Availability Of Arachidonic Acid Research Articles

The effect of oral administration of eicosapentaenoic and docosahexaenoic acids on acute inflammation and fatty acid composition in rats.

To investigate effects of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) on acute inflammation, we fed rats either of the following four diets: an EPA-rich diet for 5 or 15 days, a DHA-rich diet for 5 or 15 days, a control diet for 5 or 15 days, and standard chow for 15 days. At the end of diets, the carrageenan-induced swelling of footpads was measured. Peritoneal cells were analyzed for their fatty acids in the phospholipid fraction. The swelling was similarly reduced in the EPA and DHA groups (p < 0.05, if fed for 15 days) compared with rats fed the control diet for 15 days. The mean proportion of arachidonic acid (AA) to the sum of highly unsaturated fatty acids was correlated (r = 0.87) to the mean degree of swelling among all dietary groups (n = 7). Effects of EPA and DHA might be explained by the reduced availability of AA for eicosanoid formation represented by the proportion of AA.

Beeinflussung der Biosynthese der Eicosanoide durch die Ernährung‐ Klinische Konsequenzen

AbstractDie Biosynthese der für den Menschen bedeutenden Eicosanoide ist von der Konzentration der Ausgangssubstanz, Arachidonsäure, sowie vom Vorhandensein von Inhibitoren, wie der Eicosapentaensäure, abhängig. Eine Hemmung der Eicosanoidbildung durch Fischölfettsäuren ist umso wirksamer, je weniger Arachidonsäure in den Zellen vorhanden ist. Die Konzentration der Arachidonsäure ist von der Umwandlungsrate der Linolsäure und der Zufuhr anderer mehrfach ungesättigter Fettsäuren abhängig, für die eine Kompetition im Stoffwechsel beschrieben ist. Entsprechende diätetische Maßnahmen Können die Eicosanoidbiosynthese manipulieren, wodurch zahlreich Erkrankungen behandelt werden können. Eine gesteigerte Biosynthese der Eicosanoide konnte für die Arteriosklerose, allergische und neoplastische Erkrankungen, die Colitis ulcerosa, den Morbus Crohn, die Psoriasis und entzündliche Gelenkerkrankungen nachgewiesen werden. Eine Ernährung, die auf eine Verminderung der Eicosanoidbiosynthese ausgerichtet ist, kann den Verlauf dieser Krankheiten bessern. Ernährungstherapeutische Maßnahmen sind eine ausreichend hohe Zufuhr an Linolsäure, die Verminderung der Arachidonsäure und die Einbeziehung langkettiger n‐3‐Fettsäuren in die Kost.

Phospholipase A2 activity in endometrium from early pregnant and non-pregnant ewes

In the ewe, synthesis of the luteolytic factor, prostaglandin F 2α, increases from day 13 to the end of the estrous cycle. Availability of free arachidonic acid is usually the rate-limiting step in prostaglandin biosynthesis. Phospholipase A 2 (PLA 2) may be the key enzyme for the hydrolysis of arachidonic acid from membrane-bound phospholipids. To investigate uterine PLA 2 activity during the estrous cycle and early pregnancy, we monitored the release of [ 14C]oleic acid from the substrate 1-palmitoyl-2-[ 14C]oleoyl-phosphorylcholine by homogenates and cytosolic fractions of endometrium from ewes on days 12, 14 and 16 of the estrous cycle or pregnancy. We observed that PLA 2 activity dropped by 58% (p<0.02) in day-16 pregnant endometrium compared to day-16 non-pregnant endometrium. We then investigated whether the reduced PLA 2 activity was due to induction of a specific inhibitor. The PLA 2-inhibitor activity was determined by monitoring the inhibition of release of [ 14C]oleic acid from the radioactive substrate by porcine pancreatic PLA 2. Inhibition by endometrial homogenates of pregnant animals of the control enzyme activity was 27% and only 14% by cyclic ones. Inhibition was dose-dependent and was as high as 53% (p<0.01) with 1 mg protein from pregnant endometrial homogenates. Endometrial PLA 2 behaved as a Michaëlian enzyme in the endometrium of day-16 cyclic ewes (Km = 79.4 μmol/l). Furthermore, the inhibitory activity from pregnant endometrium had characteristics of competitive inhibition. Our results suggest that inhibition of endometrial PLA 2 activity could occur in early pregnant ewes.

Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen.

Stimulation of heparinized blood with 1 microM formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in the formation of < 30 pmol/ml plasma of 5-lipoxygenase (5-LO) products. The preincubation of blood with 1 microgram/ml of lipopolysaccharide (LPS) (Escherichia coli 0111-B4) for 30 min before stimulation with FMLP resulted in the accumulation of 250-300 pmol of 5-LO products per ml plasma. The major products detected were leukotriene B4 and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid which were produced in equivalent amounts. The priming activity was detectable with as little as 1-10 ng LPS per ml blood and was optimal using 1-10 micrograms LPS/ml blood. The priming for 5-LO product synthesis was optimal after 20-30 min of preincubation with LPS and declined at preincubation times > 30 min. The priming effect of LPS was also observed using the complement fragment C5a or interleukin 8 as agonists. Polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells accounted for 80 and 20% of the synthesis of 5-LO products, respectively. The ability of LPS to prime isolated PMN was dependent on the presence of plasma and was inhibited by the anti-CD14 antibody IOM2, indicating a CD14-dependent priming mechanism. The priming of whole blood with tumor necrosis factor alpha (TNF-alpha) and LPS was additive and the presence of mononuclear cells did not enhance the ability of LPS to prime PMN, indicating that the priming activity of LPS is independent of LPS-induced TNF-alpha synthesis. The mechanism by which LPS enhance 5-LO product synthesis in PMN was investigated. Treatment of PMN with LPS strongly enhanced the release of arachidonic acid after stimulation with FMLP. The release of arachidonic acid was optimal 2-3 min after stimulation with FMLP, attaining levels 5-15-fold greater than those observed in unprimed cells stimulated with FMLP. These results demonstrate that LPS dramatically increases the ability of blood to generate 5-LO products, and support the putative role of leukotrienes in pathological states involving LPS.

Prostacyclin release in experimental diabetes: Effects of evening primrose oil

Alterations in release of endothelium-derived vasomotor agents could underlie microvascular and neuropathic complications in diabetes. This study examined release of the potent vasodilator prostacyclin, measured as immunoreactive 6-keto prostaglandin F 1α, from rat lung, kidney and peripheral nerve. Tissues were taken from control and streptozotocin-diabetic rats which had been treated for 8 weeks with either evening primrose oil (EPO) or, as a control for lipid intake, coconut oil (CO). Lung and kidney slices were incubated in the presence of acetylcholine (ACh), the calcium ionophore 4-Br-A23187, arachidonic acid (AA) or without agonist (basal). Segments of sciatic nerve, with their epineuria punctured, were incubated with or without 4-Br-A23187. Basal prostacyclin release form the lung was significantly higher in rats treated with EPO irrespective of diabetic state (increased by 60% in controls and by 77% in diabetics). Levels were reduced in CO-diabetics compared to EPO-controls (53% reduction) and CO-controls (30% reduction), although this did not reach statistical significance in the latter. Basal prostacyclin release was also significantly reduced in the kidney from CO-diabetics (40% reduction compared to CO-controls and 56% reduction compared to EPO-controls). In the presence of AA, lung prostacyclin release was significantly lower in CO-diabetic rats compared to all other groups (40% reduction compared to EPO-diabetics and 60% compared to both control groups) but there were no differences in renal release between any group. Prostacyclin release by nerves from CO-diabetic rats was significantly reduced (by 91–93%) compared to all other groups. Under ACh- and 4-Br-A23187-stimulated conditions release from any tissue did not differ significantly from basal. These data thus indicate reduced prostacyclin release from diabetic lung, kidney and nerve, which may be due in part to reduced cyclooxygenase activity. Deficits in release were attenuated, if not prevented, by treatment with EPO. This may further suggest a decreased availability of AA for prostacyclin production in diabetes.

Prostaglandin E2 limits arachidonic acid availability and inhibits leukotriene B4 synthesis in rat alveolar macrophages by a nonphospholipase A2 mechanism.

Prostaglandin E2 (PGE2), a potent mediator of inflammation released in large amounts by endotoxin-stimulated alveolar macrophages (AM), has been shown to inhibit leukotriene B4 (LTB4) release by activated neutrophils. We investigated the hypothesis that LTB4 synthesis by AM can be regulated by PGE2 and performed experiments to determine the biochemical site of regulation. AM obtained from Sprague-Dawley rats were preincubated with PGE2 before stimulation with the calcium ionophore A23187. LTB4, platelet-activating factor (PAF), lysoPAF, (AA), and 5-hydroxyeicosatetraenoic acid were isolated from AM cells and supernatants, then quantified by gas chromatography/electron capture negative ion mass spectrometry. Stimulated AM released 30.50 +/- 4.52 ng LTB4/10(6) cells and were inhibited by PGE2 in a dose dependent manner. PGE2 (1 microM) inhibited LTB4 synthesis by 37% and decreased release of both 5-hydroxyeicosatetraenoic acid and arachidonic acid by stimulated AM, but did not alter synthesis of PAF or lysoPAF. These mass measurements suggest that PGE2 does not affect the activity of phospholipase A2, PAF acetyltransferase, leukotriene A4 hydrolase. We conclude that PGE2 attenuates LTB4 production in alveolar macrophages by altering the activity of lipases other than phospholipase A2. PGE2-mediated inhibition of LTB4 synthesis by AM may regulate the initiation of lung inflammation.

Effect of exogenous arachidonic acid and enzyme inhibitors on placental prostanoid production

Prostanoids play an important role throughout pregnancy and during the initiation and progress of labor. The human placenta, at term, produces large quantities of prostanoids, yet little is known of the rate-limiting steps regulating their biosynthesis. In these studies, the effect of exogenous arachidonic acid and of enzyme inhibitors on the release of placental prostanoids was investigated. Basal prostaglandin E (PGE), prostaglandin F (PGF), thromboxane (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) increased from the fifth hour in culture, yet the increased PGF release did not result in further 13,14-dihydro-15-keto-PGF2 alpha (PGFM) production. Addition of varying doses of arachidonic acid (0.2-10 micrograms/ml) had no significant effect on PGFM, TxB2 or 6-keto-PGF1 alpha, although the endogenous arachidonate was similar to or much less than the doses studied. Only at the 10 micrograms/ml dose was a delayed increase of PGE and PGF observed. Incubation with indomethacin resulted in an immediate inhibition of PGE, PGF, TxB2 and 6-keto-PGF1 alpha, with a delayed inhibition of PGFM. The phospholipase A2 inhibitor, quinacrine (10 microM), had no significant effect on PGE, PGFM, TxB2 or 6-keto-PGF1 alpha. PGF was inhibited within the first hour of quinacrine exposure, but no significant inhibition was observed thereafter. Ca2+ chelator, EDTA, effected an inhibition of only 6-keto-PGF1 alpha. Nordihydroguaiaretic acid, a leukotriene inhibitor, reduced PGE release as well as 6-keto-PGF1 alpha. These data demonstrate the high biosynthetic competence of the human term placenta to produce prostanoids. This capacity does not appear to be rate-limited by arachidonic acid availability. However, the metabolism of PGF to PGFM appears to be saturated. In addition, the production of placenta prostacyclin may be affected by Ca2+ levels, as chelating agent inhibited the release of 6-keto-PGF1 alpha.

Thin layer chromatographic identification of thromboxane B2 in serum, and platelet rich plasma stimulated by arachidonic acid and collagen.

The thromboxane B2(TxB2) level in platelet rich plasma (PRP), after arachidonic acid and stimulus, and its presence in serum, may help determine abnormalities in the platelet prostaglandin pathway} For example, if no TxB2 is produced in response to any aggregating agent, this could be due to cyclo-oxygenase deficiency, thromboxane synthetase deficiency or more commonly aspirin ingestion. Similarly, TxB2 is absent from serum2 h after the ingestionof aspirin. T~ is usually measured by radioimmunoassay (RIA) and by high performance liquid chromatography (HPLC). RIA is expensive and not well suited for small batch analysis. The lack of ultraviolet absorption of Tx~ means that a derivitization stage is necessary prior to analysis by HPLC.2 The difficulties encountered in the determination of TxB2, usually mean that its estimation is rarely undertaken during platelet studies. Thin layer chromatography (TLC) can be performed at reasonable cost, with minimal equipment, and has previously been used to separate TxB2 from other prostaglandins.! Since TxB2is synthesized in large amounts in response to arachidonic acid and collagen and the serum concentration of TxB2 is of the same order as that in PRP stimulated with arachidonic acid, the simplicity and general availability of TLC would make it a useful method in the identification of TxB2 during the study of arachidonic acid metabolism in platelets.

Time-Dependent Effects of Progressive Gamma -Linolenate Feeding on Hyperphagia, Weight Gain, and Erythrocyte Fatty Acid Composition During Growth of Zucker Obese Rats

Obese Zucker rats (fa/fa) have low levels of arachidonic acid (AA) in liver phospholipids (PL). We have previously shown that a 70% gamma-linolenate concentrate (GLA; an AA intermediate) fed at a fixed dose (0.07 g/day) normalized hepatic PL AA and reduced weight gain selectively in the obese animals. In a follow-up study, 16 obese (fa/fa) and 16 lean (Fa/Fa) 4-week-old male rats were randomized into 4 groups of 8 each and gavaged daily with soybean oil (SOY) containing 55% 18:2omega6 (an AA precursor) or GLA, using a progressive dose (< or = 5% of total calories) based on body weight. A defined diet with 11% of energy as SOY was fed ad libitum for 60 days. GLA obese had lower body weight (p<0.0001) and 60-day cumulative food intake (p<0.05) compared to SOY obese, but neither parameter differed between the lean groups. For the last twenty days cumulative food intake was identical for GLA obese and SOY lean, whereas SOY obese consumed 18% more (p<0.05). Thus the progressive dose of GLA selectively suppressed hyperphagia in obese Zucker rats. Erythrocytes collected at 15-day intervals showed parallel increases in AA in both genotypes over time, suggesting normal AA availability during rapid growth. Thus, the reduced PL AA in the livers from the obese rats probably reflects impaired distribution in selected tissues rather than reduced hepatic production. Due to the potential health risks of enriching tissue lipids with AA, great caution is advised in considering GLA as therapy for human obesity.

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations

The biosynthesis of leukotrienes is known to occur through a series of complex processes which in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB 4, 20-hydroxyl-LTB 4, 5-HETE and LTC 4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and Δ 6- trans-LTB 4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB 4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Hydrogen peroxide increases the availability of arachidonic acid for oxidative metabolism by inhibiting acylation into phospholipids in the alveolar macrophage.

Reactive oxygen species stimulate metabolism of arachidonic acid (AA) to eicosanoids in a variety of cells and tissues, yet the pathway(s) by which oxidants increase the availability of AA for oxidative metabolism are not known. Thus, we explored the effects of hydrogen peroxide (H2O2) on deacylation and reacylation of AA to determine the enzymatic mechanism(s) by which this oxidant increases levels of free, unesterified AA, and thereby its oxidative metabolism to eicosanoids, in the rat alveolar macrophage (AM). Over the range from 0.1 to 0.5 mM, H2O2 caused marked time- and dose-dependent inhibition of incorporation of [3H]AA into macrophage phospholipids, whereas calcium ionophore A23187 and zymosan particles did not cause such inhibition. Within this concentration range, there was an almost exact reciprocal correlation between inhibition of [3H]AA acylation and H2O2-stimulated accumulation of free [3H]AA in prelabeled AM cultures. Thimerosal, which blocks AA reacylation but spares deacylation via phospholipase A2 (PLA2), did not affect accumulation of free [3H]AA in prelabeled cells stimulated with H2O2, while markedly augmenting [3H]AA release in response to A23187 and to zymosan. Despite its ability to block AA acylation almost completely, H2O2 did not directly inhibit arachidonoyl CoA synthetase or arachidonoyl CoA:lysophosphatide acyltransferase, which catalyze AA incorporation into phospholipids. However, H2O2 (0.1 to 0.5 mM) markedly depleted AMs of ATP, required for synthesis of the acylation intermediate arachidonoyl CoA, suggesting that this was the means by which H2O2 inhibited acylation. Notably, H2O2 (0.03 to 3 mM) failed to stimulate macrophage PLA2 activity. We conclude that H2O2, in contrast to A23187 and zymosan, inhibits incorporation of AA into phospholipids, and that this represents the major mechanism by which the oxidant increases the availability of free AA for oxidative metabolism in the AM. This may be an important basis for release of eicosanoids in oxidant-induced inflammation and injury of the lung.

Phospholipase A 2 activity in reproductive tissues of the firebrat Thermobia domestica (Insecta:Thysanura)

Upon in vitro incubation with [ 14]arachidonic acid, reproductive tissues of Thermobia domestica incorporated radioactivity, mostly into phospholipids. Stimulation with calcium ionophore A 23187 modified the distribution of the labeling between the various phospholipids, and promoted a release of arachidonic acid as well as metabolites comigrating with authentic HETEs. The lipolytic activity was tested against three exogenous phosphatidylcholines. It is concluded that arachidonic acid availability for eicosanoid biosynthesis is mostly regulated by a calcium-dependent phospholipase A 2. The specific activity of PLA 2 was assayed in reproductive tissues. After insemination, the phospholipase activity strongly increased in the female seminal receptacle, but the rise was independent of the number of transfered spermatophores.

Essential fatty acid deficiency normalizes function and histology in rat nephrotoxic nephritis.

The central lipid abnormality in essential fatty acid deficiency (EFAD) is the lack of availability of arachidonic acid. To examine the role of total eicosanoid's biosyntheses in the pathology and pathophysiology of glomerulonephritis, EFAD was induced in weanling rats, which were then subjected to antiglomerular basement membrane antibody (NTS)-induced injury in adulthood. Glomerular dynamics (as assessed by micropuncture), quantitative histology, and eicosanoid generation rates were measured at two hours and two weeks post-NTS, and compared to those of standard diet-fed (STD) controls. Two hours post-NTS, and despite the occurrence of proteinuria in both EFAD and STD animals, glomerular dynamics were essentially normal in EFAD rats, whereas STD animals had reduced values for glomerular filtration rate (GFR) and renal plasma flow rate (RPF). At two weeks, severe histologic changes were observed in STD animals including mesangial and stalk hypercellularity, moderate sclerosis, and interstitial nephritis, coupled with heavy proteinuria and reduced GFR and RPF. In dramatic contrast, EFAD rats displayed totally normal glomerular structures and functions. In parallel, glomerular generation rates of prostaglandin E2 and thromboxane A2 were suppressed markedly in EFAD rats. Thus, EFAD confers complete protection against the histopathologic and functional sequelae of immune-initiated injury in the glomerulus. The data suggest that the initial wave of complement-induced neutrophil infiltration (with resultant proteinuria) is not sufficient to perpetuate injury into the more destructive chronic phases. The results provide strong impetus for the design of more specific interventional therapies targeting the various enzymes and products of arachidonic acid metabolism in the attempts to control glomerular inflammation.

Tracheal epithelial cell fatty acid composition modulates prostaglandin E2 and cAMP production.

Tracheal epithelial (TE) cells from both rabbits and humans, when cultured in defined serum-free media without lipid supplements, develop fatty acid profiles significantly different from freshly isolated epithelium, including a markedly decreased cellular content of arachidonic acid (AA). In rabbit TE cells, supplementation of media with a phospholipid-rich lipoprotein extract (Excyte III) plus 1 microM bovine serum albumin-complexed AA (Excyte/AA) restored the fatty acid composition of the cultured cells more similar to that of native airway epithelium than did supplementation of media with 5% fetal bovine serum (FBS). In human TE cells, Excyte/AA or 5% FBS increased AA content, but neither lipid supplement completely "normalized" the fatty acid profiles. Compared with lipid-unsupplemented cultures, basal production of prostaglandin E2 (PGE2) was increased by approximately four- to eightfold in rabbit and human TE cells supplemented with 5% FBS or Excyte/AA. In Excyte/AA-supplemented human TE cells, PGE2 production induced by 5 microM calcium ionophore A23187 was more than threefold greater than that of companion ionophore-stimulated unsupplemented monolayers, but PGE2 production was similar in both culture conditions in response to 10 microM exogenous AA. Thus increased cellular content and availability of AA, rather than changes in cyclooxygenase activity, appear to be responsible for the elevated PGE2 production in Excyte/AA-supplemented human TE cells. Secondary effects of lipid supplementation were also observed; Excyte/AA-supplemented human TE cells produced significantly less adenosine 3',5'-cyclic monophosphate (cAMP) in response to exogenous PGE2 and isoproterenol than did lipid-unsupplemented cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the concentration of free arachidonic acid in homogenates of human platelets.

With the intention to study the regulation of the availability of free arachidonic acid through the enzymes of the Lands cycle, we established a model system in homogenates of human platelets. Phospholipase A2, arachidonoyl-CoA synthetase and lysophosphatidyl acyltransferase proved to be simultaneously active and a steady turnover of arachidonic acid was the consequence. EGTA suppressed the deacylating activity that acted on endogenous membrane phospholipids and prevented eicosanoid formation from previously esterified exogenous arachidonoyl-CoA. The reacylating enzymes took part in the control of eicosanoid biosynthesis by re-esterification of liberated arachidonic acid. Blockade of the reacylation by apyrase made arachidonic acid completely available for further metabolization into 12-HETE and thereby induced an increase in the eicosanoid release.

Protein kinase C activation modulates arachidonic acid metabolism in cultured alveolar epithelial cells.

Cultured alveolar type II cells can liberate esterified arachidonic acid (AA) and metabolize it predominantly via the cyclooxygenase pathway, and their capacity to do so increases as they alter their phenotype over time in culture. Little is known, however, about the regulation of AA metabolism in alveolar pneumocytes. We have examined the effects of protein kinase C (PKC) activation on arachidonate metabolism in primary cultures of rat alveolar epithelial cells studied at 2 and 7 days following isolation. The potent PKC activator phorbol myristate acetate (PMA) stimulated dose-dependent increases in free AA levels in both day 2 and day 7 cultures, with optimal stimulation at 50 nM. Greater stimulation was demonstrated for day 7 cells, and this was associated with greater prostanoid synthesis in response to PMA by day 7 than by day 2 cells. The capacity of PMA to "prime" epithelial cells for augmented AA liberation and metabolism in response to calcium ionophore A23187 (5 microM) was examined also. Significant priming by PMA was observed in both day 2 and day 7 cells; once again, augmentation of both free AA levels as well as prostaglandin E2 levels was greater for day 7 cells than for day 2 cells. That the capacity of PMA to modulate AA metabolism was mediated by activation of PKC was confirmed by demonstrating that (1) phorbol didecanoate, which lacks the ability to activate PKC, failed to activate AA metabolism; (2) pretreatment for 18 h with 1 microM PMA, which depletes cellular PKC, abolished subsequent AA metabolism activated by 50 nM PMA; and (3) the PKC inhibitor staurosporine abrogated increases in the quantities of both free AA and prostaglandin E2 in response to PMA. We conclude that activation of PKC increases the availability of AA for prostanoid synthesis in alveolar pneumocytes, and that this effect is more evident as type II cell differentiation is modeled during prolonged cultivation.

Responses of Human Dermal Microvascular Endothelial Cells to Histamine and Their Modulation by Interleukin 1 and Substance P

The action of histamine on human dermal microvascular endothelial cells and modulation of its effects by the cytokine interleukin-1 and the vasoactive neuropeptide substance P have been investigated. Histamine (10(-6)-10(-3) M) induces release of prostaglandin E2 in a concentration- and time-dependent manner. Prostaglandin E2 release is facilitated principally by histamine H1 receptors as the H1 receptor antagonist pyrilamine attenuates prostaglandin E2 release whereas the H2 receptor antagonist cimetidine only slightly reduces release. In contrast to other cells, the histamine/receptor interaction is not associated with increased intracellular accumulation of the cyclic nucleotides, cyclic AMP, or cyclic GMP. Interleukin-1 induces a concentration-dependent release of prostaglandin E2 following 24 h incubation. However, substance P does not increase release of prostaglandin E2 above baseline. In cells incubated with 1 U/ml human recombinant interleukin 1 alpha for 24 h prior to stimulation with histamine (10(-5)-10(-3) M) for 30 min, there is a significant potentiation of histamine-induced release of prostaglandin E2 (p less than 0.05). Using a solubilized cell sonicate prepared from human dermal microvascular endothelial cells incubated with 1 U/ml human recombinant interleukin 1 alpha for 24 h, conversion of exogenous arachidonic acid into prostaglandin E2 increased by 60.19 +/- 18.28%. Cycloheximide partially reduces the increased conversion but completely blocks interleukin-1-induced release of prostaglandin E2 from intact cells. Substance P does not potentiate histamine-induced release of prostaglandin E2 or increase arachidonic acid conversion. These results demonstrate that human dermal microvascular endothelial cells are responsive to histamine and that interleukin-1, but not substance P, can potentiate histamine-induced release of prostaglandin E2. Interleukin-1 appears to act, at least in part, by regulating the availability of free arachidonic acid. Interactions between histamine and interleukin-1 may be important in the modulation of inflammatory reactions in skin.

Granulocyte-macrophage colony-stimulating factor increases the synthesis of leukotriene B4 by human neutrophils in response to platelet-activating factor. Enhancement of both arachidonic acid availability and 5-lipoxygenase activation.

Granulocyte-macrophage CSF (GM-CSF) primes human neutrophils for increased functional responsiveness to a variety of inflammatory agonists. In the present report, we have investigated the effect of human GM-CSF on the ability of platelet-activating factor (PAF) to induce the synthesis of 5-lipoxygenase products in human neutrophils. Human neutrophils stimulated with PAF in the range of 10(-5) to 10(-7) M for 15 min released small quantities of leukotriene B4 and its omega-oxidation products, 20-OH- and 20-COOH-leukotriene B4 in amounts that were detectable by enzyme immunoassay. Preincubation of normal peripheral blood neutrophils with human rGM-CSF enhanced the synthesis of the 5-lipoxygenase products in a time- and dose-dependent manner. Treatment with GM-CSF enabled their detection in response to lower concentrations of PAF (greater than or equal to 10(-9) M). The PAF receptor antagonist BN52021 inhibited the synthesis of 5-lipoxygenase products by GM-CSF-treated neutrophils in response to PAF. In addition to its effect on PAF-induced leukotriene synthesis, GM-CSF also augmented intracellular calcium mobilization by PAF. This observation prompted us to examine the effect of GM-CSF on two calcium-dependent events that are essential for leukotriene synthesis, arachidonic acid liberation, and 5-lipoxygenase activation. GM-CSF by itself, did not directly activate either of these two processes, however, it consistently and markedly enhanced the ability of PAF to do so. These results indicate that preincubation of peripheral blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and 5-lipoxygenase activation in response to PAF. These observations provide additional evidence of an important role for GM-CSF in the modulation of inflammatory responses to endogenous agonists through enhancement of the production of potent cellular inflammatory mediators such as leukotrienes.

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts.

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.

Polyunsaturated fatty acids and cardiac arrhythmia

lschacmic heart disease is a major cause of death in the western world. The manifestation of this disease can be chest pain, heart attack or sudden cardiac death. Coronary heart disease (CHD) mortality is positively related to dietary saturated fat [I] . Such diets are correspondingly low in polyunsaturated fatty acids, in particular linoleic acid [2]. There is now ample support from cross-sectional and longitudinal studies that diets low in polyunsaturated fatty acids, whether of plant or fish oil origin, i.e. n 6 or ti 3 , may predispose to CHD 131. Ventricular fibrillation, a chaotic disturbance of the normal rhythm of the heart, occurs during the early, and hence potentially reversible, phase of an acute heart attack and is the principal cause of sudden cardiac death. It is not possible t o predict those who are at risk of imminent sudden cardiac death, because of the nature of the disease. Most of our current knowledge of factors predisposing to ischaemic arrhythmias has been derived from animal models. Acute myocardial ischaemia is induced by ligation of a major coronary artery and the resultant early appearance of arrhythmias is similar to that in man (heart models are widely used t o test the efficacy of anti-arrhythmic treatment in vivo or in vitro). Detailed recommendations on the conduction of such experiments have been proposed to ensure adequate group sizes, uniform definition of serious arrhythmias and awareness o f the requirement to exclude the influence of confounding factors (i.e. ischaemic area, heart rate, etc.) 141. Early studies examined the effect of large dietary supplements of linoleic acid-rich oils. Linoleic acid-rich diets, fed over varying periods ( 12 weeks12 months), reduced ischaemic ventricular fibrillation in anaesthetized and conscious rats [ 5-71. The polyunsaturated to saturated fatty acid (I ' / .S) ratios were extremely high (/'/S 6) and bear little resemblance to the human diet ( / ' / .S= 0.3), and dietary vitamin and mineral intake are not always balanced. The mechanisms of the reduction in arrhythmias is assumed to be due t o increased availability of arachidonic acid in myocardial phospholipids for prostanoid synthesis [ 71.