Exogenous Arachidonic Acid Research Articles

Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S),12(S)-dihydroxyeicosatetraenoic acid and 14(R),15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5- and 15-hydroxyeicosatetraenoic acids, and 5,15-dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)-dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R),15(S)-dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 μm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)-dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 × 105 for 15-HETE, 1.4 × 105 for 12-HETE, and 0.9 × 105 for 5-HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.—Dadaian, M., and P. Westlund. Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets. J. Lipid Res. 1999. 40: 940–947.

Arachidonic Acid Is Preferentially Metabolized by Cyclooxygenase-2 to Prostacyclin and Prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.

Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells.

Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-alpha, IL-1beta, and interferon (IFN)-gamma had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1beta. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-gamma or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-gamma and 10 microM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.

Signalling Events Mediating the Activation of Protein Kinase C by Interleukin-2 in Cytotoxic T Cells

Interleukin-2 (IL-2) plays a vital role in the generation and regulation of the immune response, including important aspects of T cell survival. IL-2-mediated survival of T cells appears to be dependent on the activation of a pool of membrane-associated protein kinase C (PKC) that occurs in the absence of detectable translocation of the enzyme from the cytosol to membranes. In this report we investigate the mechanism(s) responsible for this PKC activation after IL-2 stimulation in the cytotoxic T cell line, CTLL-2. Tyrosine kinase activity, activated after IL-2 stimulation, was found not to be linked to the activation of PKC by the cytokine. On the other hand, a pertussis toxin (PTX)-sensitive G protein did appear coupled to PKC activation since PTX effectively blocked IL-2 stimulated PKC activity. Diacylglycerols (DAG), but not inositol 1,3,5-triphosphate (IP 3) and intracellular Ca 2+, increased after IL-2 stimulation suggesting that DAGs were generated via the phosphatidylcholine-phospholipase C (PC-PLC) or phosphatidylcholine-phospholipase D (PC-PLD) pathways. The increase in DAG by IL-2 was probably necessary for activation of membrane-resident PKC since exogenously applied DAG stimulated this PKC pool in both intact cells and in isolated membranes. IL-2 also increased arachidonic acid (AA) production in CTLL-2 cells, probably via phospholipase A 2 (PLA 2) since the PLA 2 inhibitors oleoyloxyethyl phosphocholine and AACOCF 3 (AACF) effectively blocked IL-2 stimulated PKC activation. Exogenous AA also increased PKC activity in intact cells and isolated membranes, suggesting that AA produced by IL-2 receptor stimulation was probably linked to PKC activation. These results suggest that the activation of membrane-resident PKC by IL-2 involves multiple second messengers, including G proteins, DAG and AA.

Region and Labour-dependent Synthesis of Prostaglandin E2 by Human Fetal Membranes

To examine the effect of region and labour upon prostaglandin synthesis in human fetal membranes, intact membranes from three regions, the cervical region, the periplacental region and a region midway between the two, were collected following spontaneous labour and delivery or at elective caesarean section prior to labour. Discs of 2-cm diameter were cut from each of three regions and incubated for 1, 2, 4, 6, 12 or 24h after which prostaglandin E2 concentration in the supernatant was measured. We found that there was an overall decrease in prostaglandin synthesis in tissues collected after labour, but that this effect could be reversed if exogenous arachidonic acid substrate was supplied. We found no differences in prostaglandin synthesis between tissues collected from each of the three regions. We conclude that prostaglandin synthesis from the fetal membranes during labour leads to depletion of arachidonic acid substrate and that regional changes in prostaglandin dehydrogenase activity do not appear to have a significant effect upon overall prostaglandin synthesis.

Synergy between cyclo-oxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy.

Prostanoids produced via the action of cyclo-oxygenase-2 (COX-2) appear central to many inflammatory conditions. Here we show in LPS-treated rats, however, that COX-2 induction alone does not greatly increase prostanoid production in vivo. For this, a second, arachidonic acid liberating stimulus is also required. Thus, only after intravenous injection of bradykinin or exogenous arachidonic acid was a marked increase in prostanoid formation seen. There is, therefore, synergy between proinflammatory mediators: both induction of COX-2 protein and an increase in the supply of arachidonic acid are required to greatly enhance prostanoid production. Second, we show that supplying arachidonic acid to increase prostanoid production reduces the effectiveness of both currently used nonsteroidal antiinflammatory drugs (NSAIDs) (diclofenac) and novel COX-2-selective inhibitors (NS-398, celecoxib) as inhibitors of COX-2 activity. Our data lead to two important conclusions. First, increased prostanoid production in inflammation is a two-component response: increased COX-2 expression and increased arachidonic acid supply. Second, the supply of arachidonic acid to COX-2 determines the effectiveness of NSAIDs. NSAIDs and selective COX-2 inhibitors, therefore, will generally be less effective at more inflamed sites, providing a rationale for the very high doses of NSAIDs required in human conditions such as rheumatoid arthritis.--Hamilton, L. C., Tomlinson, A. M., Mitchell, J. A., Warner, T. D. Synergy between cyclo-oxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy.

Transfection of an Active Cytochrome P450 Arachidonic Acid Epoxygenase Indicates That 14,15-Epoxyeicosatrienoic Acid Functions as an Intracellular Second Messenger in Response to Epidermal Growth Factor

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.

Decreased Leukotriene C4 Synthesis Accompanies Adherence-Dependent Nuclear Import of 5-Lipoxygenase in Human Blood Eosinophils

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [3H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types.

ATP-induced inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells: lack of involvement of known purinoceptor-coupled signaling pathways.

P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+]i, activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl- cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier ( approximately 50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+i, BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+, Cl- cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4, 5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+, Cl- cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl- cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl- channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl- cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl- cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism.

Hydrogenated Fat High in Trans Monoenes with an Adequate Level of Linoleic Acid Has no Effect on Prostaglandin Synthesis in Rats

Our study was designed to determine whether hydrogenated fat high in trans monoenes concentration affected prostaglandin synthesis. Corn oil (CO), butter (B), hydrogenated vegetable oil (HF) and coating fat (CF) were used in this study. These fats were fed to rats for 10 wk at 10 g/100 g diet. The phospholipid (PL) fatty acid content of platelets, aorta and heart was determined by gas liquid chromatography, and the in vitro aorta production of prostacyclin (PGI2) from exogenous or endogenous arachidonic acid (AA) was measured using the radioimmuno-assay (RIA) method. Serum thromboxane B2 (TXB2) released by platelets as thromboxane A2 (TXA2) during incubation of whole blood was also measured by this method. In the group fed CF, AA was significantly lower in the PL of aorta, platelet and heart, and the ratio 20:3(n-9)/20:4(n-6) was greater than in the groups fed CO, B or HF, indicating that the group fed CF was essential fatty acid (EFA) deficient. Although AA was significantly lower in the aorta and platelet PL of the group fed HF compared to the group fed CO, that difference did not affect the amounts of PGI2 or TXB2 produced in these groups. The group fed CF had significantly less PGI2 and TXB2 released by aorta and platelets than the other groups. This was the result of the reduced level of AA and the presence of higher amounts of 20:3(n-9) acid in the PL, which might act as a competitive inhibitor for cyclooxygenase. The aortic production of PGI2 from exogenous AA did not differ among the groups indicating that prostaglandin synthetase was not affected by the dietary fat. We conclude that the consumption of hydrogenated fats high in trans 18:1 acids with adequate amount of linoleic acid had no effect on the amount of thromboxane or prostacyclin produced by platelet or aorta in vitro.

Role of autocrine motility factor in a 12-lipoxygenase dependent anti-apoptotic pathway.

Apoptosis or programmed cell death, plays an indispensable role in embryonic development, maturation of the immune system and maintenance of tissue and organ homeostasis (1–2). A wide spectrum of molecular entities including oncogenes, tumor suppressor genes, signal transducers, cell cycle proteins, free radicals, cations and proteases have been implicated in apoptosis. Recent work from our laboratory (3) demonstrated that 12-lipoxygenase (12-LOX) may play an essential role in regulating apoptosis in rat Walker carcinosarcoma (W256) cells. Down regulation of 12-LOX gene expression by antisense oligonucleotides triggered time and dose dependent apoptosis. The W256 cell death, triggered by antisense oligos could be partially blocked by exogenous 12(S)-HETE. The antisense oligo treatment also resulted in a significant decrease in the ratio of Bcl-2/Bax proteins which are critical determinants of apoptosis. As expected, over expression of Bcl-2 protein provided partial death sparing effects (3). It also was demonstrated that exogenous arachidonic acid (AA) protected against apoptosis (4). Exogenous AA in a time and dose dependent fashion suppressed NDGA induced W256 apoptosis as well as DNA fragmentation. Serum withdrawal also caused W256 cells to undergo typical apoptosis which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation induced W256 cell apoptosis (4). Further studies demonstrated that the factor responsible for AA induced protection against apoptosis was the 12-LOX metabolite of AA 12(S)-HETE. 12-LOX has been demonstrated by our laboratory to be expressed in numerous tumor cells from human, rat and mouse (5). Factors controlling the regulation of 12-LOX are largely unknown. Several growth factors have been shown to stimulate 12-LOX activity in tumor cells. For example, in A431, human epidermoid carcinoma cells platelet type 12-LOX is expressed (6). Serum starvation induces a time and dose dependent reduction in the expression of 12-LOX protein (8). Another cytokine which has been demonstrated to regulate 12-LOX levels in tumor cells is autocrine motility factor (AMF). AMF stimulates cell motility and invasion via receptor mediated signaling pathways. AMF is also a potent growth factor. Our laboratory demonstrated that AMF induced cell motility was mediated by the lipoxygenase generation of 12(S)-HETE (7). In a subsequent report, we also demonstrated that AMF was able to increase the cellular protein levels of 12-LOX in high but not in low metastatic clones (8).

Arachidonic acid and freshly isolated human bone marrow mononuclear cells.

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis

Nodulation is the temporally and quantitatively most important cellular defense reaction to bacterial infections in insects. Inhibition of eicosanoid biosynthesis in adults of the cricket, Gryllus assimilis, immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A 2, cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a complex process involving lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 2 h of injection, and nodulation was significantly reduced, relative to control crickets, over 22 h. The dexamethasone effects were reversed by treating bacteria-injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids, and fat body preparations were shown to be competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. These findings in a hemimetabolous insect broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2.

To test the hypothesis that intracellular Ca2+ activation of large-conductance Ca2+-activated K+ (BK) channels involves the cytosolic form of phospholipase A2 (cPLA2), we first inhibited the expression of cPLA2 by treating GH3 cells with antisense oligonucleotides directed at the two possible translation start sites on cPLA2. Western blot analysis and a biochemical assay of cPLA2 activity showed marked inhibition of the expression of cPLA2 in antisense-treated cells. We then examined the effects of intracellular Ca2+ concentration ([Ca2+]i) on single BK channels from these cells. Open channel probability (Po) for the cells exposed to cPLA2 antisense oligonucleotides in 0.1 microM intracellular Ca2+ was significantly lower than in untreated or sense oligonucleotide-treated cells, but the voltage sensitivity did not change (measured as the slope of the Po-voltage relationship). In fact, a 1,000-fold increase in [Ca2+]i from 0.1 to 100 microM did not significantly increase Po in these cells, whereas BK channels from cells in the other treatment groups showed a normal Po-[Ca2+]i response. Finally, we examined the effect of exogenous arachidonic acid on the Po of BK channels from antisense-treated cells. Although arachidonic acid did significantly increase Po, it did so without restoring the [Ca2+]i sensitivity observed in untreated cells. We conclude that although [Ca2+]i does impart some basal activity to BK channels in GH3 cells, the steep Po-[Ca2+]i relationship that is characteristic of these channels involves cPLA2.

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP.

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.

Respective roles of the 14 kDa and 85 kDa phospholipase A2 enzymes in human monocyte eicosanoid formation.

Human monocytes possess both the cytosolic 85 kDa phospholipase (PLA) A2 and a 14 kDa PLA2 and are capable of simultaneously producing prostanoids (PG), leukotrienes (LT) and platelet activating factor (PAF). As the exact roles of the two enzymes in monocyte lipid mediator formation was unclear, both selective PLA2 inhibitors and antisense were used to elucidate their respective roles. Reduction in 85 kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85 kDa PLA2 inhibitor, arachidonyl trifluormethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocytes prostanoid formation but not LTC4 or PAF production. This confirmed the important role of the 85 kDa PLA2 in prostanoid formation but indicated a less significant role in LT or PAF biosynthesis. Alternatively, treatment of monocytes with the selective, active-site-directed 14 kDa PLA2 inhibitor, SB 203347, totally inhibited LT and PAF formation, while prostanoid formation was not altered. Addition of 20 uM exogenous arachidonic acid (AA) to monocytes exposed to SB 203347 did not alter A23187-induced LTC4 generation, indicating that SB 203347 had no effect on downstream AA metabolizing enzymes in this setting. Taken together, these results provide evidence that the 14 kDa PLA2 provides substrate for monocyte LT and PAF formation, while the 85 kDa PLA2 plays a more significant role in the formation of PG.

Benzydamine reduces prostaglandin production in human gingival fibroblasts challenged with interleukin-1β or tumor necrosis factor a

Benzydamine [1-benzyl-3-(3-dimethylamino)propoxy-1H-indazole] is a drug with analgesic, anesthetic, antimicrobial and anti-inflammatory activity. The purpose of the present study was to investigate the effect of benzydamine on prostaglandin production in human gingival fibroblasts. Benzydamine significantly reduced the basal production of both prostaglandin E2 (PGE2) and 6-keto-PGF1α, the stable breakdown product of prostaglandin I2(PGI2), in unstimulated human gingival fibroblasts. When the cells were treated simultaneously with benzydamine and the cytokines IL-1β or TNFα, the agent benzydamine reduced (P < 0.05) the stimulatory effect of IL-1β and TNFα respectively, on PGE2 and PGI2 production in human gingival fibroblasts. Furthermore, benzydamine reduced (P < 0.05) both the basal level and the cytokineinduced 3H-arachidonic acid release 3H-(AA) in gingival fibroblasts. The addition of exogenous arachidonic acid to the cells resulted in enhanced PGE2 production, which was reduced (P < 0.05) in the presence of benzydamine. The study indicates that benzydamine reduces the prostaglandin synthesis in gingival fibroblasts, partly at the level of phospholipase A2, by diminishing the liberation of arachidonic acid (AA) from phospholipids, and partly at the level of cyclooxygenase. The inhibitory effect of benzydamine on prostaglandin production may explain the anti-inflammatory effect of the drug in the management of patients with oral inflammatory conditions.

Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes.

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes.

Heterogeneous long chain acyl-CoA synthetases control distribution of individual fatty acids in newly-formed glycerolipids of neuronal cells undergoing neurite outgrowth.

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [3H]glycerol, [3H]oleic acid (OA) or [3H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 microM), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [3H]OA into glycerolipids by 30-90%, and increased by about 60% the accumulation of free [3H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [3H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 microM, respectively; and in cytoplasm, 6 and 0.6 nmol/min/mg protein and 38 and 60 microM, respectively. Triacsin C (2-33 microM) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC50 and apparent Ki values were 13.5 microM and 14 microM in microsomes, and 3.8 microM and 4 microM in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.

Functional Coupling Between Various Phospholipase A2s and Cyclooxygenases in Immediate and Delayed Prostanoid Biosynthetic Pathways

Several distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another.