Nitric oxide stimulated vascular smooth muscle cells undergo apoptosis induced in part by arachidonic acid derived eicosanoids

The role of eicosanoids in atherogenesis has not been thoroughly explained. This is partly due to the numerous eicosanoids and the variable effects that each has on different systems. Apoptosis of vascular smooth muscle cells has been shown to play a role in the atherosclerotic disease leading to lesion formation and further destabilization of the formed lesion. In this study, we have investigated the role of arachidonic acid derived eicosanoids in nitric oxide (NO)-stimulated vascular smooth muscle cells. We have shown previously that the nitric oxide (NO)-induced apoptosis of vascular smooth muscle cells was accompanied by arachidonic acid release via cytoplasmic phospholipase A(2) (cPLA(2)) activation. Also, arachidonic acid, but not oleic acid, induced apoptosis of these cells at low concentrations (5-10 microM). Our results revealed that the cPLA(2) specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3)), blocked NO-induced eicosanoid production, while the presence of arachidonic acid enhanced the ability of the cells to make prostaglandin E(2) (PGE(2)). Also, inhibitors of the cyclo-oxygenase (Cox) enzymes, such as N-[2-cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), a specific Cox-2 inhibitor, or indomethacin, a non-specific Cox inhibitor, blocked NO-induced PGE(2) production and apoptosis of vascular smooth muscle cells to the same extent, indicating that apoptosis might be induced by a Cox-2 metabolic product. In addition to these observations, the eicosanoids investigated, namely, PGE(2), PGI(2) LTB(4), and PGJ(2), showed different effects on vascular smooth muscle cells. Both PGJ(2) and LTB(4) decreased the percentage of viable cells and induced apoptosis of vascular smooth muscle cells, while PGE(2) and PGI(2) had no effect on cell viability and failed to induce apoptosis. These data suggest that eicosanoids, such as PGJ(2), but not PGE(2) or PGI(2), are involved in NO-induced apoptosis of vascular smooth muscle cells and that the eicosanoid synthesis pathways might be utilized for vascular therapeutic strategies.     

Prostaglandin modulation of airway inflammation and hyperresponsiveness in mice sensitized without adjuvant

As adjuvant during sensitization may cause unspecific immune reactions, the aim of the present study was to define the role of cyclooxygenase (COX) activity on airway inflammation and airway hyperresponsiveness (AHR) in an adjuvant-free allergic mouse model.Administration of diclofenac and indomethacin (non-selective COX inhibitors), FR122047 (COX-1 inhibitor) and lumiracoxib (selective COX-2 inhibitor) enhanced AHR. Only diclofenac and lumiracoxib reduced the inflammatory cell content of bronchoalveolar lavage (BAL). Moreover, levels of prostaglandins in BAL were reduced by indomethacin and FR122047 but were unaffected by lumiracoxib. However, compared with antigen controls, none of the COX inhibitors displayed major effects on the production of cytokines, smooth muscle mass, number of goblet cells and eosinophils, or collagen deposition in the airways.These data in mice sensitized without adjuvant support the fact that COX products have a general bronchoprotective role in allergic airway inflammation. Furthermore, the data suggest that COX-1 activity predominantly generates prostanoids in BAL, whereas COX-2 activity is associated with the accumulation of inflammatory cells in BAL. This study further supports that AHR on the one hand, and the inflammatory response and generation of prostanoids on the other, are dissociated and, at least in part, uncoupled events.     

Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid

In the lung, the contraction of smooth muscle, or bronchospasm, is generally caused by an immunologic insult resulting in mast cell degranulation and the release of histamine, slow reacting substances, and other mediators of inflammation (1). Although the immediate response is bronchospasm, continued activation of this sequence of events results in a chronic inflammatory disease. In the uterus, numerous conditions can result in smooth muscle contraction. One major pathophysiological syndrome associated with increased uterine tone and severe rhythmic contraction is primary dysmenorrhea (2). In this disease state, prostaglandins have been shown to play a major role in these contractions (3,4), and inhibitors of cyclooxygenase have proven beneficial in clinical practice (5). Both dysmenorrhea and cervical ripening have been likened to inflammatory reactions due to varying degrees of vasodilation, invasion by inflammatory cells, proliferation of fibroblasts and smooth muscle contraction (6,7). Metabolism of arachidonic acid (AA) via cyclooxygenase to prostaglandins and thromboxanes and via lipoxygenase to hydroxyeicosatetraenoic acids (HETEs) and leukotrienes is an integral part of both the acute and chronic inflammatory reaction in the lung or uterus. The material reviewed here examines the effect of endogenous leukotrienes on both the lung and uterus and suggests that other smooth muscles and pathophysiological states may be more involved with the lipoxygenase pathway of AA metabolism than previously believed.     

Lactacystin stimulates arachidonic acid metabolism in rat liver cells: effects of cell density on arachidonic acid release, PGI2 production and cyclooxygenase activity

Lactacystin, an inhibitor of proteasome activity, amplifies prostaglandin I2 production by rat liver cells stimulated by 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor-alpha or interleukin-1. Lactacystin also stimulates the cell's release of arachidonic acid (AA) and increases the cyclooxygenase activity in these cells. In serum deprived cells, the enhanced AA release is reduced, cyclooxygenase activity on exogenous AA is increased and endogenous production of prostaglandin I2 is unchanged. These findings suggest that, in vivo, the ratio of dividing to quiescent cells in a tissue may influence eicosanoid production. The increases in prostaglandin I2 production, AA release and cyclooxygenase activity on exogenous AA resulting from the combined lactacystin and 12-O-tetradecanoylphorbol-13-acetate treatment are inhibited by actinomycin or cycloheximide.     

The stimulations of arachidonic acid metabolism by recombinant murine interleukin 1 and tumor promoters or 1-oleoyl-2-acetyl-glycerol are synergistic

Recombinant murine IL 1 stimulated arachidonic acid metabolism by rat liver cells (the C-9 cell line) and squirrel monkey smooth muscle cells, and in the presence of tumor promoters this stimulation was synergistic. In the rat liver cells that had been prelabeled with [3H]arachidonic acid, the release of 6-keto-PGF1 alpha and arachidonic acid also was stimulated by the IL 1, and this release was synergistic in the presence of TPA. 1-Oleoyl-2-acetyl-glycerol (OAG) stimulated prostaglandin production, and IL 1 synergized the prostaglandin production in the presence of OAG. OAG and TPA mimic the endogenous activator of protein kinase C, 1,2-diacylglycerol, and therefore IL 1 may amplify arachidonic acid metabolism during signal transmission processes.     

Cholecystokinin (CCK) down regulates PGE2 and PGI2 release in inflamed Guinea pig gallbladder smooth muscle cell cultures

This study examines the hypothesis that cholecystitis down-regulates Guinea pig gallbladder (GPGB) smooth muscle cholecystokinin (CCK)-stimulated prostaglandin (PG) release. Guinea pig gallbladder from Control and 48 h bile duct ligated (BDL) animals were placed in cell culture and grown to confluence. The cultures underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacyclin Synthase (PS), or were incubated with CCK at 10(-8)M or 10(-6)M with and without indomethacin for 1h and analyzed for release of 6-keto-PGF1alpha, PGE2 and TxB2 by EIA. BDL increased Guinea pig gallbladder cell culture basal PGE2 and PGI2 release which was in part due to increased COX-2 content. CCK incubation down-regulated BDL Guinea pig gallbladder cell culture release of 6-keto-PGF1alpha and PGE2 and down-regulated COX-2 content but did not alter the Control group. The decrease in CCK-mediated BDL cell Guinea pig gallbladder release may be an endogenous mechanism to limit physiologic derangements induced by increased endogenous gallbladder PG synthesis during early acute cholecystitis.     

The role of selective cyclooxygenase isoforms in human intestinal smooth muscle cell stimulated prostanoid formation and proliferation.

Intestinal smooth muscle plays a major role in the repair of injured intestine and contributes to the prostanoid pool during intestinal inflammatory states. Cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostanoids exists in two isoforms, COX-1 and COX-2. The purpose of this study was to determine the relative contributions of COX-1 and COX-2 in the production of prostanoids by human intestinal smooth muscle (HISM) cells when stimulated by interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS). Furthermore the effects of specific COX-1 and COX-2 inhibitors on the proliferation of smooth muscle cells was also evaluated. Confluent monolayer cultures of HISM cells were incubated with IL-1beta or LPS for 0-24h while control cells received medium alone. PGE2 and PGI2 as 6-keto-PGF1alpha and LTB4 were measured by a specific radioimmunoassay. COX enzymes were evaluated by Western immunoblotting. Unstimulated and stimulated cells were exposed to the specific COX-1 inhibitor valerylsalicylic acid (VSA) and the COX-2 inhibitors NS-398 and SC-58125. The effects of serum on proliferation were then evaluated in the presence of each of the specific COX inhibitors by incorporation of 3H-thymidine into DNA. IL-1beta and LPS increased both PGE2 and 6-keto-PGF1alpha in a dose dependent fashion with enhanced production detected two hours following exposure. Neither stimulus stimulated LTB4 release. Immunoblot analysis using isoform-specific antibodies showed that both COX-1 and COX-2 were present constitutively. Furthermore, COX-1 was upregulated by each inflammatory stimulus. In a separate set of experiments cells were pretreated with either the selective COX-1 inhibitor VSA or the selective COX-2 inhibitors NS-398 or SC-58125 prior to treatment with IL-1beta or LPS. The COX-1 and COX-2 inhibitors decreased both basal and IL-1beta and LPS stimulated prostanoid release. Spontaneous DNA synthesis was present and serum consistently increased proliferation. 3H-thymidine incorporation, stimulated by serum, was inhibited by both COX-1 and COX-2 inhibitors. This study suggests that the prostanoid response stimulated by proinflammatory agents of gut-derived smooth muscle cells appears to be mediated by both COX-1 and COX-2 enzymes. Proliferation of smooth muscles cells also appears to be influenced by both COX-1 and COX-2.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.     

CLA isomers inhibit TNFalpha-induced eicosanoid release from human vascular smooth muscle cells via a PPARgamma ligand-like action

Conjugated linoleic acids (CLAs) were reported to have anti-atherogenic properties in animal feeding experiments. In an attempt to elucidate the molecular mechanisms of these anti-atherogenic effects, the modulatory potential of CLA on cytokine-induced eicosanoid production from smooth muscle cells (SMCs), which contributes to the chronic inflammatory response associated with atherosclerosis, has been investigated in the present study. cis-9, trans-11 CLA and trans-10, cis-12 CLA were shown to reduce proportions of the eicosanoid precursor arachidonic acid in SMC total lipids and to inhibit cytokine-induced NF-kappaB DNA-binding activity, mRNA levels of inducible enzymes involved in eicosanoid formation (cPLA2, COX-2, mPGES), and the production of the prostaglandins PGE2 and PGI2 by TNFalpha-stimulated SMCs in a dose-dependent manner. The effect of 50 micromol/L of either CLA isomer was as effective as 10 micromol/L of the PPARgamma agonist troglitazone in terms of inhibiting the TNFalpha-stimulated eicosanoid production by SMCs. PPARgamma DNA-binding activity was increased by both CLA isomers compared to control cells. Moreover, it was shown that the PPARgamma antagonist T0070907 partially abrogated the inhibitory action of CLA isomers on cytokine-induced eicosanoid production and NF-kappaB DNA-binding activity by vascular SMCs suggesting that PPARgamma signalling is at least partially involved in the action of CLA in human vascular SMCs. With respect to the effects of CLA on experimental atherosclerosis, our findings suggest that the anti-inflammatory effect of CLA is at least partially responsible for the anti-atherogenic effects of CLA observed in vivo.     

Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells

Eicosanoid production is reduced when the nitric oxide (NO·) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO· and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO· and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100–500 µM) and 3-morpholinosydnonimine (SIN-1; 200 µM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A₂ (cPLA₂). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA₂ activation and arachidonic acid release. Thus studies investigating the role of the NO· pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease. reactive nitrogen species; prostaglandin H₂ synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A₂     

Role of hydroxyl radical in superoxide induced microsomal lipid peroxidation: Protective effect of anion channel blocker

Treatment of bovine pulmonary artery smooth muscle microsomes with the superoxide radical generating system hypoxanthine plus xanthine oxidase stimulated iron release, hydroxyl radical production and lipid peroxidation. Pretreatment of the microsomes with deferoxamine or dime thy lthiourea markedly inhibited lipid peroxidation, and prevented hydroxyl radical production without appreciably altering iron release. The superoxide radical generating system did not alter the ambient superoxide dismutase activity. However,addition of exogenous superoxide dismutase prevented superoxide radical induced iron release,hydroxyl radical production and lipid peroxidation. Simultaneous treatment of the microsomes with deferoxamine, dimethylthiourea or superoxide dismutase prevented hydroxyl radical production and liqid peroxidation. While deferoxamine or dimethylthiourea did not appreciably alter iron release, superoxide dismutase prevented iron release. However, addition of deferoxamine, dimethylthiourea or superoxide dismutase even 2 min after treatment did not significantly inhibit lipid peroxidation, hydroxyl radical production and iron release. Pretreatment of microsomes with the anion channel blocker 4,4’- dithiocyano 2,′- disulphonic acid stilbine did not cause any discernible change in chemiluminiscence induced by the superoxide radical generating system but markedly inhibited lipid peroxidation without appreciably altering iron release and hydroxial radical production.     

Secretory and cytosolic phospholipase A(2)regulate the long-term cytokine-induced eicosanoid production in human keratinocytes

The involvement of cytosolic phospholipase A(2)(cPLA(2)) and secretory non-pancreatic PLA(2)(npPLA(2)) in release of arachidonic acid (AA) preceding eicosanoid formation in the human keratinocyte cell line HaCaT was examined. Interleukin 1beta (IL-1beta) and tumour necrosis factor-alpha (TNF), phorbol myristate acetate (PMA) and calcium ionophore A(23187)increased the extracellular AA release, and stimulated eicosanoid synthesis as determined by HPLC analysis. The main metabolites after stimulation with IL-1beta, PMA or A(23187)were PGE(2), an unidentified PG and LTB(4), while TNF stimulated HETE-production. Both cPLA(2)and npPLA(2)message and enzyme activity were detected in unstimulated HaCaT cells. IL-1beta, PMA and TNF increased both cPLA(2)enzyme activity and expression, but did not lead to any increase in npPLA(2)expression or activity. The selective npPLA(2)inhibitors LY311727 and 12-epi-scalaradial, or the cPLA(2)inhibitor arachidonyl trifluoro methyl ketone (AACOCF(3)) reduced IL-1beta-induced eicosanoid production in a concentration dependent manner. The results presented strongly suggest that both cPLA(2)and npPLA(2)contribute to the long-term generation of AA preceding eicosanoid production in differentiated, human keratinocytes. Inhibitors against npPLA2 or cPLA2 enzymes should be useful in treating inflammatory skin diseases, such as psoriasis.     

Essential fatty acid deficiency inhibits early but not late leukocyte infiltration in rabbit myocardial infarcts

Essential fatty acid (EFA) deficiency, induced by elimination of the dietary (n-6) fatty acids, has been shown to limit inflammatory cell influx and consequent enhanced eicosanoid production in experimental glomerulonephritis and hydronephrosis. To determine whether EFA-deficiency exerts anti-inflammatory effects following left ventricular myocardial infarction (LVMI), male weanling rabbits were fed EFA-deficient diet for 3 months prior to 60 minutes of distal left circumflex coronary artery occlusion followed by reperfusion. One and 4 days later, corresponding to infiltration of cardiac tissue with polymorphonuclear (PMN) and mononuclear leukocytes respectively, infarcted hearts were buffer perfused and stimulated to produce eicosanoids with f-met-leu-phe or bradykinin. One day following LVMI, the hearts of EFA-deficient rabbits demonstrated a marked suppression of PMN infiltration and eicosanoid production relative to controls. Four days following myocardial infarction, no differences were observed in mononuclear cell invasion, collagen deposition, or eicosanoid production between EFA-deficient and normal hearts. Our data show that EFA-deficiency inhibits PMN influx and consequent enhanced eicosanoid production without affecting the later appearance of mononuclear cells, collagen deposition, or eicosanoid production. Recent studies have shown that suppression of PMN invasion limits the extent of tissue damage following LVMI. Selective inhibition of PMN infiltration is possible and may be useful in the management of acute myocardial infarction.     

Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-alpha (GRO-alpha) from ASMC induced by cytokines and the role of MAPK and NF-kappaB pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1beta and TNF-alpha after growth arrest. GRO-alpha release, measured by ELISA, was increased by >50-fold after IL-1beta (0.1 ng/ml) or 5-fold after TNF-alpha (1 ng/ml) in a dose- and time-dependent manner. GRO-alpha release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1beta and TNF-alpha also induced GRO-alpha mRNA expression. Supernatants from IL-1beta-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-alpha blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-alpha release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-alpha release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1beta and TNF-alpha. By chromatin immunoprecipitation assay, IL-1beta and TNF-alpha enhanced p65 binding to the GRO-alpha promoter, which was inhibited by AS-602868. IL-1beta- and TNF-alpha-stimulated expression of GRO-alpha from ASMC is regulated by independent pathways involving NF-kappaB activation and ERK and JNK pathways. GRO-alpha released from ASMC participates in neutrophil chemotaxis.     

Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187

The release of prostanoids from the three different vascular cell types derived from rat aortic explants has been studied in vitro. Under resting conditions and when incubated with exogenous arachidonic acid (AA, 10 microM), the endothelial cells (EC) produced the highest concentration of prostacyclin (PGI2 PGE2 PGF2 alpha TxA2). In contrast, PGE2 was the major prostanoid produced by the smooth muscle cells and fibroblasts. Pretreatment of EC with aspirin (10 microM) or indomethacin (10 microM) effectively inhibited the production of prostanoids by these cells. Incubation with the calcium ionophore A23187 (10 microM) did not stimulate production of PGI2 or leukotriene B4 (LTB4) by EC. However, treatment of EC with a combination of A23187 and AA led to production of amounts of both PGI2 and LTB4 which were greater than the summed values for the different drug treatments. These findings indicate that the concentration of substrate, AA, is a limiting factor in prostanoid formation by these cultured vascular cells but that rat EC are relatively poor in the enzymes required for leukotriene formation.     

Dietary gamma-linolenic acid dose-dependently modifies fatty acid composition and immune parameters in rats.

gamma-linolenic acid (GLA) has been reported to improve several inflammatory disorders through regulation of eicosanoid production. However, since GLA is a precursor of arachidonic acid, it may bring about increasing tissue arachidonic acid levels with subsequent pro-inflammatory events. To explore this possibility, we examined the effect of high-dose GLA acid on the fatty acid profile of immune cells, leukotriene B4 production by peritoneal exudate cells and immunoglobulin productivity of mesenteric lymph node lymphocytes of Sprague-Dawley rats. Male rats were fed 10% fat diets containing graded levels, 0, 20, 40 and 60% of GLA for 3 weeks. The results showed the distinction in activity of metabolizing GLA between immune cells and liver. Thus, in immune cells such as mesenteric lymph node and spleen lymphocytes and peritoneal exudate cells, more dihomo-gamma-linolenic acid was found than in the liver. Leukotriene B4 production by peritoneal exudate cells was significantly suppressed when fed the highest level of GLA suggesting a lower risk of allergic reaction. Moreover, immunoglobulin productivity in mesenteric lymph node lymphocytes was promoted by dietary GLA. The present study indicates that a high dose of GLA may exert anti-inflammatory effects through suppression of leukotriene B4 release and strengthening of gut immune system, thus ameliorating allergic reaction.     

Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures

This study examines the hypothesis that PAF stimulates release of PGI₂ from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF_1α from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF_1α was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF_1α release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI₂ release.     

CLA isomers inhibit TNFα-induced eicosanoid release from human vascular smooth muscle cells via a PPARγ ligand-like action

Conjugated linoleic acids (CLAs) were reported to have anti-atherogenic properties in animal feeding experiments. In an attempt to elucidate the molecular mechanisms of these anti-atherogenic effects, the modulatory potential of CLA on cytokine-induced eicosanoid production from smooth muscle cells (SMCs), which contributes to the chronic inflammatory response associated with atherosclerosis, has been investigated in the present study. cis-9, trans-11 CLA and trans-10, cis-12 CLA were shown to reduce proportions of the eicosanoid precursor arachidonic acid in SMC total lipids and to inhibit cytokine-induced NF-κB DNA-binding activity, mRNA levels of inducible enzymes involved in eicosanoid formation (cPLA₂, COX-2, mPGES), and the production of the prostaglandins PGE₂ and PGI₂ by TNFα-stimulated SMCs in a dose-dependent manner. The effect of 50 μmol/L of either CLA isomer was as effective as 10 μmol/L of the PPARγ agonist troglitazone in terms of inhibiting the TNFα-stimulated eicosanoid production by SMCs. PPARγ DNA-binding activity was increased by both CLA isomers compared to control cells. Moreover, it was shown that the PPARγ antagonist T0070907 partially abrogated the inhibitory action of CLA isomers on cytokine-induced eicosanoid production and NF-κB DNA-binding activity by vascular SMCs suggesting that PPARγ signalling is at least partially involved in the action of CLA in human vascular SMCs. With respect to the effects of CLA on experimental atherosclerosis, our findings suggest that the anti-inflammatory effect of CLA is at least partially responsible for the anti-atherogenic effects of CLA observed in vivo.     

Glucocorticoid-mediated alterations in fluidity of rabbit cardiac muscle microvessel endothelial cell membranes: influences on eicosanoid release

Experiments were conducted to determine effects of the synthetic glucocorticoid, dexamethasone, on the lipid fluidity of cultured rabbit cardiac muscle microvessel endothelial cells and the possible role(s) for altered fluidity in the steroid inhibition of cellular eicosanoid production. Following a sixteen hour exposure to 10(-7) M dexamethasone, membranes prepared from treated cells exhibited a decreased fluidity compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH). Examination of the effects of temperature on the anisotropy values of DPH using Arrhenius plots revealed consistent differences in the steroid treated cells over the entire temperature range (40-5 degrees C). These dexamethasone-dependent fluidity changes were associated with increases in the cholesterol/phospholipid ratio of membrane lipids. Restoration of membrane fluidity to control values with the fluidizing agent, 2-(2-methoxyethoxy)ethyl-8-(cis- 2-n-octylcyclopropyl)octanoate (A2C), partially reversed dexamethasone induced inhibition of A23187-stimulated eicosanoid release. These observations suggest that at least part of dexamethasone's inhibitory actions on eicosanoid generation in microvessel endothelial cells are mediated by alterations in membrane composition and fluidity.