Dependent Phospholipase Research Articles

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca2+]i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca2+. Maximal level of Ca2+, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A2 and/or Ca2+/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca2+]i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca2+-sparks and Ca2+-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca2+-microdomain formation, where the “vicious circle” of Ca2+-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca2+-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na+ and Ca2+ (lipid pore).

Nonhydrolyzable analogs of phosphatidylinositol as ligands of phospholipases C

Phosphatidylinositol-specific phospholipases C (PI-PLCs) are important enzymes participating in transmembrane signal transduction. The structures of the two major species of these enzymes: bacterial Ca2+-nondependent enzyme from B. cereus and mammalian Ca2+-dependent PLCδ1 from rat brain in the complexes with the polar head groups of their substrates have been previously solved. Although these structures show few differences as compared to free enzymes, there is a compelling evidence that full catalytic activity of PI-PLC necessitates interaction of the enzyme with the entire substrate, including the hydrophobic fatty acid chains. In this work we develop new tightly binding and cleavage-resistant analogs of phosphatidylinositol, using relatively minor modifications of the structure. Two synthesized analogs, 2-amino-2-deoxy-PI (8) and the conformationally constrained analog (10) had binding affinities (Ki) in 10 μM range. 15N-1H HSQC NMR spectra of uniformly 15N-labeled bacterial Ca2+-nondependent and Ca2+-dependent phospholipases C, btPLC and saPLC1, respectively, displayed changes upon ligand binding that suggest an occurrence of a conformational change.

Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells.

Human alveolar epithelial cells actively contribute to the innate immune response in the lung and play an important role in mycobacterial dissemination during primary infection, by undergoing cell death and by releasing mycobacteria. In the present study, we report that natural lysophospholipids, such as lysophosphatidic acid or sphingosine 1-phosphate, reduce Mycobacterium tuberculosis-induced cytotoxicity and enhance anti-mycobacterial activity in the A549 cell line, used as a model of type II alveolar epithelial cells. Intracellular mycobacterial killing was strictly dependent on phagolysosome maturation, which in turn was promoted by the activation of a Ca(2+)dependent phospholipase D. Finally, the restriction of mycobacteria in highly microbiocidal compartments was associated, in vitro, with a significant decrease in mycobacterial dissemination to macrophages. Taken as whole, these results suggest that the pulmonary lysophospholipid microenvironment may play a protective role during the early phases of host-pathogen interaction by enhancing anti-mycobacterial activity in type II alveolar epithelial cells.

A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity

An extracellular phospholipase D (PLD(St)) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD(St) was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca(2+), and the maximum activity was at 2 mM CaCl(2). The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 micromol min(-1) mg(-1), respectively. More importantly, PLD(St) could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD( St ) does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD(St) may therefore be a novel type of PLD enzyme.

Chronic treatment of astrocytes with therapeutically relevant fluoxetine concentrations enhances cPLA2 expression secondary to 5-HT2B-induced, transactivation-mediated ERK1/2 phosphorylation

We have recently shown that fluoxetine, a serotonin-specific reuptake inhibitor (SSRI), has low micromolar affinity for the 5-HT(2C) receptor (but not for 5-HT(2A) and 5-HT(2B) receptors) in primary cultures of mouse astrocytes. This was determined as phosphorylation (stimulation) of extracellular-regulated kinase 1 and 2 (ERK(1/2)) by transactivation-mediated phosphorylation of the epidermal growth factor (EGF) receptor, followed by conventional EGF receptor signaling (Li et al., Psychopharmacology 194:333-334, 2007). Paroxetine has an identical effect. The present study shows that chronic fluoxetine treatment with even higher affinity (EC(50) = 0.5-2.0 microM) upregulates Ca(2+)-dependent phospholipase A(2) (cPLA(2)), which releases arachidonic acid from the sn-2 position of membrane-bound phospholipid, without effect on secretory PLA(2) (sPLA(2)) and intracellular PLA(2) (iPLA(2)). This demonstration replicates the fluoxetine-induced cPLA(2) upregulation in rat brain shown by Rao et al. (Pharmacogenomics J 6:413-420, 2006) and provides the new information that upregulation (1) occurs in astrocytes, (2) is evoked by stimulation of 5-HT(2B) receptor, and (3) requires transactivation-mediated ERK(1/2) phosphorylation. Similar upregulation of cPLA(2) in intact brain in response to 5-HT(2)-mediated signaling by elevated serotonin levels and/or an SSRI during antidepressant treatment may explain the repeatedly reported ability of SSRIs to normalize regional decreases which occur in brain metabolism during major depression, since (1) arachidonic acid strongly stimulates glucose metabolism in cultured astrocytes (Yu et al., J Neurosci Res 64:295-303, 1993) and (2) plasma concentrations of arachidonic acid in depressed patients are linearly correlated with regional brain glucose metabolism (Elizabeth Sublette et al., Prostaglandins Leukot Essent Fatty Acids 80:57-64, 2009).

Uncompetitive Phospholipase A2 Inhibition by CHEC Sequences Including Oral Treatment of Experimental Autoimmune Myeloencephalitis

Catalytic sites of metal dependent phosphatases and phospholipases are characterized by a catalytic histidine (H) and adjacent acidic metal binding residue E (or D). For the secreted phospholipases A2 (sPLA2s), the active sites are stabilized by nearby flanking cystines. CHEC-9 (CHEASAAQC), which displays this organization, is a sPLA2 inhibitor that is an effective treatment for models of traumatic and autoimmune neurodegenerative disorders. We screened modifi- cations of this sequence and found that elimination of the two central alanines followed by cyclization results in another potent sPLA2 inhibitor, CHEC-7 (CHEASQC). CHEC-7, like CHEC-9, had uncompetitive kinetic properties making it ideal for in vivo applications. Subcutaneous injections of CHEC-7 into restrained rats inhibited the transient rise in plasma sPLA2 activity suggesting that this peptide was also effective in vivo. Oral delivery of the peptide attenuated the LPS- induced increases in sPLA2 activity. Thin layer chromatography and mass spectroscopy of representative LPS-treated rats suggested that the peptide also reduced levels of lysophosphatidylcholine, a hydrolytic product of the enzyme reaction. In order to test the efficacy of CHEC-7 for neuroimmune therapies, rats were immunized with spinal cord homogenates to induce experimental autoimmune encephalomyelitis (EAE), followed by daily treatment with oral (1.5 mg/kg) or subcuta- neous (0.1, 0.5, and1.5mg/kg) CHEC-7 and CHEC-9 (n=10 for each different condition). The rats were scored for severity of disease applying a standard EAE scale. Control rats all developed disease over the 23-day course of the experiments. Both CHEC-9 and CHEC-7 produced highly significant reductions in disease severity including via oral delivery. The performance of oral CHEC-7 was notable in that this treatment completely prevented disease in half the rats. The results suggested that continued study of modifications of CHEC sequences would provide stable therapeutic peptides for a variety of inflammatory and autoimmune disorders.

Partial characterization of a fraction from bull seminal vesicle fluid that potentiates the bull sperm acrosome reaction in vitro.

A fraction from bovine seminal vesicle fluid that initiated acrosome reaction of bovine epididymal spermatozoa in vitro in the presence of heparin was prepared by sequential chromatographies on heparin-Sepharose, gel filtration (Superose 12) and reversed phase chromatography (ProRPC). Sequence analysis of the separated fraction showed that it contained the major protein (PDC-109) with 100% homology. This fraction contained also Ca(2+)-dependent phospholipase A2-like activity which hydrolysed phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position. This protein was not detected in N-terminal sequence analysis. Lysophosphatidylcholine, lysophosphatidylethanolamine, and p-bromophenacyl bromide (p-BPB) inhibited this lipolytic activity. Sulfoglycolipid (Seminolipid) had inhibitory effect at concentrations above 0.1 mM but activated slightly the enzyme at lower concentrations. Boiling destroyed acrosome initiating activity in the separated fraction.

A liquid crystal-based sensor for real-time and label-free identification of phospholipase-like toxins and their inhibitors

We report a liquid crystal (LC)-based sensor for real-time and label-free identification of phospholipase-like toxins. Beta-bungarotoxin exhibits Ca2+-dependent phospholipase A2 activity whereas alpha-bungarotoxin and myotoxin II do not exhibit any phospholipase activity. The sensor can selectively identify beta-bungarotoxin, when it hydrolyzes a phospholipid monolayer self-assembled at aqueous–LC interface, through orientational responses of LCs. As a result, optical signals that reflect the spatial and temporal distribution of phospholipids during the hydrolysis can therefore be generated in a real-time manner. The sensor is very sensitive and requires less than 5pg of beta-bungarotoxin for the detection. When phospholipase A2 inhibitors are introduced together with beta-bungarotoxin, no orientational response of LCs can be observed. In addition, the regeneration of the sensor can be done without affecting the sensing performance. This work demonstrates a simple and cost-effective LC-based sensor for identifying phospholipase-like toxins and for screening compound libraries to find potential toxin inhibitors.

Up-regulation and Cytoprotective Role of Epithelial Multidrug Resistance-associated Protein 1 in Inflammatory Bowel Disease

MRP1 (multidrug resistance-associated protein 1) is well known for its role in providing multidrug resistance to cancer cells. In addition, MRP1 has been associated with both pro- and anti-inflammatory functions in nonmalignant cells. The pro-inflammatory function is evident from the fact that MRP1 is a high affinity transporter for cysteinyl-leukotriene C4 (LTC4), a lipid mediator of inflammation. It remains unexplained, however, why the absence of Mrp1 leads to increased intestinal epithelial damage in mice treated with dextran-sodium sulfate, a model for inflammatory bowel disease (IBD). We found that MRP1 expression is induced in the inflamed intestine of IBD patients, e.g. Crohn disease and ulcerative colitis. Increased MRP1 expression was detected at the basolateral membrane of intestinal epithelial cells. To study a putative role for MRP1 in protecting epithelial cells against inflammatory cues, we manipulated MRP1 levels in human epithelial DLD-1 cells and exposed these cells to cytokines and anti-Fas. Inhibition of MRP1 (by MK571 or RNA interference) resulted in increased cytokine- and anti-Fas-induced apoptosis of DLD-1 cells. Opposite effects, e.g. protection of DLD-1 cells against cytokine- and anti-Fas-induced apoptosis, were observed after recombinant MRP1 overexpression. Inhibition of LTC4 synthesis reduced anti-Fas-induced apoptosis when MRP1 function was blocked, suggesting that LTC4 is the pro-apoptotic compound exported by epithelial MRP1 during inflammation. These data show that MRP1 protects intestinal epithelial cells against inflammation-induced apoptotic cell death and provides a functional role for MRP1 in the inflamed intestinal epithelium of IBD patients.

Involvement of calcium-independent phospholipase A2 in regulation of Ca2+ signals induced by a calmodulin inhibitor in rat thymocytes

Previous studies have shown that micromolar concentrations of calmodulin inhibitor calmidazolium induce fast activation of nonselective Ca2+ channels in plasma membranes of Ehrlich ascites carcinoma cells (Zinchenko, V.P., Kasymov, V.A., Li, V.V., and Kaimachnikov, N.P., Biofizika (Rus.), 2005, vol. 50 (6), pp. 1055–1069). In order to detect this type of Ca2+ channels in other cells and to establish common regulatory mechanisms, we studied calmidazolium effects on rat thymocytes. It was found that calmidazolium induces biphasic increases in Ca2+ content in cytosol of rat thymocytes due to Ca2+ entry from external medium and reflects the activity of nonselective Ca2+ channels permeable for Mn2+ and Ni2+ ions. The rate and the amplitude of the fast phase are decreased, while those of the slow phase are increased in the presence of specific inhibitors of Ca2+-independent phospholipase A2 (bromoenol lactone and palmitoyl trifluoromethyl ketone). The rate and the amplitude of the fast phase are also inhibited by arachidonic acid and the lipoxygenase inhibitor nordihydroguaiaretic acid, while the Ca2+-dependent phospholipase A2 inhibitor bromophenacyl bromide, the cyclooxygenase inhibitor indomethacin, the specific store-operated Ca2+ channel inhibitor gadolinium and the phospholipase C inhibitor U73122 have no such effect. The rate of the fast phase only slightly depends on temperature, while that of the slow phase shows a strong temperature dependence and increases with a rise in temperatures (Q10 = 2). The amplitude of the fast phase of the Ca2+ signal increases with a decrease of temperatures due to prolongation of the maximum activity of the Ca2+ channel. The data obtained suggest that iPLA2 is an intermediate link in the activation of calmidazolium-induced nonselective Ca2+ channels. The iPLA2 products lysophospholipids and arachidonic acid activate and inhibit Ca2+ channels, respectively. The fact that these compounds manifest different affinities for Ca2+ channels shed additional light on the mechanisms of biphasic Ca2+ elevation in thymus cell cytosol and prolongation of the active state of Ca2+ channels at low temperatures.

Expression of Ca2+-independent and Ca2+-dependent phospholipases A2 and cyclooxygenases in human melanocytes and malignant melanoma cell lines

We provide novel evidence that human melanoma cell lines (M10, M14, SK-MEL28, SK-MEL93, 243MEL, 1074MEL, OCM-1, and COLO38) expressed, at mRNA and protein levels, either Ca2+-independent phospholipase A2 (iPLA2) or cytosolic phospholipase A2 (cPLA2) and its phosphorylated form. Normal human melanocytes contained the lowest levels of both PLA2s. Cyclooxygenase-1 and -2 (COX-1 and COX-2) were also expressed in cultured tumor cells as measured by Western blots. The most pronounced overexpression of iPLA2 and COX-1 was found in two melanoma-derived cells, M14 and COLO38. Normal human melanocytes and the M10 melanoma cell line displayed no COX-2 expression. Using subcellular fractionation, Western blot and confocal microcopy analyses, in paradigmatic SK-MEL28 and SK-MEL93 cells we showed that iPLA2, COX-1 and even cPLA2 were equally located in the cytosol, membrane structures and perinuclear region while COX-2 was preferentially associated with the cytosol. Specific inhibitors of these three enzymes significantly reduced the basal proliferation rate either in melanocytes or in melanoma cell lines. These results, coupled with the inhibition of the cell proliferation by electroporation of melanoma cells with cPLA2 or COX-2 antibodies, demonstrate that a possible correlation between PLA2-COX expression and tumor cell proliferation in the melanocytic system does exist. In addition, the high expression level of both PLA2s and COXs suggests that eicosanoids modulate cell proliferation and tumor invasiveness.

All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.

Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions

Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca(2+)-dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoP-dependent since it was not transcribed in the S. coelicolor DeltaphoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoP-dependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced -10 and -35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoP.

Up-regulation of cPLA2 gene expression in astrocytes by all three conventional anti-bipolar drugs is drug-specific and enzyme-specific

Common biological effects by all three conventional anti-bipolar drugs, the lithium ion (Li(+)), carbamazepine, and valproic acid, are important because identical effects may provide information about the pathophysiology of affective disorders. It has been reported that chronic treatment with either drug in vivo down-regulates the turnover of arachidonic acid in brain. This reaction is catalyzed by Ca(2+)-dependent phospholipase A(2) (cPLA(2)), the expression of which was down-regulated by Li(+) or carbamazepine but not by valproic acid; expression of two other PLA subtypes, iPLA(2) and sPLA(2) was unaffected. cPLA(2) is amply expressed in astrocytes, and in the present study, effects of 1-4 weeks of treatment with clinically relevant concentrations of each of the three anti-bipolar drugs on cPLA(2), iPLA(2), and sPLA(2) mRNA and protein expression were determined in primary cultures of mouse astrocytes by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. Two or more weeks treatment with Li(+) concentrations below 2 mM, carbamazepine or valproic acid up-regulated mRNA and protein expression of cPLA(2), but had no effect on iPLA(2) and sPLA(2), showing enzyme specificity. The effect occurred more rapidly at higher than lower concentrations but also tended to end after 4 weeks at the higher concentrations. Two millimolar Li(+) caused an initial increase of cPLA(2) followed by a decrease after 3 and 4 weeks. Topiramate had no effect, indicating specificity for anti-bipolar drugs. Both up- and down-regulation of cPLA(2) gene expression are involved in the mechanisms of action of anti-bipolar drugs; astrocytes are a target for these drugs.

Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats

Recently, we reported that intracerebroventricularly (i.c.v.) administered histamine evokes the secretion of noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid cascade in the histamine-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by phospholipase A2 (PLA2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, histamine (27 nmol/animal, i.c.v.) -induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.), ET-18-OCH3 (phosphatidylinositol-specific PLC inhibitor) (10 and 30 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.). However, mepacrine (PLA2 inhibitor) (1.1 and 2.2 micromol/animal, i.c.v.) and D609 (phosphatidylcholine-specific PLC inhibitor) (30, 100 and 300 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain phosphatidylinositol-specific PLC and diacylglycerol lipase in the centrally administered histamine-induced activation of the adrenomedullary outflow in rats.

Impact of Aluminum on the Oxidation of Lipids and Enzymatic Lipolysis in Monomolecular Films at the Air/Water Interface

There is evidence that serious pathologies are associated with aluminum (Al). In the present work, the influence of Al on enzymatic lipolysis was studied with the aim to get more insight into the possible link between the Al-induced membrane modification and the cytotoxicity of the trivalent cation (AlIII). Lipid monolayers were used as model membranes. The monomolecular film technique allowed monitoring the Al-dependent modifications of the lipid monolayer properties and enzyme kinetics. Two enzymes, namely, Candida rugosa lipase and a calcium (CaII)-dependent phospholipase A2 from porcine pancreas, were used to catalyze the lipolysis of triglyceride and phosphoglyceride monolayers, respectively. The results obtained show that Al modifies both the monolayer structure and enzymatic reaction rates. While the enzymes used in this study can be considered as probes detecting lipid membrane properties, it cannot be excluded that in physiological conditions modulation of the enzyme action by the Al-bound membranes is among the reasons for Al toxicity.

Mitochondrial Uncoupling Protein-4 Regulates Calcium Homeostasis and Sensitivity to Store Depletion-induced Apoptosis in Neural Cells

An increase in the cytoplasmic-free Ca(2+) concentration mediates cellular responses to environmental signals that influence a range of processes, including gene expression, motility, secretion of hormones and neurotransmitters, changes in energy metabolism, and apoptosis. Mitochondria play important roles in cellular Ca(2+) homeostasis and signaling, but the roles of specific mitochondrial proteins in these processes are unknown. Uncoupling proteins (UCPs) are a family of proteins located in the inner mitochondrial membrane that can dissociate oxidative phosphorylation from respiration, thereby promoting heat production and decreasing oxyradical production. Here we show that UCP4, a neuronal UCP, influences store-operated Ca(2+) entry, a process in which depletion of endoplasmic reticulum Ca(2+) stores triggers Ca(2+) influx through plasma membrane "store-operated" channels. PC12 neural cells expressing human UCP4 exhibit reduced Ca(2+) entry in response to thapsigargin-induced endoplasmic reticulum Ca(2+) store depletion. The elevations of cytoplasmic and intramitochondrial Ca(2+) concentrations and mitochondrial oxidative stress induced by thapsigargin were attenuated in cells expressing UCP4. The stabilization of Ca(2+) homeostasis and preservation of mitochondrial function by UCP4 was correlated with reduced mitochondrial reactive oxygen species generation, oxidative stress, and Gadd153 up-regulation and increased resistance of the cells to death. Reduced Ca(2+)-dependent cytosolic phospholipase A2 activation and oxidative metabolism of arachidonic acid also contributed to the stabilization of mitochondrial function in cells expressing human UCP4. These findings demonstrate that UCP4 can regulate cellular Ca(2+) homeostasis, suggesting that UCPs may play roles in modulating Ca(2+) signaling in physiological and pathological conditions.

CpG oligodeoxynucleotides induce Ca 2+-dependent phospholipase D activity leading to phagolysosome maturation and intracellular mycobacterial growth inhibition in monocytes

Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) have been reported to induce antimycobacterial activity both in vitro and in vivo. The present study analyzes the signals leading to CpG ODN-induced antimicrobial activity in monocytes. In this context, CpG, but not GpC, ODN induced cytosolic Ca 2+ influx of extracellular origin which, in turn, activated host phospholipase D (PLD). The production of CpG-induced PLD-dependent phosphatidic acid induced the maturation of phagolysosomes and intracellular mycobacterial growth inhibition. These results show the presence of an antimicrobial pathway in monocytes, mediated by Ca 2+-dependent PLD which can be useful for the exploitation of novel anti-tuberculosis immunotherapy approaches.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

Effect of Arachidonic Acid Reacylation on Leukotriene Biosynthesis in Human Neutrophils Stimulated with Granulocyte-macrophage Colony-stimulating Factor and Formyl-methionyl-leucyl-phenylalanine

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.