Endogenous arachidonic acid metabolism by calcium ionophore A23187-stimulated lamb lungs: effect of hypoxia

We have determined eicosanoid production from endogenous arachidonic acid by neonatal lamb lungs stimulated with calcium ionophore A23187 during normoxia and hypoxia. Lungs of lambs 19 to 25 d of age were isolated and perfused with cell-free Krebs' bicarbonate buffer at a flow rate of 15 to 20 ml/kg/min. After 30 min of equilibration in a recirculating system, A23187 was added to the perfusate in a 5-microM concentration and perfusion continued for 15 min more. Eicosanoids were measured in perfusate and lung homogenate supernatant. Cyclooxygenase metabolites prostaglandin (PG) E2, thromboxane A2, and PGI2, were measured by radioimmunoassay, and 5-lipoxygenase metabolites leukotrienes (LT) B4, C4, D4, and E4 by high performance liquid chromatography. During normoxia, all three cyclooxygenase metabolites were present in perfusate, but only PGI2 and thromboxane A2 were present in lung homogenate supernatant. Prostacyclin constituted 50% of all the cyclooxygenase products measured. LTC4 and LTD4 were detected in both perfusate and lung homogenate supernatant with little production of LTE4 and LTB4. During hypoxia, the profile of cyclooxygenase products was unchanged and prostacyclin production was not increased. However, the profile of leukotriene metabolites was altered. LTC4 synthesis was markedly reduced. The synthesis of LTE4 and LTB4 was increased 10-fold, with most of the leukotrienes being retained in lung tissue. We conclude that hypoxia significantly alters leukotriene metabolism of endogenous arachidonic acid by calcium ionophore-stimulated lungs. The increased production by stimulated lungs during hypoxia of LTE4, a substance that may increase lung capillary permeability, and that of LTB4, a powerful chemoattractant, may be important contributing factors to lung injury.     

Phospholipase A2 (PLA2) activity in rabbit chondrocytes

The effects of recombinant interleukin-1 (rIL-1), recombinant tumor necrosis factor (rTNF) and two growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on PLA₂ activity and prostaglandin E₂ (PGE₂) release were investigated using rabbit chondrocytes. Cellular PLA₂ activity increased 2–10×above controls in the presence of 8×10^–12 M (5 U) rIL-1 or 5×10^–9 M rTNF after 20 hr incubation. PLA₂ activity remained constant with 1–50 ng/ml of either growth factor. PGE₂ release significantly increased (p<0.05) when the chondrocytes were incubated with rIL-1, bFGF and EGF alone, but not with rTNF above. These data suggest PLA₂ activity and PGE₂ release are not coordinately regulated in rabbit chondrocytes.     

Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1, sn-2 deacylation of phospholipids

Previously, we showed that arachidonic acid (AA) stimulates Ca2+ currents in pathogenic Trypanosoma brucei (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid. Biochem. J 1998;336:659-666). Here we examine the mechanism used by T. brucei to release AA from the sn-2 position of diacyl glycero-phospholipids. We report that T. brucei accomplishes this feat in the apparent absence of phospholipase A2 (PLA2). Instead, deacylation is initiated at the sn-1 position, followed by acyl migration and hydrolysis with LysoPLA. Neither whole cell homogenates nor enriched protein fractions could release AA from substrates whose sn-1 position contained a non-hydrolyzable alkyl ether linkage. These same fractions however, released AA from ester linked phospholipids, and TLC analysis of the reaction products supported the sequential deacylation process. The release of sn-2 AA from 1-palmitoyl-2-[1-14C]arachidonyl-sn-glycero-3-PC was linear up to 90 min at an average rate of 50 nmol x min(-1) x mg(-1). sn-2 AA was processed more efficiently than sn-2 palmitate. The reaction was also greatest for: LysoPC>diacyl-PC (sn-1 labeled)>diacyl-PC (sn-2 labeled). Product formation was sensitive to polar head group, and PI was processed at less than 10% the rate of PC or PE. The enzymatic deacylation was inhibited by the serine specific reagent, methyl arachidonyl fluorophosphonate (MAFP) and the cysteine reagent N-ethylmaleimide (NEM). Both NEM and MAFP inhibited LysoPLA activity under conditions where there was little effect on PLA1 activity. Overall, we conclude that T. brucei can release AA from diacyl glycero-phospholipids by a sequential deacylation process. Two independent active sites appear to be involved. Interestingly, a high percentage of inner leaflet phospholipids are protected from degradation since they occur in the non-hydrolyzable 1-alkyl ether form.     

Cellular interactions and exaggerated arachidonic acid metabolism in rabbit renal injury

Cell cultures from explants of the rabbit hydronephrotic kidney (HNK) cortex consisted of fibroblasts and an esterase-positive cell that phagocytizes zymosan. Cortical cell cultures from the contralateral kidney (CLK) contained only the fibroblast. The HNK cultures exhibited an endotoxin-induced prostaglandin (PG) E2 (three - fourfold) release indicative of the presence of macrophages, whereas no response was observed in the CLK cultures. At bradykinin concentrations as low as 10(-9)M there was a 20-fold stimulation of PGE2 from the HNK cultures and a sevenfold stimulation in the CLK cultures. The heterogeneous population of cells in the HNK cultures was separated using a mild trypsin treatment which permits passage of only the fibroblasts. The HNK-passaged cultures contained no phagocytic cells and did not release PGE2 in response to endotoxin. The passaged HNK cultures released less PGE2 in response to bradykinin as compared to primary cultures and had a decreased cyclooxygenase activity as determined by exogenous arachidonic acid conversion to PGE2. Conditioned media from adherent rabbit peripheral blood mononuclear cells stimulated basal PGE2 production (two - threefold) from both the HNK and CLK cultures. These findings demonstrated the similarity of the PGE2 production by cultured HNK cortical cells as compared to the ex vivo perfused HNK.     

Regulation of arachidonic acid release and cytosolic phospholipase A2 activation

The 85-kDa cytosolic PLA2 (cPLA2) mediates agonist-induced arachidonic acid release in many cell models, including mouse peritoneal macrophages. cPLA2 is regulated by an increase in intracellular calcium, which binds to an amino-terminal C2 domain and induces its translocation to the nuclear envelope and endoplasmic reticulum. Phosphorylation of cPLA2 on S505 by mitogen-activated protein kinases (MAPK) also contributes to activation. In macrophages, zymosan induces a transient increase in intracellular calcium and activation of MAPK, which together fully activate cPLA2 and synergistically promote arachidonic acid release. There are alternative pathways for regulating cPLA2 in macrophages because PMA and okadaic acid induce arachidonic acid release without increasing calcium. The baculovirus expression system is a useful model to study cPLA2 activation. Sf9 cells expressing cPLA2 release arachidonic acid to either A23187 or okadaic acid. cPLA2 is phosphorylated on multiple sites in Sf9 cells, and phosphorylation of S727 is preferentially induced by okadaic acid. However, the phosphorylation sites are non-essential and only S505 phosphorylation partially contributes to cPLA2 activation in this model. Although okadaic acid does not increase intracellular calcium in Sf9 cells, calcium binding by the C2 domain is necessary for arachidonic acid release. A23187 and okadaic acid activate cPLA2 by different mechanisms, yet both induce translocation to the nuclear envelope in Sf9 cells. The results demonstrate that alternative regulatory pathways can lead to cPLA2 activation and arachidonic acid release.     

Internalization of interleukin 1 (IL 1) correlates with IL 1-induced IL 2 receptor expression and IL 2 secretion of EL4 thymoma cells

The cytokine interleukin 1 (IL 1) plays an important role in the induction of IL 2 secretion and high-affinity IL 2 receptor (IL 2R) expression by T cells. The events that follow binding of IL 1 to IL 1R, however, are still unknown. In this study we describe two variants of the murine thymoma EL4 (5D3 and D6/76) that express comparable numbers of cell surface IL 1 receptors and bind IL 1 with the same affinity, but show distinct IL 1-dependent IL 2 secretion and IL 2R expression. In the presence of the tumor promoter phorbol 12-myristate 13-acetate IL 1 augments IL 2 secretion and IL 2R expression of EL4 5D3 but not of EL4 D6/76 cells. Comparison of the internalization of IL 1 by both clones revealed that EL4 D6/76 was unable to transport cell surface-bound IL 1 to the cytoplasm. These findings suggest that internalization of receptor-bound IL 1 is required for the action of this cytokine.     

Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin-3-dependent macrophage-like cell line.

An interleukin 3 (IL-3)-dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)-prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca(2+)-dependent and alkaline pH-optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5-10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane-associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL-3-dependent M phi-like cells.     

Regulation and metabolism of arachidonic acid

Summary The metabolism of AA reflects a carefully balanced series of biochemical pathways. The level of free arachidonate in a cells is controlled byde novo synthesis, dietary uptake, and transcellular metabolism. Lysophospholipids are key controlling substrates for a variety of acyl transferase and transacylase reactions, whose combined effect is to remodel cellular membranes placing AA in up to 20 different molecular species of phospholipids. PLA₂ enzymes, both cytosolic and secretory, can release AA for subsequent metabolism via lipoxygenase, COX, and cytochrome P450 enzymes into a variety of eicosanoid products. Reactions are often tissue- and cell-specific, and provide a spectrum of inflammatory mediator release in which many of the molecular details remain to be elucidated.     

Differential release of mediators from human basophils: differences in arachidonic acid metabolism following activation by unrelated stimuli

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n = 16) challenged with 0.1 micrograms/ml anti-IgE released 38 +/- 4% of their available histamine and 39 +/- 12 ng LTC4/10(6) basophils within 15-30 min. F-Met peptide (n = 8) caused the release of 54 +/- 8% histamine and 42 +/- 25 ng LTC4/10(6) basophils within a period of 2-5 min. C5a caused the release of 22 +/- 3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67 +/- 8 and 82 +/- 11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti-IgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 microM [3H]AA incorporated 290 pmol/10(6) cells, or 32 +/- 5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73 +/- 5%), with 20 +/- 3% as neutral lipid, and only 5 +/- 2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24 +/- 2%), phosphatidylinositol, PI (22 +/- 1%), and phosphatidylethanolamine, PE (15 +/- 3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 micrograms/ml anti-IgE revealed [3H]LTC4 (2.4 +/- 1.0%), [3H]5HETE (1.0 +/- 0.1%), unmetabolized [3H]AA (91 +/- 2%), and an unidentified peak (3.4 +/- 1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil-derived prostaglandin.     

Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids

A group of flavonoids isolated from medicinal plants and which are selective inhibitors of lipoxygenase activityin vitro: sideritoflavone, cirsiliol, hypolaetin-8-O--d-glucoside, hypolaetin, oroxindin, quercetagetin-7-O--d-glucoside, gossypin, hibifolin and gossypetin, besides leucocyanidol, have been studied for their effects on acute responses induced by carrageenin in mice. The oral administration of flavonoids to mice inhibited dose-dependently the development of paw oedema at 1, 3 and 5 h after carrageenin injection. A similar administration of flavonoids induced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenin into mice. Some of the flavonoids exhibited a potency against leukocyte infiltration similar to that seen for inhibition of carrageenin oedema at 3 h of induction. In agreement with data reported in rats, indomethacin was much more effective on inhibition of prostaglandin E₂ (PGE₂) formation than on leukocyte infiltration in mice. The selectivity of flavonoids towards lipoxygenase is not retainedin vivo since they behave as dual inhibitors of PGE₂ and leukotriene B₄ (LTB₄) formation in peritoneal exudates. Our data support the inhibition of arachidonic acid metabolism as one of the mechanisms by which flavonoids exert their anti-inflammatory effects.     

Cell type-dependent activation of 5-lipoxygenase by arachidonic acid

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. We show that stimulation of polymorphonuclear leukocytes (PMNL), rat basophilic leukemia (RBL)-1, or transfected HeLa cells with arachidonic acid (AA) caused prominent 5-LO product formation that coincided with the activity of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase. 5-LO product formation in AA-stimulated PMNL and RBL-1 cells was independent of Ca2+. However, in HeLa cells expressing a 5-LO mutant lacking potential 5-LO phosphorylation sites, removal of Ca2+ caused a prominent loss of 5-LO activity. For Mono Mac 6 (MM6) cells, AA failed to activate ERKs, and AA-induced 5-LO product formation was only minute. Also, activation of ERKs by phorbol esters did not lead to prominent 5-LO product synthesis. Instead, 5-LO activation in MM6 cells required Ca2+ or alternative signaling pathways induced by hyperosmotic stress. In summary, mechanisms for activation of 5-LO differ considerably between cell types.     

Synergistic activation of human T cells by interleukin 1 and interleukin 6

Purified human interleukin 6 (IL 6) was found to stimulate the proliferation of human tonsillar and peripheral rosetting T cells subliminally activated with phytohemagglutinin (PHA). This response seemed independent of IL 2 but highly dependent on the presence of accessory cells. Indeed, when accessory cell-depleted tonsillar T cells were activated with PHA and exposed to IL 6, only minimal proliferations were observed. A similar result was obtained with IL 1. However, a combination of these two cytokines induced strong proliferations, indicating that IL 1 and IL 6 plays a synergistic role in the interactions between accessory cells and T lymphocytes.     

Dysregulation of arachidonic acid release and metabolism by atopic mononuclear cells

We studied the ability of monocytes to metabolize [3H]arachidonic acid (AA) provided exogenously by activated T cells, and the extent to which dexamethasone suppressed eicosanoid production by normal and atopic cells. [3H]AA metabolites were identified using a reverse-phase high pressure liquid chromatography system (HPLC). Unstimulated and PHA-stimulated T cells from normal and atopic subjects exhibited a similar uptake and time-dependent release of radiolabel, 90% of which was identified as free AA. The addition of autologous normal and atopic monocytes to these cultures enhanced the release of radiolabel, even in the absence of stimulation with mitogen. Atopic T cell/monocyte cultures released significantly (P = 0.046) more radiolabel than normal cells when stimulated with PHA. Furthermore, the monocytes from both normal and atopic subjects metabolized T cell derived [3H]AA into cyclo-oxygenase (CO) and lipoxygenase (LO) products. Under unstimulated conditions, atopic cells produced significantly (P = 0.04) less CO products than normal cells. In contrast, under PHA and calcium ionophore-stimulated conditions, the atopic cells produced significantly (P = 0.048) more prostaglandins than normal donor cells. Furthermore, although the total release of radioactivity was comparable in both groups, significantly less (P = 0.02) free AA remained in ionophore-stimulated culture supernatants from atopic cells. In order to study the regulation of AA release by normal and atopic T cells, dexamethasone (1 microM) was added to T cell cultures. Dexamethasone inhibited the release of [3H]AA from normal T cells to a significantly (P = 0.003) greater extent than it did to atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of arachidonic acid and inhibitors of arachidonic acid metabolism on phagocyte-induced sister chromatid exchanges

Stimulated human phagocytes produce toxic oxygen radicals which induce sister chromatid exchanges in cultured mammalian cells. Oxidative damage to membranes initiates lipid peroxidation chain reactions and stimulation of the arachidonic acid cascade. The products of these reactions may mediate the genetic toxicity of oxygen radicals. Arachidonic acid significantly augmented the number of sister chromatid exchanges in target cells exposed to stimulated phagocytes. This genetic damage was abrogated in radical-treated cells preincubated with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (nordihydroguaiaretic acid) or both (piroxicam) pathways.     

Interleukin-1 inhibits keratan sulfate production by rabbit chondrocytes: Possible role of prostaglandin E2

We investigated the importance of prostaglandin E2 (PGE2) release in interleukin-1 (IL-1)-induced inhibition of aggrecan synthesis by chondrocytes. Keratan sulfate (KS) production was measured in parallel with PGE2 release in chondrocytes. IL-1 inhibited KS production and stimulated PGE2 release. In the presence of PGE2, there was a dosedependent decrease in baseline KS production. Indomethacin and dexamethasone partially blocked the IL-1-induced PGE2 release while KS production recovered. Our results suggest that IL-1 inhibits KS production, in part, by stimulating the release of PGE2.     

Immunorecognition of chondrocytes in articular cartilage activated by synovial interleukin 1.

A polyclonal antiserum which recognizes surface epitopes on IL1-activated pig chondrocytes has been used to immunolocalize chondrocytes responding to IL1 produced during co-culture of pig synovium and articular cartilage. Activation of the chondrocytes by the cytokine was restricted to the articular and subarticular region of the cartilage adjacent to the synovium. Chondrocyte activation was also seen when human rheumatoid synovium was co-cultured with the cartilage. The presence of IL1 in some synovial cells was confirmed by immunolocalization using antisera specific for IL1 alpha and IL1 beta.