Differential effects of arachidonic and eicosapentaenoic Acid-derived eicosanoids on polymorphonuclear transmigration across endothelial cell cultures.

The beneficial effects of fish oil on inflammation have been attributed to the content of eicosapentaenoic (EPA)/docosahexaenoic acid. EPA is also a substrate for arachidonic acid (AA) cascade enzymes, but it induces the production of alternative eicosanoids such as 3-series prostanoids and 5-series leukotrienes, which are considered to be less proinflammatory than AA metabolites. However, the molecular basis of this action is poorly understood. In this study, we compared the effects of prostaglandin (PG) E(2) and PGE(3) on endothelium permeability, and the effects of leukotriene (LT) B(4) and LTB(5) on endothelium permeability and mononuclear adhesion and migration. In our study, both prostaglandins increased trans-endothelial Evans blue-albumin (EBA) permeability in a concentration-dependent manner. It is interesting that the effect of PGE(3) was significantly more pronounced than the effect of PGE(2), and both were antagonized by EP(1) and EP(2) antagonists. LTB(4) and LTB(5) had a slight effect on EBA extravasation. However, we observed the enhancement of endothelial permeability in the presence of polymorphonuclear (PMN) cells, probably a consequence of an interplay between leukotriene and prostanoid effects. LTB(4) caused significant increases in the number of PMN cells adhering to endothelial cells, whereas LTB(5) did not induce a significant effect. This effect of LTB(4) appears BLT1 receptor-dependent and was mediated through the enhancement of lymphocyte function-associated antigen-1, membrane attack complex-1, E-selectin, and intercellular adhesion molecule-1 expression. Finally, we observed that, unlike LTB(5), which had a weak effect, LTB(4) was a highly potent chemoattractant. An understanding of the differences in the effects of LTB(4)/LTB(5) on PMN cell adhesion and migration may help to explain the beneficial impact of omega-3 fatty acids in inflammatory processes.

Anti-atherosclerotic effects of dihomo-gamma-linolenic acid in ApoE-deficient mice.

AIM: Dihomo-gamma-linolenic acid (DGLA) is an n-6 polyunsaturated fatty acid that is mainly metabolized to an anti-inflammatory eicosanoid, prostaglandin (PG) E1, via the cyclooxygenase (COX) pathway. We evaluated the effect of DGLA on atherosclerosis in apoE-deficient mice and studied the mechanism of the anti-atherosclerotic effect. METHODS: ApoE-deficient mice were fed a normal diet supplemented with 0.5% DGLA or vehicle for 6 months. ApoE-deficient mice were also fed a high-cholesterol diet supplemented with 0.5% DGLA or vehicle for 1 month. To clarify the influence of a COX inhibitor, naproxen, on the anti-atherosclerotic effect of DGLA, age-matched apoE-deficient mice fed a high-cholesterol diet supplemented with 0.5% DGLA were given oral naproxen for 1 month. RESULTS: In normal diet-fed mice, acetylcholine-induced vascular relaxation was significantly greater in the DGLA group than in the vehicle group. NADPH oxidase subunits, p22phox and gp91phox, intercellular adhesion molecule-1, and vascular cellular adhesion molecule-1 were significantly lower in the DGLA group than in the vehicle group, and DGLA significantly prevented atherosclerosis. In high-cholesterol diet-fed mice, DGLA also significantly prevented atherosclerosis, but the anti-atherosclerotic effect was attenuated by naproxen. CONCLUSION: DGLA may have an anti-atherosclerotic effect in apoE-deficient mice via PGE1 formation.

The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases.

Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids, and their subsequent metabolism by cyclooxygenases (COXs) and lipoxygenases (LOXs) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and, for 72 h, examined expression of proinflammatory and anti-inflammatory eicosanoids using LC/ESI-MS/MS, and examined immunohistochemical expression of COX-2, 12-LOX, 15-LOX, and leukocyte markers, while quantifying clinical erythema. We show that vasodilatory prostaglandins (PGs) PGE(2), PGF(2alpha), and PGE(3) accompany the erythema in the first 24-48 h, associated with increased COX-2 expression at 24 h. Novel, potent leukocyte chemoattractants 11-, 12-, and 8-monohydroxy-eicosatetraenoic acid (HETE) are elevated from 4 to 72 h, in association with peak dermal neutrophil influx at 24 h, and increased dermal CD3(+) lymphocytes and 12- and 15-LOX expression from 24 to 72 h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72 h. Sunburn is characterized by overlapping sequential profiles of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution.

Effects of firocoxib and tepoxalin on healing in a canine gastric mucosal injury model.

BACKGROUND: Little is known about the effect of dual cyclooxygenase (COX) and lipoxygenase inhibition on canine gastric mucosal healing. OBJECTIVE: This study compares the effects of putative dual COX and 5-lipoxygenase inhibition with that of COX-2 selective inhibition on gastric mucosal lesion healing in dogs. ANIMALS: Six normal adult mixed-breed research dogs. METHODS: Gastric body and pyloric lesions were induced by endoscopic biopsy. Dogs were treated with tepoxalin, firocoxib, or placebo for 7 days in a randomized 3-way crossover study design. Healing was evaluated on days 2, 4, and 7 of treatment by endoscopic lesion scoring. Eicosanoid concentrations in plasma and at the lesion margins were determined on days 2, 4, and 7. Repeated measures analyses were performed. All hypothesis tests were 2-sided with P < .05. Multiple comparisons were adjusted using Tukey's test. RESULTS: Significant treatment differences were noted in the pyloric lesion area measurements. Overall, the firocoxib group had larger lesions than the placebo (P= .0469) or tepoxalin (P= .0089) groups. Despite larger pyloric lesions in the firocoxib group, mucosal prostaglandin production did not differ significantly from placebo. In contrast, the tepoxalin group had significantly lower pyloric mucosal prostaglandin production compared with the firocoxib (P < .0001) or the placebo (P < .0001) groups but pyloric lesions were not significantly larger than those of the placebo group (P= .7829). CONCLUSION: COX-2 inhibition by firocoxib slowed wound healing by a mechanism independent of prostaglandin synthesis. Suppression of mucosal prostaglandin production by tepoxalin did not alter mucosal lesion healing compared with placebo.

Omega-3 fatty acids (fish oil) as an anti-inflammatory: an alternative to nonsteroidal anti-inflammatory drugs for discogenic pain.

BACKGROUND: The use of NSAID medications is a well-established effective therapy for both acute and chronic nonspecific neck and back pain. Extreme complications, including gastric ulcers, bleeding, myocardial infarction, and even deaths, are associated with their use. An alternative treatment with fewer side effects that also reduces the inflammatory response and thereby reduces pain is believed to be omega-3 EFAs found in fish oil. We report our experience in a neurosurgical practice using fish oil supplements for pain relief. METHODS: From March to June 2004, 250 patients who had been seen by a neurosurgeon and were found to have nonsurgical neck or back pain were asked to take a total of 1200 mg per day of omega-3 EFAs (eicosapentaenoic acid and decosahexaenoic acid) found in fish oil supplements. A questionnaire was sent approximately 1 month after starting the supplement. RESULTS: Of the 250 patients, 125 returned the questionnaire at an average of 75 days on fish oil. Seventy-eight percent were taking 1200 mg and 22% were taking 2400 mg of EFAs. Fifty-nine percent discontinued to take their prescription NSAID medications for pain. Sixty percent stated that their overall pain was improved, and 60% stated that their joint pain had improved. Eighty percent stated they were satisfied with their improvement, and 88% stated they would continue to take the fish oil. There were no significant side effects reported. CONCLUSIONS: Our results mirror other controlled studies that compared ibuprofen and omega-3 EFAs demonstrating equivalent effect in reducing arthritic pain. omega-3 EFA fish oil supplements appear to be a safer alternative to NSAIDs for treatment of nonsurgical neck or back pain in this selective group.

Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion.

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE(1) synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE(1) synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.

Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofibroblasts.

BACKGROUND: The mucus layer protects the gastrointestinal mucosa from mechanical, chemical, and microbial challenge. Mucin 2 (MUC-2) is the most prominent mucin secreted by intestinal epithelial cells. There is accumulating evidence that subepithelial myofibroblasts regulate intestinal epithelial cell function and are an important source of prostaglandins (PG). PG enhance mucin secretion and are key players in mucoprotection. The role of bacterial fermentation products in these processes deserves further attention. AIMS: We therefore determined whether the effect of short chain fatty acids (SCFA) on MUC-2 expression involves intermediate PG production. METHODS: Both mono- and cocultures of epithelial cells and myofibroblasts were used to study the effects of SCFA on MUC-2 expression and PG synthesis. Cell culture supernatants were used to determine the role of myofibroblast derived prostaglandins in increasing MUC-2 expression in epithelial cells. RESULTS: Prostaglandin E(1) (PGE(1)) was found to be far more potent than PGE(2) in stimulating MUC-2 expression. SCFA supported a mucoprotective PG profile, reflected by an increased PGE(1)/PGE(2) ratio in myofibroblast supernatants and increased MUC-2 expression in mono- and cocultures. Incubation with indomethacin revealed the latter to be mediated by PG. CONCLUSIONS: SCFA can differentially regulate PG production, thus stimulating MUC-2 expression in intestinal epithelial cells. This mechanism involving functional interaction between myofibroblasts and epithelial cells may play an important role in the mucoprotective effect of bacterial fermentation products.

Early events of the exogenously provided L-Carnitine in murine macrophages, T- and B-lymphocytes: modulation of prostaglandin E1 and E2 production in response to arachidonic acid.

L-carnitine is an essential energy-providing compound to the cell since it transports long chain fatty acids through the mitochondrial membrane and delivers them to the beta-oxidation pathway for catabolism and/or entrance to biosynthetic pathways. Some of the early events taking place in immune cells after L-carnitine inoculation in vitro are defined in this report. Using arachidonic acid as a fatty acid source, we determined the utilization rate of L-carnitine by murine T-, B-lymphocytes and macrophages within two hours of cell culture, its effect on prostaglandin E1 and E2 production and the levels of beta-hydroxy-butyrate. The results show that although all immune cells consume a small portion of L-carnitine, beta-hydroxy-butyrate decreases upon addition of arachidonic acid and/or L-carnitine indicating that active biosynthetic pathways are induced. L-carnitine is shown to increase the arachidonic acid-induced production of prostaglandins E1 and E2 in macrophages, while their secretion from T- and B-lymphocytes is decreased. These findings indicate the L-carnitine may very rapidly alter the activation state of immune cells and lead to the development of various reactions, beneficial or not to the organism.

Effect of linoleic acid and gamma-linolenic acid on tear production, tear clearance and on the ocular surface after photorefractive keratectomy.

BACKGROUND: The aim of this study was to evaluate the effect of linoleic acid (LA) and gamma-linolenic acid (GLA), both precursors of PGE(1), on tear production, tear fluorescein clearance and on the ocular surface after photorefractive keratectomy (PRK). METHODS: Sixty subjects (age 25+/-10 years; refractive error -3+/-2 diopters (spherical equivalent), mean +/- standard deviation) undergoing PRK were enrolled. The inclusion criteria were: Schirmer 1 test >10 mm/5 min, no corneal fluorescein staining, low irritation symptoms (questionnaire score <5), standardised visual scale (to evaluate tear fluorescein clearance) score <3. Patients were randomly divided into two groups. One group of 31 subjects was treated once daily orally with tablets containing LA (28.5 mg) and GLA (15.1 mg) (from 3 days before PRK to 1 month after PRK). The control group (29 subjects) underwent PRK and received no treatment with LA and GLA. A symptoms questionnaire, Schirmer 1 test and fluorescein clearance test (FCT) using the standardised visual scale were performed before starting therapy (T(0)) and 30 days after PRK (T(1)). RESULTS: All patients completed the study. The Schirmer 1 test varied from 16.3+/-6.9 (T(0)) to 17.6+/-7.2 (T(1)) for the treated group and from 18.3+/-6.2 (T(0)) to 15.7+/-7.4 (T(1)) for the untreated group ( P<0.0001, two-tailed unpaired t-test). FCT was 1.9+/-0.6 at T(0) and 1.6+/-0.8 at T(1) for the treated group and 1.7+/-0.7 at T(0) and 2.0+/-0.9 at T(1) for the untreated group ( P<0.0001). The symptoms score was 4.7+/-1.9 at T(0) and 7.6+/-7.2 at T(1) for the treated group and 4.2+/-2.0 at T(0) and 10.1+/-7.6 at T(1) for the untreated group ( P<0.05). CONCLUSION: Reduced corneal sensitivity has already been proved after PRK. This could be the main reason for a decrease in tear production and for a reduced blinking rate leading to delayed tear clearance. The oral precursors of PGE(1), LA and GLA, could be helpful in increasing tear production and clearance after PRK.

Differential metabolism of dihomo-gamma-linolenic acid and arachidonic acid by cyclo-oxygenase-1 and cyclo-oxygenase-2: implications for cellular synthesis of prostaglandin E1 and prostaglandin E2.

Prostaglandin (PG) E(1) has been shown to possess anti-inflammatory properties and to modulate vascular reactivity. These activities are sometimes distinct from those of PGE(2), suggesting that endogenously produced PGE(1) may have some beneficial therapeutic effects compared with PGE(2). Increasing the endogenous formation of PGE(1) requires optimization of two separate processes, namely, enrichment of cellular lipids with dihomo-gamma-linolenic acid (20:3 n-6; DGLA) and effective cyclo-oxygenase-dependent oxygenation of substrate DGLA relative to arachidonic acid (AA; 20:4 n-6). DGLA and AA had similar affinities (K(m) values) and maximal reaction rates (V(max)) for cyclo-oxygenase-2 (COX-2), whereas AA was metabolized preferentially by cyclo-oxygenase-1 (COX-1). To overcome the kinetic preference of COX-1 for AA, CP-24879, a mixed Delta(5)/Delta(6) desaturase inhibitor, was used to enhance preferential accumulation of DGLA over AA in cells cultured in the presence of precursor gamma-linolenic acid (18:3 n-6). This protocol was tested in two cell lines and both yielded a DGLA/AA ratio of approx. 2.8 in the total cellular lipids. From the enzyme kinetic data, it was calculated that this ratio should offset the preference of COX-1 for AA over DGLA. PGE(1) synthesis in the DGLA-enriched cells was increased concurrent with a decline in PGE(2) formation. Nevertheless, PGE(1) synthesis was still substantially lower than that of PGE(2). It appears that employing a dietary or a combined dietary/pharmacological paradigm to augment the cellular ratio of DGLA/AA is not an effective route to enhance endogenous synthesis of PGE(1) over PGE(2), at least in cells/tissues where COX-1 predominates over COX-2.

Statins inhibit A beta-neurotoxicity in vitro and A beta-induced vasoconstriction and inflammation in rat aortae.

Freshly solubilized A beta peptides synergistically increase the magnitude of the constriction induced by endothelin-1 (ET-1), via the activation of a pro-inflammatory pathway. We report that mevinolin and mevastatin, two inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are able to completely abolish the vasoactive properties of A beta in rat aortae. Mevinolin also appears to oppose the increased vascular reactivity to ET-1 induced by interleukin 1-beta and phospholipase A(2) suggesting that statins display some anti-inflammatory properties. We show that freshly solubilized A beta stimulates prostaglandin E(2) and F(2 alpha) production (by 6 and 3.6 times, respectively) in isolated rat aortae and that mevinolin completely antagonizes this effect confirming the anti-inflammatory action of mevinolin ex vivo in rat aortae. In addition, we observed that A beta vasoactivity is not mediated nor modulated by mevalonic acid suggesting that the anti-inflammatory action of the statins are not related to an inhibition of HMG-CoA reductase activity. Differentiated human neuroblastoma cells (IMR32) were used to assess the neurotoxic effect of pre-aggregated A beta by quantifying the release of lactate dehydrogenase (LDH) in the cell culture medium. A beta appears to enhance LDH release by 30% in IMR32 cells, an effect that can be completely opposed by mevastatin. Taken together these data show that statins can antagonize the effect of A beta in different assays and provide new clues to understand the prophylactic action of the statins against Alzheimer's disease.

Porphyromonas gingivalis lipopolysaccharide activated bone resorption of osteoclasts by inducing IL-1, TNF, and PGE.

AIM: To study the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on inducing interleukin-1 (IL-1), tumor necrosis factor (TNF), prostaglandin E (PGE), and activating osteoclasts, in order to understand mechanism of osteoclast activation. METHODS: Pg-LPS was prepared by phenol-water method. IL-1, TNF, and PGE induced by Pg-LPS were isolated by chromatography. Ca2+ concentration was detected by atomic absorption spectrophotometry. Acid phosphatase and carbonic anhydrase in periodontal membranes were examined by histochemistry. RESULTS: Pg-LPS was able to stimulate peripheral blood mononuclear cells (PBMC) or the cells from human periodontal tissue secreting IL-1, TNF, and PGE. The outputs of these cytokines were increased in pace with the enhancement of Pg-LPS at the dose range of 0.5 - 5.0 mg/L. All of the three cytokines showed activities of accelerating Ca2+ release from rat calvarial bones, and the activity of PGE was the strongest. The amounts of both the acid phosphatase and carbonic anhydrase in the periodontal membranes of Pg-LPS injected rats were obviously increased (P < 0.01). In the periodontal membranes of Pg-LPS injected rats, the amount of activated osteoclasts were obviously increased in pace with Pg-LPS injection times (P < 0.01). However, the activating rates of osteoclasts were stable to approximately 65 % because of the increase of inactivated osteoclasts. CONCLUSION: Pg-LPS possessed strong activities to induce human PBMC and the cells from human periodontal tissue to produce IL-1, TNF, and PGE in a dose-dependent m anner within a certain concentration range of the LPS. Pg-LPS could efficiently activate osteoclasts, and the mechanism of osteoclast activation was probably associated with the increase of acid phosphatase and carbonic anhydrase.

Borage oil reduction of rheumatoid arthritis activity may be mediated by increased cAMP that suppresses tumor necrosis factor-alpha.

Recent double blind studies have shown some benefit of borage oil in treatment of rheumatoid arthritis. Tumor necrosis factor-alpha has been shown to be a central mediator of inflammatory and joint destructive processes in rheumatoid arthritis. In this paper, evidence from published research is reviewed that indicates gamma linolenic acid component of borage oil increases prostaglandin E levels that increase cAMP levels that in turn suppress tumor necrosis factor-alpha synthesis. If this biochemical path of borage oil is correct then (1) concomitant non-steroidal anti-inflammatory drug use would tend to undermine borage oil effects, and (2) borage oil would be contraindicated in pregnancy given the teratogenic and labor inducing effects of prostaglandin E agonists.

3alpha-hydroxysteroid dehydrogenase messenger RNA transcription in the immature rat ovary in response to an ovulatory dose of gonadotropin.

The ovulatory process in mammals involves a substantial increase in the metabolism of steroids and eicosanoids in response to a surge in LH or to an injection of hCG into experimental animals. This study provides evidence that the ovulatory stimulus causes induction of the gene for 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), an enzyme that belongs to several oxidoreductase superfamilies that affect steroid and eicosanoid metabolism. Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction (PCR) differential display to detect gene expression in the stimulated ovarian tissue. One of the PCR primer sets differentially amplified a cDNA fragment that is 52.3% homologous with a 3alpha-HSD gene in rat liver. Northern analyses revealed that maximum transcription was at 8 h after the animals had been treated with hCG. The Northerns also indicated that the 3alpha-HSD cDNA probe cross-hybridized with as many as six different bands of mRNA on the blots. In situ hybridization localized 3alpha-HSD mRNA in the granulosa and thecal layers of mature follicles and in newly formed corpora lutea at 24 h after the ovulatory stimulus. In conclusion, gene(s) for 3alpha-HSD are transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin. A possible function of the oxidoreductase enzyme that is translated from the 3alpha-HSD mRNA may be to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation.

Cloning and characterization of CYP4F21: a prostaglandin E2 20-hydroxylase of ram seminal vesicles.

Ram semen contains high concentrations of PGE1, PGE2, 20-hydroxy-PGE1, and 20-hydroxy-PGE2, which mainly originate from the ram seminal vesicles. The 20-hydroxy-PGE compounds are formed by a tentatively identified cytochrome P450, designated PGE2 20-hydroxylase. Our aim was to clone the enzyme and express it in yeast. Total RNA was isolated from ram seminal vesicle. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers for the CYP4 family yielded a novel cDNA sequence of a cytochrome P450. The full coding region (1584 bp) was cloned by RT-PCR and designated CYP4F21. The deduced protein sequence of CYP4F21 contained 528 amino acids and showed 74% amino acid identity with CYP4F8 of human seminal vesicles. CYP4F21 was expressed in yeast, and its catalytic properties were studied by liquid chromatography-mass spectrometry. Recombinant CYP4F21 oxidizes three stable PGH2 analogs (U44069, U46619, and U51605) and PGE2 to their 20-hydroxy metabolites, whereas PGH1, PGH2, PGE1, and PGF2alpha appeared to be poor substrates. The apparent Km for hydroxylation of PGE2 was 0.05 mM. Microsomes of ram seminal vesicles and NADPH metabolized PGE2 and the three PGH2 analogs essentially in the same way as CYP4F21. Our results suggest that CYP4F21 might be a sheep homolog to CYP4F8 of human seminal vesicles. The reproductive function of CYP4F21 is likely to biosynthesize 20-hydroxy-PGE1 and 20-hydroxy-PGE2, which is excreted by the seminal vesicles.

Evidence for the formation of dinor isoprostanes E1 from alpha-linolenic acid in plants.

The free radical oxidation of arachidonic acid is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins and exert potent receptor-mediated biological activities. In the present study, we show that alpha-linolenic acid can undergo a similar oxidation process, resulting in a series of isomeric dinor isoprostanes E1. E-ring dinor isoprostane formation from linolenate was found to be catalyzed by soybean lipoxygenase. The main enzymatic products were 13- and 9-hydroperoxylinolenate but in addition, two dinor isoprostane E1 regioisomers were formed with a yield of 0.31%. Identification and quantification of two dinor isoprostane E1 regioisomers in plant cell cultures was achieved by a negative chemical ionization gas chromatography-mass spectrometry method using [18O]dinor isoprostanes E1 as internal standards. Endogenous levels of these compounds were determined in four taxonomically distant plant species and found to be in the range of 4.5 to 60.9 ng/g of dry weight. Thus analogous pathways in animals and plants exist, each leading to a family of prostaglandin-like compounds derived from polyunsaturated fatty acids. It remains to be shown whether the dinor isoprostanes exert biological activities in plants as has been demonstrated for their C20 congeners in mammals.

Production of prostaglandins E1 and E2 by adult human red blood cells.

We showed that human adult red blood cells (RBCs) produce prostaglandin E1 (PGE1) and E2 (PGE2). RBCs that were mechanically stressed in the presence of extracellular Ca2+ by being injected rapidly through a fine needle produced PGE1 and PGE2 within 30 min after this mechanical stress. The amounts of PGE1 and PGE2 produced by 1 x 10(9) mechanically stressed RBCs were approximately 50 pg and 100 pg, respectively, which were determined in the cytosolic fraction from sonicated RBCs using a competitive enzyme immunoassay method. A Western blot analysis using anti-cyclooxygenase-2 antibody revealed a band at the 70-kDa position in the samples from RBCs producing PGE1 and PGE2. Treatment with 10 micrograms/mL indomethacin completely inhibited the productions of PGE1 and PGE2. The present results may indicate a new role of RBCs in microcirculation.

Eicosanoid production by placental and amnion tissues from control and non-insulin-dependent diabetic rats. Influence of oxytocin in the incubating medium.

Eicosanoid production by intrauterine tissues from control and neonatal-streptozotocin induced diabetic rats during late pregnancy was evaluated. In diabetic placenta the release of 6-keto-PGF1alpha was found diminished when compared to controls. In addition, LTB4 generation was increased in diabetic placenta. No alterations in the production of TXA2, PGE2, PGE1 and PGF2alpha was found when diabetic and control placenta were compared. In amnion tissue a decreased generation of 6-keto-PGF1alpha was observed in the diabetic group, but no alteration in any other eicosanoid evaluated was found. Oxytocin (5 mU/ml, in vitro), which increases prostaglandin synthesis in rabbit and human amnion tissues, did not modify eicosanoid generation in control rat amnion. In contrast, in diabetic amnion the presence of oxytocin further decreased the release of 6-keto-PGF1alpha and diminished PGE1 generation. The present results suggest that this mildly diabetic state induces alterations in eicosanoid production in intrauterine tissues, abnormalities probably enhanced during parturition, when endogenous concentrations of oxytocin are elevated.

In vivo formation of prostaglandin E1 and prostaglandin E2 in atopic dermatitis.

Immunological and biochemical alterations in atopic dermatitis have been attributed to a deficient conversion of omega-6 fatty acids (i.e. linoleic acid, gamma-linolenic acid, and dihomo-gamma-linolenic acid) to prostaglandin (PG) E1. In patients with atopic dermatitis, however, the formation of PGE1 has not been evaluated so far. We therefore measured plasma concentrations of 15-keto-13,14-dihydro-PGE1, which reflects endogenous PGE1 release, by gas chromatography-mass spectrometry in 31 patients with atopic dermatitis (aged 18-41 years, median 26 years) and in 31 healthy, age- and sex-matched control subjects. In order to exclude a metabolic shift from PGE1 to PGE2, we also measured the plasma levels of 15-keto-13,14-dihydro-PGE2. There was no difference between patients and control subjects with respect to plasma concentrations of 15-keto-13,14-dihydro-PGE1 (3.9-49.6, median 10.3 pg/ml vs. 3.2-80.4, median 8.3 pg/ml, P = 0.22), 15-keto-13,14-dihydro-PGE2 (11.6-201.0, median 24.8 pg/ml vs. 8.6-201.0, median 19.6 pg/ml, P = 0.10), and the ratio of 15-keto-13,14-dihydro-PGE1 to 15-keto-13,14-dihydro-PGE2 (0.17-1.39, median 0.41 vs. 0.2-1.17, median 0.45, P = 0.29). These results indicate that the endogenous formation of both PGE1 and PGE2 is normal in our patients. The results do not confirm the pivotal role that other authors have attributed to a deficient PGE1 formation in the pathogenesis of atopic dermatitis.