Release Of 3H-arachidonic Acid Research Articles

Mechanism of Human Platelet Activation by Endotoxic Glycolipid-Bearing Mutant Re595 of Salmonella minnesota

The mechanism through which human blood platelets interact with gram-negative bacteria with well-defined structural variations in endotoxic lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation of platelets separated from plasma proteins were observed on challenge with rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking heptose phosphate in the core and O-polysaccharide in its outer portion. Both 14C-serotonin secretion and platelet aggregation were concentration-dependent, with a half-maximum response at the ratio of one bacterial colony-forming unit (CFU) to two platelets. The aggregation of human platelets induced by mutant Re595 was divalent cation-dependent and required secretion of ADP and fibrinogen from platelet storage granules because it was inhibited by chelators, by the ADP-splitting enzyme apyrase, and by monospecific antifibrinogen Fab fragments. The synthetic peptide analog of the platelet receptor recognition site on the gamma chain of fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected. Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha 572-575) and to the cell adhesion site of fibronectin, also inhibited aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-serotonin was preceded by a very rapid phosphorylation of a platelet protein of mol wt 47,000, which is associated with protein kinase C activation. Myosin light chain (mol wt 20,000) was also phosphorylated. Both phosphoproteins were dephosphorylated while secretion was reaching maximum. Furthermore, release of 3H-arachidonic acid from platelet phospholipids and generation of thromboxane B2 via the cyclooxygenase pathway were observed. Inhibition of this pathway with acetylsalicylic acid (10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C-serotonin secretion and platelet aggregation. The role of Lipid A in the interaction of mutant Re595 with human platelets was deduced from the inhibitory effect of the Lipid A-binding protein present in Limulus amebocyte lysate. Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The reactivity of mutant Re595 toward platelets was attenuated by mild acid hydrolysis, during which KDO was dissociated from the glycolipid, and by alkaline hydrolysis, which breaks ester-linked fatty acids in Lipid A. In contrast to mutant Re595, strain S218 of S minnesota bearing "complete" endotoxic lipopolysaccharide did not induce secretion and aggregation of human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Advanced glycation end products inhibit Na + K + ATPase in proximal tubule epithelial cells: Role of cytosolic phospholipase A 2α and phosphatidylinositol 4-phosphate 5-kinase γ

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. In diabetes, renal Na + K + ATPase (NKA) activity is downregulated and phosphoinositide metabolism is upregulated. We examined the effects of AGEs on NKA activity in porcine LLC-PK1 and human HK2 proximal tubule epithelial cells. AGE-BSA increased cellular phosphoinositol 4,5 bisphosphate (PIP2) production as determined by immunofluorescence microscopy and thin layer chromatography. AGE-BSA (40 μM) induced 3H-arachidonic acid release and reactive oxygen species (ROS) production via cytosolic phospholipase A 2 (cPLA 2) activation. Within minutes, AGE-BSA significantly inhibited NKA surface expression and activity in a dose- and time-dependent manner as determined by immunofluorescence staining and [ 86Rb +] uptake, respectively, suggesting AGEs inhibit NKA by stimulating its endocytosis. The AGE-BSA-induced decrease in cell surface NKA was reversed by a cPLA 2α inhibitor, neomycin, a PIP2 inhibitor, and PP2, a Src inhibitor. AGE-BSA increased binding of NKA to the α-adaptin but not β2- or μ2-adaptin subunits of the AP-2 clathrin pit adaptor complex. Transfection of HK2 cells with PIP5Kγ siRNA prevented AGE-BSA inhibition of NKA activity. AGEs may stimulate PIP5Kγ to increase PIP2 production, which may enhance AP-2 localisation to clathrin pits, increase clathrin pit formation, enhance NKA cargo recognition by AP-2 and/or stimulate cPLA 2α activity. These results suggest AGEs modulate arachidonic acid and phosphoinositide metabolism to inhibit NKA via clathrin-mediated endocytosis. Elucidation of new intracellular AGE signaling pathways may lead to improved therapies for diabetic nephropathy.

The anti-apoptotic effects of nordihydroguaiaretic acid: Inhibition of cPLA 2 activation during TNF-induced apoptosis arises from inhibition of calcium signaling

Nordihydroguaiaretic acid (NDGA) is a plant lignan produced by Larrea tridentata, the creosote bush of the American southwest. In this report we examine the mechanism underlying the ability of NDGA to inhibit TNF-induced apoptosis. Our results show that NDGA blocks many key indicators of apoptosis. Caspase cleavage, mitochondrial inactivation, externalization of phosphatidyl serine, and 51Cr-release were all blocked by low micromolar concentrations of NDGA. NDGA also inhibited the cPLA 2-dependent release of 3H-arachidonic acid. We investigated this activity and found that NDGA prevented the rise in intracellular calcium necessary for the apoptotic activation of cPLA 2. On the other hand, NDGA did not interfere with the TNF-induced phosphorylation of cPLA 2, indicating that NDGA does not block all TNF-dependent signaling. Finally, we asked whether the anti-apoptotic effect of NDGA could be attributed to its anti-oxidant activity. Comparison with the effects of butylated hydroxyanisole (BHA) did not completely support this hypothesis. While BHA strongly inhibited caspase activation and partially blocked the release of 51Cr, it was unable to significantly block the calcium response or the release of 3H-arachidonic acid associated with TNF-induced apoptosis. The anti-oxidant activity of NDGA may, therefore, explain some but not all of its anti-apoptotic activity.

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death.

The purpose of this study was to determine the roles of calcium-dependent phospholipase A2 (cPLA2) and calcium-independent phospholipase A2 (iPLA2) in thapsigargin-induced membrane susceptibility to secretory phospholipase A2 (sPLA2) and programmed cell death. 3H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA2 (BEL) and completely by an inhibitor of both cPLA2 and iPLA2 (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA2 in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA2. In conclusion, the results suggested that cPLA2 and iPLA2 are active during thapsigargin-induced apoptosis in S49 cells and that cPLA2 tempers the tendency of the cells to become susceptible to sPLA2 during apoptosis.

Regulation of the Na+-K+(NH4+)-2Cl- cotransporter of rat submandibular glands.

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH4Cl, and the variations of the intracellular concentration of calcium ([Ca2+]i) and the intracellular pH (pHi) were measured using fura-2 and 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH4Cl significantly increased the [Ca2+]i without affecting the response to 100 micromol/l carbachol. When exposed to 1 and 5 mmol/l NH4Cl, the cells acidified immediately. At 30 mmol/l, NH4Cl first alkalinized the cells and the pHi subsequently dropped. This drop reflects the uptake of NH4+ ions that dissociate to NH3 and H+ in the cytosol. These protons are exchanged for extracellular sodium by the Na+/H+ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH4Cl. Ouabain partly blocked the uptake of NH4+. In the combined presence of ouabain and bumetanide (an inhibitor of the Na+-K+-2Cl- cotransporter), 1 mmol/l NH4Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na+-K+-2Cl- cotransporter in the uptake of NH4+ was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH4+ by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH4+ by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPbetaS. ATP only slightly inhibited the increase of cyclic AMP (-22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the beta-adrenergic agonist. ATP increased the release of 3H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na+-K+-2Cl- cotransporter.

Signal transduction pathways involved in the synergistic stimulation of prostaglandin production by interleukin-1beta and tumor necrosis factor alpha in human gingival fibroblasts.

Accumulating evidence indicates that prostaglandins play an important role in the pathogenesis of periodontal disease. In this study, the effects and interactions between IL-1beta and TNFalpha on prostaglandin production and its regulation were investigated. The cytokines IL-1beta and TNFalpha stimulated prostaglandin E2 (PGE2) and prostacyclin (PGI2) production in gingival fibroblasts. Simultaneous treatment of the cells with IL-1beta and TNFalpha resulted in a synergistic stimulation of PGE2 and PGI2 formation. IL-1beta and, to a lesser extent, TNFalpha stimulated the release of 3H-arachidonic acid (3H-AA), and simultaneous addition of IL-1beta and TNFalpha further increased the release of 3H-AA from pre-labeled gingival fibroblasts. Furthermore, IL-1beta and, to a lesser extent, TNFalpha induced the expression of cyclooxygenase-2 (COX-2) mRNA. Simultaneous addition of IL-1beta and TNFalpha synergistically enhanced COX-2 mRNA levels, accompanied by a corresponding stimulation of PGE2 synthesis. Neither IL-1beta, TNFalpha, nor the combination of these two cytokines affected COX-1 mRNA levels. PMA, known to activate protein kinase C (PKC), enhanced the stimulatory effect of IL-1beta, TNFalpha, and the combination on COX-2 mRNA levels accompanied by a corresponding increase in PGE2 production. The phospholipase A2 (PLA2) inhibitor, BPB, and the PKC inhibitor, BIS, reduced PGE2 production, whereas dexamethasone, indomethacin, and NS-398 completely abolished PGE2 production induced by IL-1beta, TNFalpha, and the combination. The study indicates that the synergistic stimulation of prostaglandin production by IL-1beta, and TNFalpha is mediated partly at the level of COX-2 and partly at the level of PLA2 and that PKC is involved in the signal transduction of the synergy between the two cytokines. The synergy between IL-1beta and TNFalpha may play an important role in the inflammatory processes in gingival tissue in vivo.

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.

Effect of Turmeric, Turmerin and Curcumin on H 2O 2-Induced Renal Epithelial (LLC-PK 1) Cell Injury

Cell injury by oxidative stress is an important mechanism for renal epithelial cell destruction. This study observed the protective effect of turmeric and its constituents on H 2O 2-induced injury. Turmeric consists of a water soluble turmerin and lipid soluble curcumin with potent antioxidant properties. Confluent LLC-PK 1 cells were labelled with 3H-arachidonic acid at 0.1 μCi/ml over 18 h and then further labelled with 51Cr. Turmeric (100 μg/ml-0.1 μg/ml), turmerin (800 ng/ml-0.8 ng/ml), curcumin (100 μg/ml-0.1 μg/ml), vitamin E (100 μM) and 21-aminosteroid (20 μM) were added and incubated for 3 h at 37°C in 24-well plate. The adherent cells were washed and incubated for 3 h with 1.5 mM H 2O 2 at 37°C. 3H-arachidonic acid release, 51Cr release and lipid peroxidation by the thiobarbituric acid reaction was determined. Turmeric (100 μg/ml) and curcumin (100 μg/ml, 10 μg/ml) gave as much protection as did vitamin E in both chromium release assay and lipid degradation while Turmeric (100 μg/ ml) and curcumin (100 μg/ml) gave comparable inhibition of lipid peroxidation. Turmerin and 21-aminosteroid showed no protection. These findings provide evidence that turmeric and curcumin provide protection against oxidative stress in a renal cell line.

Inhibition of leukotriene synthesis by azelastine.

Azelastine, oxatomide, and ketotifen are used for patients with allergic diseases. These drugs inhibit the release of chemical mediators including the leukotrienes; however, the mechanism involved is unclear. To clarify the mechanism of inhibition, we investigated the effects of three drugs on the function of phospholipase A2, 5-lipoxygenase, leukotriene C4 synthase, and leukotriene A4 hydrolase, which are all catabolic enzymes involved in synthesizing leukotriene C4 and leukotriene B4 in rat basophilic leukemia (RBL)-1 cells. The production of leukotriene C4 and leukotriene B4 was measured by high performance liquid chromatography (HPLC). All three drugs inhibited the production of leukotriene C4 and leukotriene B4 when cells were stimulated with A23187. All three drugs also inhibited the A23187-stimulated release of 3H-arachidonic acid from membrane phospholipids. Azelastine inhibited the production of leukotriene C4, but not leukotriene B4, when either arachidonic acid or leukotriene A4 free acid was used as the substrate in our cell free system. Oxatomide and ketotifen did not inhibit the synthesis of either leukotriene C4 or leukotriene B4 in the same cell free study. Results indicated that oxatomide and ketotifen inhibit the production of leukotriene C4 and leukotriene B4 by inhibiting phospholipase A2 activity, whereas, azelastine inhibits the leukotriene C4 production by inhibiting phospholipase A2 and leukotriene C4 synthase.

Inhibition of leukotriene synthesis by honokiol in rat basophilic leukemia cells.

The effects of honokiol, a diphenyl compound extracted from a Chinese herbal medicine, on leukotriene (LT) synthesis were evaluated in rat basophilic leukemia (RBL) cells. The production of LTC4 and LTB4 stimulated by the Ca2+ ionophore A23187 was measured in RBL-1 cells by high-performance liquid chromatography. Honokiol inhibited the production of LTC4 and LTB4 stimulated by A23187 in RBL-1 cells. Honokiol did not inhibit either phospholipase A2 activity, measured by the release of 3H-arachidonic acid (AA), or LTC4 synthase and LTA4 hydrolase activities, measured with LTA4-free acid as substrate. The synthesis of LTC4 and LTB4 from AA in RBL-1 cell lysates in the presence of Ca2+ was inhibited by honokiol. These results indicate that honokiol blocks LT synthesis by inhibiting 5-lipoxygenase activity. Honokiol also inhibited immunoglobulin E-mediated production of these LTs in RBL-2H3 cells, which was measured by a specific radioimmunoassay (RIA). These results suggest that honokiol may exhibit antiallergic actions by inhibiting LT synthesis in immediate-type hyperreactivity.

Modulation of the glutamate-evoked release of arachidonic acid from mouse cortical neurons: involvement of a pH-sensitive membrane phospholipase A2

Excitatory synaptic transmission is associated with changes in both extracellular and intracellular pH. Using mouse cortical neurons in primary cultures, we studied the sensitivity of glutamate-evoked release of 3H-arachidonic acid (3H-AA) to changes in extracellular pH (pHo) and related intracellular pH (pHi). As pHo was shifted from 7.2 to 7.8, the glutamate-evoked release of 3H-AA was enhanced by approximately threefold. The effect of alkaline pHo on the glutamate response was rapid, becoming significant within 2 min. 3H-AA release, evoked by both NMDA and kainate, was also enhanced by pHo alkalinization. NMDA- and kainate-induced increase in free intracellular Ca2+ was unaffected by changing pHo from 7.2 to 7.8, indicating that the receptor-induced Ca2+ influx is not responsible for the pHo sensitivity of the glutamate-evoked release of 3H-AA. Alkalinization of pHi obtained by incubating neurons in the presence of HCO3- or NH4 enhanced the glutamate-evoked release of 3H-AA, while pHi acidification obtained by blockade of Na+/H+ and Cl-/HCO3- exchangers decreased the glutamate response. Membrane-bound phospholipase A2 (mPLA2) activity was stimulated by Ca2+ in a pH-dependent manner, increasing its activity as pH was shifted from 7.2 to 7.8. This pH profile corresponds to the pH profile of the glutamate-, NMDA- and kainate-evoked release of 3H-AA. Taken together, these results indicate that the glutamate-evoked release of 3H-AA may be mediated by the pH-sensitive mPLA2. Since excitatory neurotransmission mediated by glutamate results in both pHo and pHi changes and since AA enhances glutamatergic neurotransmission at both pre- and postsynaptic levels, the data reported here reveals a possible molecular mechanism whereby glutamate can modulate its own signalling efficacy in a pH-dependent manner by regulating the release of AA.

Arachidonic acid as a possible negative feedback inhibitor of nicotinic acetylcholine receptors on neurons

Neuronal acetylcholine receptors, being highly permeable to calcium, are likely to regulate calcium-dependent events in neurons. Arachidonic acid is a membrane-permeant second messenger that can be released from membrane phospholipids by phospholipases in a calcium-dependent manner. We show here that activation of neuronal acetylcholine receptors triggers release of 3H-arachidonic acid in a calcium-dependent manner from neurons preloaded with the fatty acid. Moreover, low concentrations of arachidonic acid reversibly inhibit the receptors and act most efficiently on receptors likely to have the highest permeability to calcium, namely receptors containing alpha 7 subunits. Low concentrations of arachidonic acid also reversibly inhibit alpha 7-containing receptors expressed in Xenopus oocytes following injection of alpha 7 cRNA. The oocyte results indicate following injection of alpha 7 cRNA. The oocyte results indicate that the inhibition is a feature of the receptors rather than a consequence of neuron-specific machinery. The inhibition is not mediated by specific metabolites of arachidonic acid because the effects can be mimicked by other fatty acids; their effectiveness correlates with their content of double bonds. In contrast to arachidonic effects on calcium currents, inhibition of neuronal nicotinic receptors by the fatty acid cannot be prevented by blocking production of free radicals or by inhibiting protein kinase C. An alternative mechanism is that arachidonic acid binds directly to the receptors or perturbs the local environment in such a manner as to constrain receptor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of leukotriene production by FK506 in rat basophilic leukemia-1 cells.

We investigated the inhibitory action of FK506 (0.0005-5 micrograms/ml) on the metabolism of arachidonate 5-lipoxygenase in rat basophilic leukemia-1 cells. Cells were stimulated with A23187 (10(-5) mol/l) for 15 min in the absence or presence of various concentrations of FK506. Arachidonate 5-lipoxygenase metabolites, peptide leukotrienes (LTs), leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) were measured by high-performance liquid chromatography. FK506 inhibited A23187-stimulated production of peptide LTs, LTB4 and 5-HETE in intact cells by up to 77, 73 and 60%, respectively. Phospholipase A2 activity, measured by the release of 3H-arachidonic acid (AA), was not significantly inhibited by FK506. The synthesis of peptide LTs and LTB4 was not inhibited by FK506 when leukotriene A4-free acid was added to the culture medium. The synthesis of peptide LTs, LTB4 and 5-HETE was not affected by FK506 in a cell lysate study using AA as the substrate. These results indicate that FK506 inhibits the production of peptide LTs, LTB4 and 5-HETE by inhibiting 5-lipoxygenase activity in intact cells. The inhibition is not a direct action on 5-lipoxygenase but results from the activating processes of this enzyme.

Cyclosporine nephrotoxicity: attenuation by an antioxidant-inhibitor of lipid peroxidation in vitro and in vivo.

The exact mechanisms by which cyclosporine (CsA) causes renal injury is not known. The possibility that reactive oxygen species (ROS) may play a role, since CsA induces renal microsomal lipid peroxidation and reduces glutathione levels, was investigated by examining whether administration of an antioxidant attenuates CsA-induced nephrotoxicity. One of three groups of uninephrectomized rats received vehicles, another CsA 25 mg/kg/day and the third, CsA plus antioxidant lazaroid (U-74389 G) 20 mg/kg/day, for 8 weeks. CsA-induced functional and structural derangements were accompanied by a significant induction of renal cortical lipid peroxidation (thiobarbituric acid reactive substances and conjugated diene). Administration of lazaroid significantly suppressed CsA-induced lipid peroxidation and provided significant functional and structural protection. That lazaroid affords renal functional protection against CsA in the rat was again demonstrable in a crossover study. To examine the relation between CsA nephrotoxicity and lipid peroxidation, cell culture studies were undertaken. CsA induced renal epithelial (LLC-PK1) cell injury (LDH release) as well as lipid peroxidation and degradation (prelabeled 3H arachidonic acid release). Lazaroid prevented CsA-induced cell injury and limited lipid alterations. These new findings--that an antioxidant inhibitor of lipid peroxidation limits CsA-induced renal toxicity in vitro and in vivo--suggest a pathogenic role for ROS-mediated lipid peroxidation in CsA-induced renal toxicity. Antioxidant therapy may minimize CsA-induced renal toxicity in humans.

Induction by chemokines of lipid mediator synthesis in granulocyte-macrophage colony-stimulating factor-treated human neutrophils.

In recent years, there has been a growing body of evidence suggesting that IL-8 and granulocyte-macrophage CSF (GM-CSF) play an important role in inflammatory processes. We show that after GM-CSF treatment, the exposure of human neutrophils to IL-8 results in the synthesis of leukotriene (LT)B4 and platelet-activating factor. In GM-CSF-treated cells, IL-8 induced a concentration-dependent synthesis of both lipid mediators, with a threshold at 10 to 30 nM, suggesting that IL-8 could stimulate phospholipase A2 activity, an enzyme essential for both syntheses. Accordingly, IL-8 induced a substantial release of 3H-arachidonic acid in GM-CSF-treated PMN. It was also found that IL-8 activates the neutrophil 5-lipoxygenase (5-LO), the other key enzyme in LT biosynthesis. IL-8 induced 5-LO activation in a time- and concentration-dependent manner, with a threshold at 1 nM, and prior treatment of neutrophils with GM-CSF enhanced this effect of IL-8 over the 1 to 300 nM range. Neutrophil-activating peptide-2 and the melanoma growth-stimulatory activity, two peptides that are closely related to IL-8, also had the ability to activate the 5-LO and stimulate LT synthesis, albeit less potently than IL-8. Finally, pertussis toxin and the 5-LO translocation inhibitor, MK-886, both blocked the IL-8-elicited 5-LO activation. Taken together, our results raise the possibility that the combined presence of IL-8 and of GM-CSF at inflammatory foci could result in the synthesis of platelet-activating factor and LTB4 by neutrophils, thereby contributing to the amplification of the inflammatory response.

Epidermal growth factor potentiates interleukin 1 and tumour necrosis factor-induced prostaglandin biosynthesis in human gingival fibroblasts

The effects of and interactions between epidermal growth factor (EGF), transforming growth factor α (TGF-α) interleukin 1 (IL-1) and tumour necrosis factor α (TNF-α) on arachidonic acid release and prostaglandin biosynthesis in human gingival fibroblasts were studied. IL-1α, IL-1β and TNF-α, but not EGF nor TGF-α, stimulated prostaglandin E 2 (PGE 2) formation in the gingival fibroblasts. The effect of IL-1α, IL-1β and TNF-α on PGE 2 formation was significantly potentiated by EGF in a dose-dependent manner. Similary, TGF-α synergistically potentiated IL-1β stimulated PGE 2 formation. IL-1β but not EGF stimulated the release of 3H-arachidonic acid ( 3H-AA) from prelabelled gingival fibroblasts. In contrast to the effect on PGE 2 formation, no synergistic interaction between EGF and IL-1 was seen on arachidonic acid (AA) release. Addition of unlabelled exogenous AA, in the presence of EGF, resulted in a significant increase in PGE 2 formation compared to that seen in fibroblasts not exposed to EGF. The results demonstrate that EGF and IL-1 as well as EGF and TNF-α act in concert to enhance prostanoid formation in gingival fibroblasts. Data indicates that EGF potentiates the IL-1 and TNF-α induced PGE 2 formation at the level of prostaglandin endoperoxide synthase (cyclooxygenase). The synergistic effects of inflammatory cytokines and growth factors may be of physiological importance for regulation of regenerative tissue growth during inflammation and repair.

The adenovirus e3 region 14.7 kDa protein, heat and sodium arsenite inhibit the TNF-induced release of arachidonic acid

In this report we show that the adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite, which have been defined previously as inhibitors of cytolysis, inhibit the tumor necrosis factor-α (TNF)-induced release of 3H-arachidonic acid from cycloheximide-sensitized C3HA fibroblasts. Since the A23187-induced release of 3H-a.a. was unaffected, our results suggest that these inhibitors provide resistance to lysis by selectively interfering with the lytic response pathway. Our results also show that heat and sodium arsenite can themselves induce the release of 3H-arachidonic acid. These results raise the possibility that stressor-induced resistance to TNF results from the selective desensitization of phospholipase A 2.

Acute and chronic suppression of leukotriene B4 synthesis ex vivo in neutrophils from patients with rheumatoid arthritis beginning treatment with methotrexate.

To compare the cumulative effects of oral methotrexate (MTX) therapy (after 6-8 weeks) with the acute effects (24 hours after a dose) on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from patients with active rheumatoid arthritis (RA) who were beginning therapy with MTX. Neutrophils and monocytes were isolated from whole blood from 7 patients with RA, immediately before and 24 hours after their first weekly dose of 7.5 mg of MTX, and again after their dose at 6-8 weeks. Total immunoreactive leukotriene B4 (LTB4) formation in neutrophils activated ex vivo with calcium ionophore A23187 was significantly suppressed (by 33%) before the 6-8-week dose, compared with the level before the first dose (mean +/- SEM 8.29 +/- 1.24 ng/10(6) cells at predose 6-8 weeks versus 12.29 +/- 2.13 ng/10(6) cells at predose 1; P = 0.03). Reductions were also observed after the first dose (27%; P = 0.07) and after the 6-8-week dose (43%; P = 0.05) compared with the respective predose levels. MTX treatment produced significant reductions in the total generation of 5-LO pathway products (5-hydroxyeicosatetraenoic acid + 6-trans-LTB4 + LTB4 + omega-oxidation products of LTB4) by calcium ionophore-activated neutrophils, as quantitated by integrated optical density after resolution on reverse-phase high-performance liquid chromatography. Decreases were observed after the first dose (26%; P = 0.025), immediately before the 6-8-week dose (23%; P = 0.05), and after the 6-8-week dose (47%; P = 0.0033) compared with levels before the first dose, and after the 6-8-week dose compared with the level before it (32%; P = 0.04). The generation of LTB4 by calcium ionophore-activated monocytes was not significantly affected by MTX therapy. The significant decreases in the formation of omega-oxidation products of LTB4 and in the total generation of neutrophil 5-LO pathway products in the absence of a significant change in the release of 3H-arachidonic acid or the generation of platelet-activating factor suggest that the activity of the 5-LO enzyme in neutrophils is inhibited. We conclude that weekly oral MTX therapy in patients with active RA inhibits neutrophil 5-LO pathway product generation in a pattern consistent with inhibition of the activity of the 5-LO enzyme; an effect is observed after the first dose. The inhibition of 5-LO is cell-selective and cumulative, with a superimposed incremental inhibition observed after the weekly MTX dose.

Bradykinin‐2 receptor‐mediated release of 3H‐arachidonic acid and formation of prostaglandin E2 in human gingival fibroblasts

Bradykinin stimulated production of prostaglandin E2 (PGE2) and the release of 3H-arachidonic acid by gingival fibroblasts in a time- and dose-dependent manner. The effect on PGE2 biosynthesis was seen already after 15 seconds and was maximal after 5 minutes. Several structurally unrelated inhibitors of arachidonic acid metabolism via the cyclooxygenase pathway totally abolished the PGE2 response to bradykinin. The stimulation of PGE2 formation was seen at and above 10 nmol/l of bradykinin. Des-Arg9-bradykinin was 100-fold less potent compared to bradykinin. Des-Arg9-Leu8-bradykinin did not antagonize bradykinin-induced PGE2 formation. Met-Lys-bradykinin and Lys-bradykinin also enhanced PGE2 formation in gingival fibroblasts. The stimulatory action of bradykinin on 3H-arachidonic acid release was observed after 30 s and progressively increased for at least 15 min. The stimulatory effect on 3H-arachidonic acid release by bradykinin was seen at and above 10 nmol/l, whereas des-Arg9-bradykinin was without effect up to a concentration of 1 mumol/l. Indomethacin did not affect bradykinin-induced 3H-arachidonic acid release. These data show that bradykinin, via a B2-receptor-mediated pathway, can stimulate arachidonic acid release and subsequent prostanoid formation in gingival fibroblasts. Consequently, gingival fibroblasts may contribute, by a bradykinin-regulated reaction, to the enhanced amounts of prostanoids found in gingival tissues and crevicular fluids in patients with periodontal diseases.

Calcium Ionophore but Not Phorbol Ester Promotes Eicosanoids Release by Proliferating Interleukin-3-Dependent Bone Marrow Cells

Eicosanoid release during multilineage hematopoiesis was assessed using freshly isolated mouse bone marrow cells cultured in the presence of interleukin-3 (IL-3) (10% WEHI-3 culture-conditioned medium). Cells that could release prostaglandin E2 (PGE2) when stimulated with calcium ionophore A23187, but not with phorbol ester (PMA), appeared within 4 days. The cells harvested on day 10 released 42 ng of PGE2/10(6) cells/mL after A23187 stimulation. Leukotriene B4 (LTB4) (4 ng/mL) was also detected after A23187 stimulation, but there was no detectable LTC4 (less than 0.5 ng/mL). Nonadherent bone marrow cells were isolated from 28-day cultures and cloned. All clones were strongly IL-3-dependent. Although other growth factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and CSF-1 failed to promote survival or support proliferation of the cells, three clones (11-1-A6, 3-2-D5, and 11-1-A1) showed significant increases in 3H-thymidine incorporation, respectively, after PMA treatment for 24 hours. Surviving cells displayed dominantly myeloid type morphology and phenotypic characteristics. The data suggest that IL-3 is important in the formation of PGE2-producing cells. In contrast to many macrophages (MO), neither the IL-3-dependent cell lines nor the IL-3-cultured bone marrow cells released significant amounts of PGE2 when stimulated with PMA or IL-3, although PMA and IL-3 both induced translocation of protein kinase C (PKC) to the membrane fraction. The lack of production of PGE2 and other eicosanoids by the PMA- and IL-3-stimulated cell lines was confirmed by measuring the release of 3H-arachidonic acid. The data suggest that in IL-3-dependent bone marrow cell lines the activation of eicosanoid metabolism requires elevated cellular Ca2+; PKC activation alone does not appear to be a sufficient stimulus.