Inhibition of human neutrophil leukotriene B4 synthesis in essential fatty acid deficiency: Role of leukotriene a hydrolase

A female subject dependent on long-term total parenteral nutrition developed an aversion and noncompliance to a prescribed weekly lipid infusion designed to meet essential fatty acid (EFA) requirements. Fatty acids (FA) in the subject's plasma and isolated peripheral blood neurophils were analyzed in search of biochemical evidence of EFA deficiency. Neutrophil 5-lipoxygenase metabolism was examined to assess the possible effects of EFA deficiency on neutrophil eicosanoid metabolism. EFA deficiency was confirmed by marked depletion of linoleic acid (18∶2n−6) and accumulation of eicosatrienoic acid (ETrA; 20∶3n−9) in plasma and neutrophil phospholipids. In the neutrophils, ETrA comprised 5.2% of phospholipid FA (normal reference values <0.1%), and arachidonic acid (AA; 20∶4n−6) comprised 8.6% of phospholipid FA (normal reference range 10–16%). When stimulated by A23187in vitro on three separate occasions, the subject's neutrophils displayed impaired synthesis of leukotriene B₄ (LTB₄), but produced normal amounts of 5-hydroxyeicosatetraenoic acid andall-trans isomers of LTB₄ formed nonenzymatically from leukotriene A₄ (LTA₄). This pattern of synthesis suggested inhibition of LTA hydrolase and was also seen in neutrophils from healthy subjects by addition of exogenous ETrAin vitro. Comparative studies of the effects of ETrA and eico-sapentaenoic acid (20∶5n−3) on neutrophilsin vitro suggested that ETrA is the more potent inhibitor. Accumulation of ETrA, rather than depletion of AA, appears principally responsible for the observed impairment of neutrophil LTB₄ synthesis seen in this EFA-deficient subject.     

Synthesis of arachidonic acid metabolites by syrian hamster platelets and peritoneal cells

In this study, the metabolism of arachidonic acid by hamster platelets and peritoneal macrophages was assessed. Peritoneal macrophages stimulatedin vitro with the calcium ionophore A23187 or stimulatedin vivo by intraperitoneal injections of opsonized zymosan produced prostaglandin E₂, thromboxane B₂ (TxB₂) and 6-keto-prostaglandin F_1α, as determined by radioimmuno assays. Leukotriene B₄ (LTB₄), and 11- and 15-hydroxyeicosatetraenoic acids (HETE), which were identified by reverse-phase highperformance liquid chromatography coupled with diode array detection, were produced by peritoneal cells stimulatedin vitro with A23187 but were not found in the peritoneal exudate followingin vivo stimulation with opsonized zymosan. Synthesis of 11- and 15-HETE, but not LTB₄, was inhibited by 1 μM indomethacin but not by 10 μM nordihydroguaiaretic acid, which did inhibit LTB₄ synthesis. Washed hamster platelets were prepared and shown to synthesize TxB₂, 12-HETE and 12-hydroxyheptadecatrienoic acid following stimulation with thrombin. This paper is the first to report on eicosanoid metabolism in tissues related to atherosclerosis, thrombosis and inflammation in hamsters.     

Leukotriene B4 promotes phospholipid acylation in human neutrophils

The present study was undertaken to test the hypothesis that leukotriene B₄ (LTB₄) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB₄ (1.5 to 150 nM) for 1 for 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferases catalyzing acylation of the respective lysophospholipids. LTB₄ stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid but not by arachidonic acid. LTB₄ and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB₄ or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB₄-activated neutrophils reflects activation of phospholipase A₂ and acyltransferases as well as ofde novo phospholipid synthesis.     

Differential effects of lipoxygenase products on FMLP and LTB4 evoked neutrophil aggregation

There is evidence that the endogenous biosynthesis of LTB₄ is involved in the aggregation of human neutrophils induced by the chemotactic peptide f-met-leu-phe (FMLP). If LTB₄ mediates this aggregatory response, then agents which desensitize neutrophils to LTB₄ should inhibit the cellular response to FMLP. Since many lipoxygenase products modulate other neutrophil responses to LTB₄ and FMLP, we have investigated the effects of lipoxygenase products on LTB₄- and FMLP-initiated aggregation. Prior exposure to low concentrations of LTB₄ (0.5–10nM) inhibited subsequent aggregation to the same agent (50nM), but it did not influence the response to FMLP (10⁻⁷ M). Relatively high concentrations of 5-HETE (5–50μM) inhibited aggregation initiated by either stimulus. Although the hydroperoxy derivative 5-HPETE also inhibited the response to LTB₄, in the relatively narrow concentration range of 1–4μM it stimulated FMLP-induced aggregation. This latter effect was confirmed using 12 cell preparations from six separate donors; it (the activity of 5-HPETE) was not mimicked by other 5-lipoxygenase products, including LTB₄, nor the dihydroperoxide 8,15-DiHPETE. Our results indicate that neutrophil aggregation in response to LTB₄ or FMLP can be selectively potentiated or inhibited. On the basis of these data we conclude that the endogenous synthesis of LTB₄ is not directly involved in the neutrophil aggregatory response to FMLP, although the hydroperoxy intermediate 5-HPETE may act to enhance the cellular response.     

Protectin DX, a Double Lipoxygenase Product of DHA, Inhibits Both ROS Production in Human Neutrophils and Cyclooxygenase Activities

Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits in an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX)—a docosahexaenoic acid di-hydroxylated product which inhibits blood platelet aggregation—on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B₄ (LTB₄). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases.     

Plasma Phospholipid Fatty Acid and Ex Vivo Neutrophil Responses are Differentially Altered in Dogs Fed Fish- and Linseed-Oil Containing Diets at the Same n-6:n-3 Fatty Acid Ratio

The effect of diets containing either 18-carbon n-3 fatty acids (FA) or 20/22-carbon n-3 FA on canine plasma and neutrophil membrane fatty acid composition, superoxide and leukotriene B₄ and B₅ production when fed at the same n-6:n-3 fatty acid ratio was investigated. Four groups of ten dogs each were fed a low fat basal diet supplemented with safflower oil (SFO), beef tallow (BTO), linseed oil (LSO), or Menhaden fish oil (MHO) for 28 days. Dietary fat provided 40.8% of energy and the n-6:n-3 of the diets were ~100:1, 9.7:1, 0.38:1, and 0.34:1 for the SFO, BTO, LSO and MHO groups, respectively. The MHO and LSO groups had increased incorporation of EPA and DPA in both the plasma and neutrophil membranes compared to the BTO and SFO groups. DHA was observed in the MHO but not in the LSO group. Neutrophils from the MHO diet fed dogs had less LTB₄ and greater LTB₅ than the other three groups. The LSO group also showed a reduction in LTB₄ and greater LTB₅ production compared to the SFO and BTO groups. Both LSO and MHO groups had lower superoxide production compared to the SFO and BTO groups. Diets containing 18 or 20/22 carbon n-3 FA fed at the same n-6:n-3 resulted in differential incorporation of long chain n-3 FA into neutrophil membranes. Thus, fatty acid type and chain length individually affect neutrophil membrane structure and function and these effects exist independent of dietary total n-6:total n-3 FA ratios.     

Conjugated linoleic acid modulates tissue levels of chemical mediators and immunoglobulins in rats

The effects of conjugated linoleic acid (CLA) on the levels of chemical mediators in peritoneal exudate cells, spleen and lung, and the concentration of immunoglobulins in mesenteric lymph node and splenic lymphocytes and in serum were examined in rats. After feeding diets containing either 0 (control), 0.5 or 1.0% CLA for 3 wk, there was a trend toward a reduction in the release of leukotriene B₄ (LTB₄) from the exudate cells in response to the dietary CLA levels. However, CLA did not appear to affect the release of histamine. A similar dose-response pattern also was observed in splenic LTB₄, lung LTC₄ and serum prostaglandin E₂ levels, and the differences in these indices between the control and 1.0% CLA groups were all statistically significant. The reduction by CLA of the proportions of n−6 polyunsaturated fatty acids in peritoneal exudate cells and splenic lymphocyte total lipids seems to be responsible at least in part for the reduced eicosanoid levels. Splenic levels of immunoglobulin A (IgA), IgG, and IgM increased while those of IgE decreased significantly in animals fed the 1.0% CLA diet. This was reflected in the serum levels of immunoglobulins. The levels of IgA, IgG, and IgM in mesenteric lymph node lymphocytes increased in a dose-dependent manner, while IgE was reduced in those fed the higher CLA intake. However, no differences were seen in the proportion of T-lymphocyte subsets of mesenteric lymph node. These results support the view that CLA mitigates the food-induced allergic reaction.     

Oral Administration of Oleic or Linoleic Acids Modulates the Production of Inflammatory Mediators by Rat Macrophages

Oleic (OLA) and linoleic (LNA) acids are commonly consumed fatty acids and they can modulate the inflammatory response, in which macrophages play an important role. The aim of this study was to investigate the effects of these two fatty acids on the production of inflammatory mediators by macrophages. Rats received oral administration of water (control), OLA or LNA (0.22 g/kg body weight) daily for 10 days and peritoneal resident macrophages were then isolated. Subsequently, they were seeded in culture plates and the production of various inflammatory mediators was assessed. Oral administration with OLA decreased the production of IL-1β, IL-6 and CINC-2αβ by resident macrophages and LNA decreased the production of IL-1β, IL-6 and VEGF in the absence of lipopolysaccharide (LPS), although it accelerated IL-1β release and decreased IL-10 synthesis when cells were stimulated with LPS. Neither fatty acid affected the production of superoxide anion, hydrogen peroxide, nitrite, TNF-α, PGE₂, LTB₄ or 15(S)-HETE. Thus, OLA and LNA influence the production of several inflammatory mediators by macrophages.     

Dietary Free Oleic and Linoleic Acid Enhances Neutrophil Function and Modulates the Inflammatory Response in Rats

The high ingestion of oleic (OLA) and linoleic (LNA) acids by Western populations, the presence of inflammatory diseases in these populations, and the importance of neutrophils in the inflammatory process led us to investigate the effects of oral ingestion of unesterified OLA and LNA on rat neutrophil function. Pure OLA and LNA were administered by gavage over 10 days. The doses used (0.11, 0.22 and 0.44 g/kg of body weight) were based on the Western consumption of OLA and LNA. Neither fatty acid affected food, calorie or water intake. The fatty acids were not toxic to neutrophils as evaluated by cytometry using propidium iodide (membrane integrity and DNA fragmentation). Neutrophil migration in response to intraperitoneal injection of glycogen and in the air pouch assay, was elevated after administration of either OLA or LNA. This effect was associated with enhancement of rolling and increased release of the chemokine CINC-2αβ. Both fatty acids elevated l-selectin expression, whereas no effect on β₂-integrin expression was observed, as evaluated by flow cytometry. LNA increased the production of proinflammatory cytokines (IL-1β and CINC-2αβ) by neutrophils after 4 h in culture and both fatty acids decreased the release of the same cytokines after 18 h. In conclusion, OLA and LNA modulate several functions of neutrophils and can influence the inflammatory process.     

Biochemical,functional, and histochemical effects of essential fatty acid deficiency in rat kidney

The present study was designed to examine the effects of EFA deficiency (EFAD) on biochemical, functional, and structural aspects of the kidney in growing and adult rats fed a normal or EFAD diet for 9 wk after weaning. Food and fluid intake (FI), urine volume, and Na⁺ and K⁺ excretions were measured weekly from weeks 4 to 8 by placing the rats in individual metabolic cages for 24 h. At week 9, Li⁺ and a 5% water load, respectively, were administered at 14 and 1.5 h prior to glomerular and proximal tubular function studies, as assessed by 3-h creatinine (C_Cr) and Li⁺ (C_Li+) clearances. Hematocrit and urine volume; serum and urine [Cr], [Li⁺], [Na⁺], and [K⁺]; and renal FA distribution were also measured. Data [corrected to 100 g/body weight (bw) and presented as means ±SEM] were significant, at P<-0.05. Despite a similar ingestion of solids from weeks 4 to 7 (weeks 7 to 10 of life), the rats on the EFAD diet showed a decreased body weight from week 5. From weeks 4 to 8, Fl and urine volume were similar for both groups, but the Fl increased at week 6 in the EFAD group; 24-h Na⁺ and K⁺ excretions were similar at all weeks, except for an increase in the EFAD group for both ions at week 7. In the EFAD group, C_Cr and C_Li+ decreased by 27 and 56.3%, respectively (385.7±33.4 vs. 280±21.1, and 21.0±2.1 vs. 9.2±1.1 μL/min/100 g; n=9 vs. 10), the latter result suggesting increased proximal reabsorption. The 3-h Na⁺ and K⁺ excretions were similar, but the Li⁺ decreased (0.78±0.06×10⁻² vs. 0.32±0.03×10⁻² μeq/min/100 g) in the EFAD group, giving additional support to the suggestion. Renal structure was normal and similar for both groups, but the EFAD group showed a more prominent proximal tubule brush border, together with heavier periodic acid-Schiff staining in all specimens from weeks 5 to 9. In the EFAD group, FA of the n−9 and n−7 series were higher, but most of the n−6 series were lower as a percentage of total lipids in the medulla and cortex. Medullary levels of 20∶4n−6 were maintained, 22∶4n−6 declined twice, arachidonic acid was maintained, and 20∶5n−3 was lower. The EFAD diet affected glomerular function, proximal tubular structure and function, and FA distribution in the rat kidney.     

Platelet-activating factor regulates phospholipid metabolism in human neutrophils

This study extended the earlier finding that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) promotes arachidonic acid incorporation into neutrophil phosphatidylinositol (PI) and phosphatidylcholine (PC). In the present study the effect of PAF on fatty acid uptake by human neutrophils and the incorporation of extracellular linoleic acid and palmitic acid into phospholipids were investigated. Incubation of 10⁻⁷ M PAF with neutrophils and radiolabeled arachidonic acid or linoleic acid or palmitic acid for 1–10 min resulted in an increased rate of loss of label from the incubation medium. PAF stimulated the incorporation of linoleic acid and palmitic acid most significantly into PI and PC. The magnitude of stimulation was greater in PI than in PC for the incorporation of linoleic acid, and vice versa for the incorporation of palmitic acid. The positional distribution of linoleic acid and palmitic acid in PI and PC and the mass of these phospholipids were not altered in PAF-stimulated neutrophils. An increased incorporation of all three fatty acids into both diacyl and alkylacyl species of PC was demonstrated after a two minute incubation of cells with PAF. While more radioactivity was recovered in the diacyl species, the magnitude of increase of radioactivity in the alkylacyl species was more pronounced than that in the diacyl species of PC. These results suggest that both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation induced by PAF.     

Modulation of adjuvant-induced arthritis by dietary arachidonic acid in essential fatty acid-deficient rats

Controlled feeding of linoleic acid (LA) or arachidonic acid (AA) to essential fatty acid-deficient (EFAD) rats was used to define the relationship between dietary AA and the inflammatory response evoked during adjuvant-induced arthritis. Based on energy percentage, EFAD rats were fed AA at the human daily equivalent (1×; 5.5 mg/day) or 10 times that amount (10×; 55 mg/day) or, alternatively, 0.5× of LA (273 mg/day). Feeding of 0.5×LA restored the plasma level of AA to that in chow-fed controls. In contrast, feeding of 1×AA only partially restored the plasma level of AA; 10×AA was required to fully replete AA. In parallel to the degree of repletion of AA in plasma, there were accompanying decreases in the levels of palmitoleic acid, oleic acid, and Mead acid. Compared to rats fed the standard laboratory chow diet (Control), edema in the primary hind footpads was decreased by 87% in EFAD, 71% in EFAD+1×AA, 45% in EFAD+10×AA, and 30% in EFAD+0.5×LA. The decrease in edema in the footpads of EFAD rats was nearly identical to the decrease in edema in the footpads of Control rats dosed with indomethacin. Hind footpad edema correlated with the final AA plasma level and eicosanoid levels extracted from hind footpad tissue, but not with neutrophil infiltration. The data showed that 0.5×LA and 10×AA, but not 1×AA, could quickly replete AA, accompanied by the synthesis of AA-derived eicosanoids and restoration of edema. These results suggest that in humans consumption of the average daily amount of AA without concurrent ingestion of LA would not alleviate an EFAD state.     

Platelet-activating factor may participate in signal transduction processes in rabbit leukocytes

The bacterial chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), induces the generation of platelet-activating factor (PAF), the mobilization of arachidonic acid and generation of superoxide anion (O₂ ⁻) in rabbit polymorphonuclear leukocytes (PMNs). The PAF receptor antagonists, WEB 2086 (10–100 μM) and CV 6209 (1–10 μM), reduced the mobilization of arachidonic acid and the O₂ ⁻ generation in response to fMLP but not that in response to A23187. Pretreatment of PMNs with the phospholipase A₂ inhibitor, chloroquine, or the serine protease inhibitor, tosyl-phenylalanine chloromethyl ketone, reduced the fMLP-stimulated generation of PAF and also reduced the generation of O₂ ⁻. The respiratory burst induced by a submaximal concentration of phorbol myristate acetate was not affected by these compounds. These data are consistent with the suggestion that endogenous PAF may contribute to the signal transduction cascade initiated by fMLP. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May, 1989.     

Regulation of phosphatidylinositol turnover,calcium metabolism and enzyme secretion by phorbol dibutyrate in neutrophils

The action of the tumor promoter, phorbol 12,13-dibutyrate (PDBu), on rabbit peritoneal and human neutrophils is associated with stimulation of¹⁴C-arachidonic acid incorporation into phospholipids within 1–2 min. Stimulated¹⁴C-arachidonate incorporation was relatively selective for phosphatidylinositol (PI) in rabbit neutrophils. In contrast, the secretory response of human neutrophils to PDBu coincided with stimulated label incorporation into phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and PI. Significant increases in label incorporation were observed with PDBu concentrations as low as 2 nM, and the dose response of stimulated label incorporation paralleled that of evoked lysozyme secretion. A parallel, but partial, inhibition of PDBu-stimulated PI labeling and enzyme release was observed after exposing rabbit neutrophils to calcium-deprived medium, whereas calcium deprivation failed to significantly depress either of these stimulant actions of PDBu in human neutrophils. Further, in rabbit neutrophils PDBu elicited an increase in cell associated⁴⁵Ca. However, PDBu was unable to promote the incorporation of³²P orthophosphate into PI or enhance phospholipase A₂ activity in broken cells. These findings suggest that one expression of the interaction between phorbol esters and their receptors on neutrophils involves the turnover of arachidonic acid in phospholipids. This stimulated turnover of arachidonate may be a critical step in the cascade of events associated with neutrophil activation.     

Sphingosine-1-Phosphate Receptor Type 4 (S1P4) Is Differentially Regulated in Peritoneal B1 B Cells upon TLR4 Stimulation and Facilitates the Egress of Peritoneal B1a B Cells and Subsequent Accumulation of Splenic IRA B Cells under Inflammatory Conditions

Background: Gram-negative infections of the peritoneal cavity result in profound modifications of peritoneal B cell populations and induce the migration of peritoneal B cells to distant secondary lymphoid organs. However, mechanisms controlling the egress of peritoneal B cells from the peritoneal cavity and their subsequent trafficking remain incompletely understood. Sphingosine-1-phosphate (S1P)-mediated signaling controls migratory processes in numerous immune cells. The present work investigates the role of S1P-mediated signaling in peritoneal B cell trafficking under inflammatory conditions. Methods: Differential S1P receptor expression after peritoneal B cell activation was assessed semi‑quantitatively using RT-PCR in vitro. The functional implications of differential S1P₁ and S1P₄ expression were assessed by transwell migration in vitro, by adoptive peritoneal B cell transfer in a model of sterile lipopolysaccharide (LPS)‑induced peritonitis and in the polymicrobial colon ascendens stent peritonitis (CASP) model. Results: The two sphingosine-1-phosphate receptors (S1PRs) expressed in peritoneal B cell subsets S1P₁ and S1P₄ are differentially regulated upon stimulation with the TLR4 agonist LPS, but not upon PMA/ionomycin or B cell receptor (BCR) crosslinking. S1P₄ deficiency affects both the trafficking of activated peritoneal B cells to secondary lymphoid organs and the positioning of these cells within the functional compartments of the targeted organ. S1P₄ deficiency in LPS-activated peritoneal B cells results in significantly reduced numbers of splenic innate response activator B cells. Conclusions: The S1P-S1PR system is implicated in the trafficking of LPS-activated peritoneal B cells. Given the protective role of peritoneal B1a B cells in peritoneal sepsis, further experiments to investigate the impact of S1P₄-mediated signaling on the severity and mortality of peritoneal sepsis are warranted.     

Anti-Allergic Activity of a Platycodon Root Ethanol Extract

Platycodon grandiflorum (Campanulaceae) is used as traditional medicine in Asian countries. In Korean traditional medicine, Platycodon root has been widely used since ancient times as a traditional drug to treat cold, cough and asthma. However, its effects on bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanisms remain unknown. In this study, the biological effect of Platycodon root ethanol extract (PE) was evaluated in BMMC after induction of allergic mediators by phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187 (A23187) stimulation. The effect of PE on the production of several allergic mediators, such as interleukin-6 (IL-6), prostaglandin D₂ (PGD₂), leukotriene C₄ (LTC₄), β-Hexosaminidase (β-Hex) and cyclooxygenase-2 (COX-2) protein, was investigated. The results demonstrate that PE inhibits PMA + A23187 induced production of IL-6, PGD₂, LTC₄, β-Hexosaminidase and COX-2 protein. Taken together, these results indicate that PE has the potential for use in the treatment of allergy.     

Effects of phorbol esters,A23187 and vasopressin on oleate metabolism in isolated rat hepatocytes

Studies were conducted to compare the metabolic effects of vasopressin, 4β-phorbol-12-myristate-13-acetate (PMA) and A23187 on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats. Vasopressin inhibited the formation of acid-soluble products from [1-¹⁴C]oleate (0.25 mM, 0.5 mM and 1 mM), the inhibition being most marked at low (0.25 mM) concentration of oleate. Conversion of [1-¹⁴C]oleate into¹⁴CO₂ and esterified products was stimulated by vasopressin. The stimulatory effect of this hormone on¹⁴CO₂ production was most marked at high (1 mM) concentration of oleate, whereas that on [1-¹⁴C]-oleate esterification was most marked at low (0.25 mM) concentration of oleate. These vasopressin actions were abolished when hepatocytes were incubated in the absence of calcium in the medium. Our results strongly suggest that both increase in esterification and increase in oxidation to CO₂ contribute to the anti-ketogenic action of vasopressin when oleate is added as substrate, although the relative extent of their contribution varies according to the oleate concentration. The anti-ketogenic action of vasopressin was mimicked by PMA but not by A23187. PMA also caused a stimulation of [1-¹⁴C]oleate esterification although the effect was diminished at 1 mM [1-¹⁴C]oleate. A23187 failed to affect [1-¹⁴C]oleate esterification. The metabolic effects of PMA were elicited in the absence of extracellular calcium, too. Conversion of [1-¹⁴C]oleate into¹⁴CO₂ was only slightly increased by both PMA and A23187 when 1 mM [1-¹⁴C]oleate was added as substrate. The marked stimulatory effect of vasopressin on¹⁴CO₂ production from [1-¹⁴C]oleate was not reproduced even by the combination of PMA and A23187. The possible involvement of protein kinase C and calcium mobilization in the regulation of oleate metabolism is discussed.     

Functional and ultrastructural effects of essential fatty acid deficiency in kidney epithelial cells

Madin-Darby canine kidney (MDCK) epithelial cells were grown in culture medium supplemented with 1% fetal bovine serum (FBS) to provide a cell culture model of essential fatty acid deficiency (EFAD). 5,8,11-Eicosatrienoic acid (20∶3n−9) accumulated in cellular phospholipids, and arachidonic acid (20∶4) decreased. A large increase in cellular cholesterol/phospholipid ratio was observed. Hemicyst formation was greatly reduced from normal levels in the EFAD-MDCK cells. Scanning and transmission electron microscopy revealed that EFAD-MDCK were much flatter than their normal counterparts. They had much less dense surface microvilli, mitochondria and other organelles were very sparse, except in the perinuclear area, and much of the peripheral cytoplasm was amorphous. The EFAD was rapidly reversed by the addition of as little as 10 μM linoleic or arachidonic acid to the medium. Cells supplemented with 10% FBS, the usual culture condition, displayed borderline EFAD, with intermediate levels of 20∶3n−9 and 20∶4 and hemicyst formation. These studies suggest that EFAD reduces water and electrolyte transport in renal tubular epithelium.     

γ-linolenic acid-containing diet attenuates bleomycin-induced lung fibrosis in hamsters

Although bleomycin (BLM), an antineoplastic drug, is used in the treatment of a variety of tumors, the mechanism(s) that contribute to its induced lung injury and fibrosis are not fully elucidated. Since alterations in the levels of certain fatty acid metabolites have been associated with BLM-induced lung injury, we tested the effects of dietary γ-linolenic acid (GLA)-containing evening primrose oil on BLM-induced morphological alterations in the hamster lung, the marked elevation of tissue hydroxyproline (a marker for collagen synthesis), and elevated generation of arachidonic acid metabolites (marker of inflammatory mediators). Our data revealed that after 14 d of dietary GLA-containing oil (i) BLM-induced elevation of lung hydroxyproline was suppressed (P<0.05), (ii) the marked BLM-induced elevation of lung leukotriene B₄ (LTB₄) (a marker of polymorphonuclear generation of proinflammatory LTB₄) was significantly suppressed (P<0.05). The decrease in LTB₄ was accompanied by marked elevations (P<0.05) of lung prostaglandin E₁ (PGE₁) and 15-hydroxyeicosatrienoic acid (15-HETrE), both with known antiinflammatory properties. Taken together, data from these studies suggest that dietary GLA-containing oil contributes to tissue elevation of PGE₁ and 15-HETrE, which in vivo may attenuate lung inflammation and fibrosis.     

Docosahexaenoic acid and other dietary polyunsaturated fatty acids suppress leukotriene synthesis by mouse peritoneal macrophages

The efficacy of individual ω-t-3 polyunsaturated fatty acids (PUFA) in altering eicosanoid synthesis in peritoneal macrophages was studied by feeding mice for 10 days a diet containing 2 wt% fat, which included 0.5 wt% ethyl esters of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linolenic acid (LNA). Upon stimulation with calcium ionophore A23187, macrophages from these animals produced significantly lower amounts of leukotriene C₄, leukotriene B₄ and 12-hydroxyeicosatetraenoic acid, prostaglandin E₂ and 6-keto prostaglandin F_1α compared with those obtained from animals on the diets containing olive oil or safflower oil. The decrease in leukotriene synthesis was similar in the animals fed DHA, EPA or LNA diets. This depression of eicosanoids by DHA and EPA was associated with decreased levels of arachidonic acid (AA); however, LA that altered eicosanoids did not have the same effect on AA levels.