthesis of protective factors to cause liver injury.     

Increases in Tumor Necrosis Factor-α in Response to Thyroid Hormone-induced Liver Oxidative Stress in the Rat

Thyroid hormone-induced calorigenesis contributes to liver oxidative stress and promotes an increased respiratory burst activity in Kupffer cells, which could conceivably increase the expression of redox-sensitive genes, including those coding for cytokines. Our aim was to test the hypothesis that l -3,3',5-triiodothyronine (T₃)-induced liver oxidative stress would markedly increase the production of TNF- α by Kupffer cells and its release into the circulation. Sprague-Dawley rats received a single dose of 0.1 mg T₃/kg or vehicle (controls) and determinations of liver O₂ consumption, serum TNF-α, rectal temperature, and serum T₃ levels, were carried out at different times after treatment. Hepatic content of total reduced glutathione (GSH) and biliary glutathione disulfide (GSSG) efflux were measured as indices of oxidative stress. In some studies, prior to T₃ injection animals were administered either (i) the Kupffer cell inactivator gadolinium chloride (GdCl₃), (ii) the antioxidants α-tocopherol and N-acetyl-L-cysteine (NAC), or (iii) an antisense oligonucleotide against TNF-α (ASO TJU-2755). T₃ elicited an 80-fold increase in the serum levels of TNF-α at 22h after treatment, which coincided with the onset of thyroid calorigenesis. Pretreatment with GdCl₃ , α-tocopherol, NAC, and ASO TJU-2755 virtually abolished this effect and markedly reduced T₃-induced liver GSH depletion and the increases in biliary GSSG efflux. It is concluded that the hyperthyroid state in the rat increases the circulating levels of TNF-α by actions exerted at the Kupffer cell level and these are related to the oxidative stress status established in the liver by thyroid calorigenesis.     

Selective Protection of Human Liver Tissue in TNF-Targeting of Cancers of the Liver by Transient Depletion of Adenosine Triphosphate

Background

Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF''s detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials.

Methods

Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity.

Results

PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues.

Conclusion

Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.     

Proteinase-activated receptor-2 mediated inhibition of TNFα-stimulated JNK activation — A novel paradigm for Gq/11 linked GPCRs

In this study we examined the potential for PAR₂ and TNFα to synergise at the level of MAP kinase signalling in PAR₂ expressing NCTC2544 cells. However, to our surprise we found that activation of PAR₂ by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFα stimulated IκBα loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR₄, however inhibition was reversed by pre-incubation with the novel PAR₂ antagonist K14585, suggesting that the effect is specific for PAR₂ activation. SLIGKV-OH was found to be more potent in inhibiting TNFα-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel G_q/11 inhibitor YM25480 also reversed PAR₂ mediated inhibition. Activation of PAR₂ was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR₂ or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to G_q/11, in mediating inhibition of TNFα-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.     

Activation of fluoride-stimulated adenylate cyclase by phospholipase A2 in the caudate nucleus of the rat brain

Phospholipase A₂ (PLA₂) increases adenylate cyclase (AC) activity in the rat caudate nucleus in a dose-dependent manner. After maximal stimulation by fluoride, PLA₂ treatment further increases AC activity 2.4 fold. Adenylate cyclase activity is maximal after 45% hydrolysis of the phospholipids. Of the products of PLA₂ treatment only lysophosphatidylcholine (LPC) produces such an increase in AC activity. In contrast to PLA₂ treatment, LPC solubilizes the enzyme, decreases the Km value for ATP, and requires much larger amounts of LPC than that produced by lipase treatment. After maximal stimulation with fluoride and PLA₂, removal of most of the LPC does not reduce the activity of adenylate cyclase. These findings suggest that removal of membrane lipid rather than generation of LPC is responsible for the activation of brain adenylate cyclase by phospholipase A₂.     

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.     

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro

Cytosolic phospholipase A₂ alpha (cPLA₂α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA₂α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA₂α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA₂α. Such promiscuous binding would explain the previous finding that cPLA₂α has both PLA₁ and PLA₂ activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA₂α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA₂α.     

Cytosolic phospholipase A2 and lysophospholipid acyltransferases

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of ester bonds at sn-2 positions of glycerophospholipids (PL), producing free fatty acids and lysophospholipids. In mammals, the PLA₂ superfamily comprises more than 30 known enzymes, including various structurally and biochemically different enzymes with diverse biological functions. Some of the enzymes are involved in the production of lipid mediators, including eicosanoids and lysophospholipid-related lipid mediators. Among them, cytosolic PLA₂α (cPLA₂α), a member of cPLA₂ family, is one of the most important intracellular PLA₂s. Upon cell activation, cPLA₂α is activated and involved in eicosanoid production under various physiological and pathological conditions. PLA₂s also play a role in membrane PL remodeling by coupling with re-acylation processes mediated by lysophospholipid acyltransferases (LPLATs) to generate sn-1/sn-2 fatty acid asymmetry of PLs. This review summarizes the biochemical and in vivo roles of cPLA₂ enzymes and LPLATs, including results from animal and human studies.This article is part of a Special Issue entitled Novel functions of phospholipase A₂ Guest Editors: Makoto Murakami and Gerard Lambeau.     

Suppression of murine endotoxic shock by sPLA2 inhibitor,indoxam, through group IIA sPLA2-independent mechanisms

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A₂ (sPLA₂) have been implicated in this process. Group IB sPLA₂ (PLA₂-IB) binds to the PLA₂ receptor (PLA₂R), and PLA₂R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-α. Group IIA sPLA₂ (PLA₂-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA₂ inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-α with a similar potency in PLA₂-IIA-expressing and PLA₂-IIA-deficient mice after LPS challenge. In PLA₂-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1β, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA₂-IB binding to murine PLA₂R with a high potency (K_i=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-α levels could not be observed in mice deficient in PLA₂R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA₂-IIA-independent mechanisms, possibly via blockade of the PLA₂R function.     

Role of cellular calcium homeostasis in toxic liver injury induced by the pyrrolizidine alkaloid senecionine and the alkenal trans-4-OH-2-hexenal

The pyrrolizidine alkaloid senecionine has been shown to be hepatotoxic, genotoxic, and cytotoxic. However, the biochemical mechanism by which senecionine produces hepatocellular toxicity remains to be elucidated. The role of calcium homeostasis in toxic liver injury was examined in isolated rat hepatocytes treated with senecionine and trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and appropriate cofactors. Hepatocytes treated with senecionine and t-4HH demonstrated greater cytotoxicity (leakage of lactate dehydrogenase) when incubated in the absence of extracellular Ca²⁺ than in its presence. Both compounds elicited an increase in cytosolic Ca²⁺ levels of isolated hepatocytes in the presence of extracellular Ca²⁺ In the following study, senecionine and t-4HH depleted intracellular glutathione levels and induced lipid peroxidation and cytotoxicity in isolated hepatocytes. Pretreatment with the thiolgroup reducing agent dithiothreitol prevented depletion of intracellular glutathione and protected hepatocytes against senecionine and t-4HH-induced lipid peroxidation and cytotoxicity. Both compounds also depleted intracellular ATP and NADPH levels. These results suggest that hepatotoxocity induced by senecionine and t-4HH is not dependent on the influx of extracellular Ca²⁺; however, alterations in intracellular Ca²⁺, possibly associated with depletion of intracellular glutathione, NADPH, and ATP, may play a critical role.