Addition Of Exogenous Arachidonic Acid Research Articles

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

Dormant 5-lipoxygenase in inflammatory macrophages is triggered by exogenous arachidonic acid

The differentiation of resident tissue macrophages from embryonic precursors and that of inflammatory macrophages from bone marrow cells leads to macrophage heterogeneity. Further plasticity is displayed through their ability to be polarized as subtypes M1 and M2 in a cell culture microenvironment. However, the detailed regulation of eicosanoid production and its involvement in macrophage biology remains unclear. Using a lipidomics approach, we demonstrated that eicosanoid production profiles between bone marrow-derived (BMDM) and peritoneal macrophages differed drastically. In polarized BMDMs, M1 and M2 phenotypes were distinguished by thromboxane B2, prostaglandin (PG) E2, and PGD2 production, in addition to lysophospholipid acyltransferase activity. Although Alox5 expression and the presence of 5-lipoxygenase (5-LO) protein in BMDMs was observed, the absence of leukotrienes production reflected an impairment in 5-LO activity, which could be triggered by addition of exogenous arachidonic acid (AA). The BMDM 5-LO regulatory mechanism was not responsive to PGE2/cAMP pathway modulation; however, treatment to reduce glutathione peroxidase activity increased 5-LO metabolite production after AA stimulation. Understanding the relationship between the eicosanoids pathway and macrophage biology may offer novel strategies for macrophage-associated disease therapy.

Cytosolic phospholipase A2α increases proliferation and de-differentiation of human renal tubular epithelial cells

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme controls the release of arachidonic acid from membrane bound phospholipids and is the rate-limiting step in production of eicosanoids. A variety of different kidney injuries activate cPLA2α, therefore we hypothesized that cPLA2α activity would regulate pathologic processes in HK-2 cells, a human renal tubular epithelial cell line, by regulating cell phenotype and proliferation. In two lentiviral cPLA2α-silenced knockdowns, we observed decreased proliferation and increased apoptosis compared to control HK-2 cells. cPLA2α-silenced cells also demonstrated an altered morphology, had increased expression E-cadherin, and decreased expression of Ncadherin. Increased levels of E-cadherin were associated with increased promoter activity and decreased levels of SNAIL1, SNAIL2, and ZEB1, transcriptional repressors of E-cadherin expression. Addition of exogenous arachidonic acid, but not PGE2, reversed the phenotypic changes in cPLA2α-silenced cells. These data suggest that cPLA2α may play a key role in renal repair after injury through a PGE2-independent mechanism.

Exogenous Addition of Arachidonic Acid to the Culture Media Enhances the Functionality of Dendritic Cells for Their Possible Use in Cancer Immunotherapy

The development of dendritic cell based vaccines is a promising approach in cancer immunotherapy. For their successful use in the clinics, the propagation and functionality of DCs is crucial. We earlier established a two-step method for the large scale generation of DCs from umbilical cord blood derived MNCs/CD34+ cells. This work aims at improving their functionality based on the following observations: in vitro generated DCs can be less efficient in migration and other functional activities due to lower eicosanoid levels. The production of eicosanoids from Arachidonic Acid (AA) can be hampered due to suppression of the enzyme phospholipase A2 by IL-4, an essential cytokine required for the differentiation of DCs. We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality. DCs were generated with and without AA. The two DC sets were compared by phenotypic analysis, morphology and functional assays like antigen uptake, MLR, CTL assay and in vitro and in vivo migration. Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA+ DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2. AA+ DCs also show a favorable Th1 cytokine profile than AA- DCs. Thus addition of AA to the culture media is skewing the DCs towards the secretion of more IL-12 and less of IL-10 along with the restoration of eicosanoids levels in a COX-2 mediated pathway thereby enhancing the functionality of these cells to be used as a potent cellular vaccine. Taken together, these findings will be helpful in the better contriving of DC based vaccines for cancer immunotherapy.

MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles

Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny.

A ceramide analog inhibits cPLA 2 activity and consequent PGE 2 formation in LPS-stimulated macrophages

Prostaglandin E 2 (PGE 2) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE 2 production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE 2 production in macrophages. Inhibition of PGE 2 production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA 2α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA 2α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE 2 production in LPS-stimulated macrophages by direct interaction with cPLA 2, and suggest that ceramide may similarly counteract C1P effect on cPLA 2 activity in cells. The suppression of PGE 2 production is suggested to contribute to the anti-inflammatory action of PCERA-1.

Phospholipase A 2-activating protein (PLAA) enhances cisplatin-induced apoptosis in HeLa cells

Phospholipase A 2 (PLA 2)-activating protein (PLAA) is a novel signaling molecule that regulates eicosanoid production and participates in inflammatory responses. In our current study, we revealed that PLAA production was induced by the chemotherapeutic drug cisplatin in HeLa cervical carcinoma cells. To determine the potential pro-apoptotic effects of PLAA induction by cisplatin, we utilized HeLa (Tet-off) cells overexpressing the plaa gene ( plaa high ) and compared them with control ( plaa low ) cells, which produce endogenous plaa from the chromosome. Cisplatin-stimulated plaa high cells contained significantly higher levels of DNA fragmentation, caspase 3, 8 and 9 activities, PLA 2 enzyme activity, and cytochrome c leakage from mitochondria than did the cisplatin-stimulated plaa low cells. Importantly, siRNA against PLAA (siRNA–PLAA) reduced the levels of cisplatin-induced PLAA, DNA fragmentation, and PLA 2 activation, while promoting cell viability in both plaa high and plaa low cells. Cisplatin-induced-cytochrome c leakage in plaa high cells was reduced by siRNA–PLAA and restored by the addition of exogenous arachidonic acid (AA), suggesting to us that PLAA induction by cisplatin promoted cytochrome c leakage/mitochondrial damage partially by accumulating AA. In addition, cisplatin-stimulated plaa high cells produced less cytoprotective clusterin than did the cisplatin-stimulated plaa low cells, and siRNA–PLAA promoted clusterin production from both plaa high and plaa low cells. We showed that clusterin reduced DNA fragmentation in cisplatin-stimulated plaa high and plaa low cells, which is consistent with the notion that clusterin promotes cancer chemoresistance. Furthermore, cisplatin-stimulated plaa high cells produced more IL-32 (a pro-apoptotic protein) than did cisplatin-stimulated plaa low cells, and siRNA–PLAA reduced IL-32 production from both plaa high and plaa low cells. Finally, our proteomic analysis revealed that cisplatin-stimulated plaa high cells contained higher levels of phosphorylated JNK/c-Jun and FasL than did plaa low cells treated the same way. In summary, our data indicated that PLAA induction enhanced cisplatin-induced-apoptosis through four pathways, namely by: 1) accumulation of AA and mitochondrial damage, 2) downregulation of the cytoprotective clusterin, 3) upregulation of the pro-apoptotic IL-32, and 4) induction of JNK/c-Jun signaling and FasL expression.

Regulation of MUC5AC expression by arachidonic acid metabolites

Background Cytosolic phospholipase A2 (cPLAb2) is a rate-limiting key enzyme controlling the release of arachidonic acid (AA) from membrane phospholipids. Conversion of AA by cyclooxygenases (COX) and lipoxygenases (LOX) generates prostaglandins and leukotrienes, respectively. These AA metabolites play an important role in the control of inflammation. We have previously observed that cPLA2 is involved in mucus hyper-secretion and expression of the mucin Muc5ac, in mice deficient in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (Dif et coll. submitted). Muc5ac is a major mucin present in bronchial secretions of CF patients (Thorax. 2007. 62:153-61). Aim Identify the signaling pathways involved in the mucin Muc5ac expression mediated by cPLA2 in the bronchial epithelial cell line NCI-H292. We also evaluated the effect of an inhibitor of CFTR chloride channel function (Inh172 inhibitor) on Muc5ac expression. Methods Production of Muc5ac was measured in cell lysates and supernatants by specific ELISA, after stimulation with PMA or TGF-alpha. cPLA2 activity was analyzed in cell lysates, after hydrolysis of phosphatidyl choline labeled with radioactive AA. The role of cPLA2, COX and LOX in Muc5ac production was examined after treatment with specific inhibitors. Activation of transcription factors was followed using EMSA. Results Both PMA- and TGF-alpha induced Muc5ac expression were mimicked by addition of exogenous AA and inhibited after pre-treatment of the cells with the cPLA2 inhibitor MAFP. Further, Muc5ac expression was inhibited by a LOX inhibitor (NDGA) but not by COX inhibitors (aspirin, NS398). cPLA2 induced Muc5ac expression though PPAR-β-dependent process. Indeed, MAFP abrogated PPAR-β activation and PPAR-β agonists induced Muc5ac production. However, NF-kB inhibition failed to interfere in Muc5ac production. On the other hand, Inh172 inhibitor failed to increase Muc5ac production in stimulated cells. Conclusion cPLA2 plays an important role in the production of Muc5ac mucin. This involves LOX metabolites rather than COX, and a signaling pathway resulting in the activation of the transcription factor PPAR-β. In addition, we showed that induced Muc5ac secretion does not depend on CFTR chloride channel function. Acknowledgments: Work supported by “Vaincre la Mucoviscidose”

Group VIA Calcium-independent Phospholipase A2 Mediates Endothelial Cell S Phase Progression

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis.

Inhibitors of arachidonic acid metabolism act synergistically to signal apoptosis in neoplastic cells

Arachidonic acid (AA) and its metabolites are intimately linked to carcinogenesis. Inhibitors of AA metabolic enzymes have demonstrated anti-carcinogenic effects in vivo and induce apoptosis of many cancer cell lines in vitro. The mechanism by which AA influences carcinogenesis, however, remains unresolved. The current study explores the growth inhibitory potential of Triacsin C, PLT-98625, and NS-398 which inhibit three distinct metabolic enzymes that control intracellular AA levels: fatty acid coenzyme-A ligase 4 (FACL-4), coenzyme-A independent transacylase (CoA-IT), and cyclooxygenase (COX), respectively. Results reveal the anti-proliferative effects of these inhibitors in a number of human cancer cell lines. Further studies in the SK-MES-1 cell line demonstrate that all three inhibitors induce accumulation of unesterified AA which correlates with induction of apoptosis. Addition of exogenous AA also induces apoptosis. Furthermore, in combination, these inhibitors act cooperatively to induce AA accumulation which correlates to a synergistic reduction in cell viability. Taken together, these results suggest that accumulation of unesterified AA is a common mechanism in the induction of cancer cell apoptosis by various inhibitors of AA metabolism, confirm that previously described AA remodeling pathways are valid in cancer cells, and indicate that combination treatment strategies utilizing these inhibitors may represent a novel approach to blocking cancer cell growth. Further study is required to determine the downstream pathway(s) whereby high cellular burdens of unesterified AA promote apoptosis.

Silencing the expression of mitochondrial acyl‐CoA thioesterase I and acyl‐CoA synthetase 4 inhibits hormone‐induced steroidogenesis

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

STAT-3-dependent Cytosolic Phospholipase A2 Expression Is Required for Thrombin-induced Vascular Smooth Muscle Cell Motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).

An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2 Axis in Platelet-derived Growth Factor BB-induced Vascular Smooth Muscle Cell Motility

Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A(2) (cPLA(2)). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA(2) in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA(2) prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA(2) rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA(2) signaling axis.

Inhibition of Nitric-oxide Synthase-I (NOS-I)-dependent Nitric Oxide Production by Lipopolysaccharide plus Interferon-γ Is Mediated by Arachidonic Acid: EFFECTS ON NFκB ACTIVATION AND LATE INDUCIBLE NOS EXPRESSION

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFkappaB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NFkappaB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFkappaB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.

Arachidonic Acid Regulation of Steroid Synthesis: New Partners in the Signaling Pathway of Steroidogenic Hormones

Although the role of arachidonic acid (AA) in trophic hormone‐stimulated steroid production in various steroidogenic cells is well documented , the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl‐CoA thioesterase (MTE‐I). We have shown that recombinant MTE‐I hydrolyses arachidonoyl‐CoA to release free AA. An acyl‐CoA synthetase specific for AA, acyl‐CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2‐mediated pathway. Inhibition of ACS4 and MTE‐I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH‐stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl‐CoA synthetase and the acyl‐CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2‐mediated pathway that involves a hormone‐induced acyl‐CoA synthetase and a hormone‐regulated acyl‐CoA thioesterase.

Activation of Na+-Independent Mg2+ Efflux by 20-Hydroxyeicosatetraenoic Acid in Rat Renal Epithelial Cells

Renal epithelial cells may have Mg(2+) transport pathways that regulate intracellular free Mg(2+) concentration ([Mg(2+)](i)) and reabsorption into the body. In mag-fura 2 fluorescent measurement, extracellular Mg(2+) removal induced a Na(+)-independent [Mg(2+)](i) decrease. The [Mg(2+)](i) decrease was suppressed by methyl arachidonyl fluorophosphonate, a cytosolic and Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, and bromoenol lactone, an iPLA(2) inhibitor, but it was not suppressed by a secretory phospholipase A(2) inhibitor. On the contrary, the [Mg(2+)](i) decrease was enhanced by the addition of exogenous arachidonic acid (AA). Next, we examined the effect of AA metabolite inhibitors on the [Mg(2+)](i) decrease. 17-octadecynoic acid inhibited the [Mg(2+)](i) decrease, but indomethacin and nordihydroguaiaretic acid did not. In the 17-octadecynoic acid-treated cells, 20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (20-HETE) recovered the [Mg(2+)](i) decrease. Nicardipine inhibited both the basal and the 20-HETE-enhanced [Mg(2+)](i) decrease. These results suggest that 20-HETE is a key mediator in the activation of Na(+)-independent Mg(2+) efflux.

Arachidonic acid-activated Na +-dependent Mg 2+ efflux in rat renal epithelial cells

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg 2+ deficiency. We examined whether the extracellular Mg 2+ concentration affects AA production and whether AA regulates a putative Na +-dependent Mg 2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg 2+-containing medium for 2 days because they enable us to detect Na +-stimulated Mg 2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg 2+ increased AA release both in the absence and presence of extracellular Na +. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 μM), an inhibitor of cytosolic phospholipase A 2 (cPLA 2) and Ca 2+-independent phospholipase A 2 (iPLA 2), and bromoenol lactone (BEL, 10 μM), an inhibitor of iPLA 2. However, LY-311727 (10 μM), a secretory phospholipase A 2 (sPLA 2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA 2 and iPLA 2 mRNAs, but not sPLA 2. In the mag-fura 2 fluorescence measurements, extracellular Mg 2+ removal caused slight decrease in the intracellular free Mg 2+ concentration ([Mg 2+] i) in the Na +-free condition. The addition of Na + caused a rapid decrease in [Mg 2+] i, indicating the presence of a Na +-dependent Mg 2+ efflux pathway. The Na +-dependent [Mg 2+] i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 μM), indomethacin (10 μM) and 17-octadecynoic acid (ODYA) (10 μM), enhanced the Na +-dependent [Mg 2+] i decrease. Furthermore, the addition of exogenous AA (30 μM) enhanced the Na +-dependent [Mg 2+] i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na +/Mg 2+-exchanger inhibitor. These results suggest that extracellular Mg 2+ removal elevates AA release mediated mainly by iPLA 2 and that AA upregulates the Na +-dependent Mg 2+ efflux in NRK-52E cells.

Platelets Abrogate Leukotriene B4 Generation by Human Blood Neutrophils Stimulated with Monosodium Urate Monohydrate or f-Met-Leu-Phe In Vitro

Neutrophils are physiologically associated with platelets in whole blood. Inflammatory reactions can be modulated by the presence of platelets. To investigate the influence of platelets on neutrophil activity, we studied the 5-lipoxygenase (5-LOX) metabolic pathway in normal human blood neutrophils stimulated with f-Met-Leu-Phe (fMLP) or monosodium urate monohydrate (MSUM) in the presence of autologous platelets. Platelets inhibited by more than 90% the synthesis of leukotriene B4 and 5-HETE in neutrophils activated with fMLP or MSUM. The addition of exogenous arachidonic acid did not reverse the inhibitory effect of platelets on 5-LOX–generated metabolites in fMLP- or MSUM-activated neutrophils. Preincubation of neutrophils with adenosine deaminase reversed the inhibitory effect of platelets in fMLP-treated neutrophils, indicating that adenosine was responsible for the platelet inhibition of leukotriene B4 and 5-HETE formation. In contrast, adenosine deaminase had no influence on the inhibitory effects of platelets in MSUM-stimulated cells. These results suggest that platelets can inhibit the synthesis of 5-LOX products (a) by acting mainly downstream to phospholipase A2 in cells stimulated by fMLP or MSUM, (b) through adenosine when neutrophils are activated with fMLP, and (c) by an adenosine-independent mechanism in MSUM-activated neutrophils by an as-yet-unidentified mediator.

Expression of functional prostaglandin D (DP) receptors in human corpus cavernosum smooth muscle.

Prostaglandin D(2) (PGD(2)) binds to specific G-protein coupled receptors (DP) and induces smooth muscle relaxation by stimulating the synthesis of intracellular cAMP. In this study, we examined the role of PGD(2) and DP receptors in regulating human penile smooth muscle contractility. We determined that human corpus cavernosum tissue and smooth muscle cells in culture expressed functional DP receptor and lipocalin-like prostaglandin D synthase by reverse-transcribed polymerase chain reaction (RT-PCR). Functional PGD synthase activity was confirmed by the synthesis of PGD(2) in human corpus cavernosum smooth muscle cells upon addition of exogenous arachidonic acid. Organ bath preparations of human corpus cavernosum tissue strips, contracted with phenylephrine, relaxed in a dose-dependent fashion to either PGD(2) or the DP selective agonist BW245C. Cultures of human corpus cavernosum smooth muscle cells treated with BW245C showed a two-fold increase in cAMP synthesis. These data are consistent with the expression of functional DP receptors in human corpus cavernosum. This suggests the presence of an intact prostanoid autocrine system that may play a role in regulating penile erectile function.

Role of three isoforms of phospholipase A2 in capacitative calcium influx in human T-cells.

The present study was conducted on human Jurkat T-cell lines in order to elucidate the role of phospholipase A2 in capacitative calcium entry. We have employed thapsigargin (TG) that induces increases in [Ca2+]i by emptying the calcium pool of endoplasmic reticulum, followed by capacitative calcium entry. We designed a Ca2+ free/Ca2+ reintroduction (CFCR) protocol for the experiments, conducted in Ca2+-free medium. By employing CFCR protocol, we observed that addition of exogenous arachidonic acid (AA) stimulated TG-induced capacitative calcium influx. The liberation of endogenous AA and its autocrine action seems to be implicated during TG-induced capacitative calcium influx: TG potentiates the induction of constitutively expressed mRNA of four PLA2 isoforms (type 1B, IV, V, VI), the inhibitors of the three PLA2 isotypes (type 1B, V, VI) inhibit TG-induced release of [3H]AA into the extracellular medium, and finally, these PLA2 inhibitors do curtail TG-stimulated capacitative calcium entry in these cells. These results suggest that stimulation of three isoforms of PLA2 by thapsigargin liberates free AA that, in turn, induces capacitative calcium influx in human T-cells.