Phosphatidylserine inhibits inflammatory responses in interleukin-1ß-stimulated fibroblast-like synoviocytes and alleviates carrageenan-induced arthritis in rat.

Recently, phosphatidylserine (PS) has received attention for its anti-inflammatory effect; however, the molecular mechanisms of its action have not been fully understood. Thus, we hypothesized that PS might have antiarthritic and anti-inflammatory effects. To test this hypothesis, the in vitro anti-inflammatory effect of soybean-derived PS was tested on interleukin (IL)-1ß-stimulated fibroblast-like synoviocytes from rheumatoid arthritis patients (RA-FLS) by measuring the levels of IL-6, IL-8, prostaglandin E(2), and vascular endothelial growth factor by enzyme-linked immunosorbent assay. The analgesic and antiarthritic activities of PS were investigated in rat models of carrageenan-induced acute paw pain and arthritis. The former was evaluated with a paw pressure test; the latter, by measuring paw volume and weight distribution ratio. In addition, the participation of mitogen-activated protein kinase signaling in the anti-inflammatory and antiarthritic effects of PS was investigated in RA-FLS. Phosphatidylserine inhibited the production of inflammatory mediators IL-6; IL-8; vascular endothelial growth factor; and, in particular, prostaglandin E(2) in IL-1ß-stimulated RA-FLS. These effects were associated with abrogation of inhibitor of nuclear factor-κBα phosphorylation and suppression of p38 and c-jun amino terminal kinase but not extracellular signal-regulated kinase 1/2 phosphorylation. In rats, PS also showed a significant inhibitory effect on arthritic and nociceptive symptoms induced by carrageenan. These findings suggest that PS has anti-inflammatory and antiarthritic effects in vitro and in in vivo animal models; thus, PS should be further studied to determine its potential use as either a pharmaceutical or dietary supplement for alleviating arthritic symptoms.

Inhibitory effects of panduratin A on allergy-related mediator production in rat basophilic leukemia mast cells.

Immediate-type hypersensitivity is characterized by elevated levels of immunoglobulin E (IgE) and activated mast cell plays a crucial role by releasing granule contents, lipid-derived mediators, cytokines, and chemokines. To evaluate the antiallergic effects of panduratin A isolated from Boesenbergia pandurata Roxb., we determined its effects on calcium (Ca(2+)) influx, degranulation, and inflammatory mediators in calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA)-stimulated rat basophilic leukemia (RBL-2H3) cells. Panduratin A (20 µM) inhibited secretion of ß-hexosaminidase (46.69 ± 9.6 %), histamine (34.32 ± 2.1 %), and Ca(2+) influx (43.84 %). Panduratin A reduced the production of prostaglandin E(2) (PGE(2), 47.58 ± 3.4 %), leukotriene B(4) (LTB(4), 98.15 ± 1.6 %), and the mRNA expression of cyclooxygenase-2, 5-lipoxygenase, interleukin (IL)-4, IL-13, and tumor necrosis factor-α. Furthermore, panduarin A attenuated phosphorylation of Akt, the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. These results indicate that panduratin A might be useful as an agent against immediate-type hypersensitivity.

Paracoccidioides brasiliensis uses endogenous and exogenous arachidonic acid for PGE x production.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids during fungal infections plays a critical role on fungal biology as well as on host immune response modulation. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, Bt79, Pb192) produce prostaglandin E(x) (PGE(x)). Moreover, we asked if P. brasiliensis could use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) enzyme, as reported for mammalian cells. A possible association between this prostanoid and fungus viability was also assessed. Our results showed that all strains, independently of their virulence, produce high PGE(x) levels on 4 h culture that were reduced after 8 h. However, in both culture times, higher prostanoid levels were detected after supplementation of medium with exogenous AA. Treatment with indomethacin, a COX inhibitor, induced a reduction on PGEx, as well as in fungus viability. The data provide evidence that P. brasiliensis produces prostaglandin-like molecules by metabolizing either endogenous or exogenous AA. Moreover, the results suggest the involvement of these mediators on fungal viability.

Detrimental effects of hyperandrogenism on uterine functions.

The aim of the present work was to study some of the adverse effects produced by hyperandrogenism on the uterine function. Daily injection of dehydroepiandrosterone (DHEA: 6 mg/ 100 g body weight, sc) for 20 consecutive days induced polycystic ovaries in BALB/c mice. In this model, we found that DHEA produced alterations on uterine histology closely related to the development of tumour structures. In addition, hyperandrogenism induced a pro-inflammatory and a pro-oxidant condition represented by increased levels of prostaglandin F2 alpha production and uterine nitric oxide synthase (NOS) activity and by a decrease in both superoxide dismutase (SOD) and catalase (CAT) activities together with a decrease in the levels of the antioxidant metabolite glutathione (GSH). DHEA also induced an increase in CD4+ together with a decrease in the CD8+ T lymphocytes that infiltrate the uterine tissue. We conclude that this intricate network of regulators could be responsible for the low rate of implantation observed in women with polycystic ovary syndrome.

Dose-dependent effects of dietary gamma-linolenic acid on rat spleen lymphocyte functions.

Feeding rodents a diet rich in evening primrose oil (EPO), which contains 5-10 g gamma-linolenic acid (GLA)/100 g total fatty acids, has been shown to decrease lymphocyte proliferation and natural killer cell activity. However, EPO contains a very high level of linoleic acid which itself can affect lymphocyte functions and it is not clear to what extent the effects of EPO can be attributed to GLA. The current study investigated the effect of two levels of GLA in the rat diet upon immune cell functions; the level of linoleic acid was maintained below 30 g/100 g total fatty acids. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g or 10 g GLA/100 g total fatty acids in place of a proportion of linoleic acid. The total polyunsaturated fatty acid content and the n-6 to n-3 polyunsaturated fatty acid ratio of the diet were maintained at 35 g/100 g total fatty acids and 7, respectively. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet, with an increase in the proportions of GLA and dihomo-gamma-linolenic acid when the diets containing GLA were fed; these diets also increased the proportion of arachidonic acid in spleen leukocytes. Spleen lymphocyte proliferation in response to concanavalin A was significantly reduced (by 60%) by feeding the diet containing the higher level of GLA, but not by the diet containing the lower level of GLA. Spleen natural killer cell activity and prostaglandin E (PGE) production by spleen leukocytes were not significantly affected by inclusion of GLA in the diet, although there was a tendency towards decreased natural killer cell activity by cells from rats fed the high GLA diet. Thus, this study shows that dietary GLA is capable of altering the fatty acid composition of cells of the immune system and of exerting some immunomodulatory effects, but that the level of GLA in the diet must exceed 4.4 g/100 g total fatty acids for these effects to become apparent.

Effects of dietary gamma-linolenic acid-rich borage oil combined with marine fish oils on tissue phospholipid fatty acid composition and production of prostaglandins E and F of the 1-, 2- and 3-series in a marine fish deficient in delta5 fatty acyl desaturase.

The effects of gamma-linolenic acid-rich borage oil (BO), in combination with different marine oils, namely an eicosapentaenoic acid (EPA) rich oil (MO) or a DHA-rich oil (TO), on tissue fatty acid composition and prostaglandin production were investigated in turbot, a species which lacks appreciable delta5 fatty acyl desaturase activity. The juvenile turbot grew well on the experimental diets and there were no significant differences in final weights between dietary treatments. Irrespective of the marine oil component, both the BO-containing diets increased tissue phospholipid levels of 18:2n-6 and 18:3n-6, and their respective elongation products, 20:2n-6 and 20:3n-6, compared to fish fed a control diet containing a standard Northern hemisphere fish oil. Both the BO-containing diets increased the production of 1-series prostaglandins (PG), this being observed across all tissues investigated with PGF and especially PGE. The BO/MO diet also reduced 20:4n-6 in tissue phospholipids without affecting 20:5n-3, whereas the BO/TO combination decreased 20:5n-3 but increased 20:4n-6. The production of 2-series and 3-series PGs was also altered by the dietary treatments but the changes were less dependent upon the tissue levels of their respective precursor fatty acids, 20:4n-6 and 20:5n-3. The BO-containing diets had very significant effects on gross fatty acid compositions of the phospholipids including increased proportions of saturated fatty acids and n-6 polyunsaturated fatty acids (PUFA) and decreased proportions of monounsaturated fatty acids and n-3 PUFA. Overall, this study shows that eicosanoid production in turbot tissues can be influenced by dietary fatty acids, not only by changes in the absolute and relative levels of specific eicosanoid precursor PUFA in tissue phospholipids, but also by general effects on membrane composition, structure and function induced by gross fatty acid compositional changes.

Diets rich in eicosapentaenoic acid and gamma-linolenic acid affect phospholipid fatty acid composition and production of prostaglandins E1, E2 and E3 in turbot (Scophthalmus maximus), a species deficient in delta 5 fatty acid desaturase.

Duplicate groups of juvenile turbot, (Scophthalmus maximus), were fed diets containing either Marinol K (MO), a marine fish oil rich in eicosapentaenoic acid (EPA; 20:5, n-3) or borage oil (BO), rich in gamma-linolenic acid (GLA; 18:3, n-6), for a period of 12 weeks. Individual phospholipid fatty acid compositions from hearts of fish fed BO had significantly more 18:2, n-6, GLA, 20:2, n-6, dihomo-gamma-linolenic acid (DHGLA; 20:3, n-6) and total n-6 polyunsaturated fatty acids (PUFA), but significantly less arachidonic acid (AA; 20:4, n-6), compared to fish fed MO. In both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from heart, the DHGLA was increased by over 50-fold in fish fed BO while AA was reduced by over two-thirds, compared to fish fed MO. In brain, EPA was the major C20 PUFA, i.e. potential eicosanoid precursor in all phospholipids from fish fed MO, with the EPA level being twice that of AA in brain phosphatidylinositol (PI). DHGLA was the major C20 PUFA in all phospholipid classes from fish fed BO. In kidney and gill, EPA was the predominant C20 PUFA in all phospholipid classes, except PI, in fish fed MO. In kidney of fish fed BO, DHGLA was the major C20 PUFA in all phospholipid classes, except PE. In gill of fish fed BO, DHGLA was the major C20 PUFA in all phospholipid classes, including PI, where DHGLA was over 2.5-fold greater than AA. In homogenates of heart, kidney and gill from BO-fed fish the prostaglandin E1 (PGE1) concentration was significantly increased compared to MO-fed fish. In heart and kidney homogenates from fish fed MO the PGE3 concentration was significantly increased compared to fish fed BO. The ratio of PGE2/PGE1 was significantly reduced in brain, heart, kidney and gill homogenates from fish fed BO compared to those fed MO.

Effect of supplementation with 20:3(n-6), 20:4(n-6) and 20:5(n-3) on the production of prostaglandins E and F of the 1-, 2- and 3-series in turbot (Scophthalmus maximus) brain astroglial cells in primary culture.

The production of prostaglandins E and F of the 1-, 2- and 3-series was determined in primary cultures of turbot (Scophthalmus maximus) brain astroglial cells after supplementation with 25 microM dihomo-gamma-linolenic (20:3(n-6)), arachidonic (20:4(n-6)) and eicosapentaenoic (20:5(n-3)) acids. Supplementation by 20:3(n-6), 20:4(n-6) and 20:5(n-3) for 4 days significantly increased the percentages of the respective acids in the total cellular lipid of the turbot astrocytes. The predominant prostaglandins formed by turbot astrocytes in response to stimulation with calcium ionophore A23187 were prostaglandin E2 and prostaglandin F2 alpha under all experimental conditions. The production of prostaglandin E2 was stimulated 2.6-fold, but prostaglandin F2 alpha production was unaffected after supplementation of cultures with 20:4(n-6). However, prostaglandin E2 production in astrocytes was significantly inhibited 3- and 4-fold, and prostaglandin F2 alpha was inhibited 1.6- and 14.6-fold by supplementation with 20:3(n-6) and 20:5(n-3), respectively. Supplementation with 20:3(n-6) also significantly increased the production of prostaglandin E1 (almost 4-fold) and prostaglandin F1 alpha (2.2-fold) whereas supplementation with 20:5(n-3) did not significantly increase the production of prostaglandin E3. Prostaglandin F3 alpha but not prostaglandin E1 were significantly reduced in 20:5(n-3)-supplemented cultures.

Breast milk from mothers of children with newly developed atopic eczema has low levels of long chain polyunsaturated fatty acids.

BACKGROUND: Infants at risk of atopic dermatitis have lower than normal levels of long chain polyunsaturated fatty acids. These fatty acids are normally present in substantial quantities in human breast milk. METHODS: Because of the equivocal evidence concerning the ability of breastfeeding to delay the onset or reduce the severity of atopic dermatitis, we have analyzed the fatty acid composition of breast milk from the mothers of children with newly developed disease with the use of gas chromatography. RESULTS: Breast milk lipids from mothers of children with newly developed atopic dermatitis had increased proportions of linoleic acid and significantly decreased proportions of its long chain polyunsaturated derivatives compared with a control group. The ratio of linoleic acid to the sum of its metabolites, gamma-linolenic acid, dihomo-gamma-linolenic acid, and arachidonic acid was 11.78 in the atopic group and 9.02 in the control group (p < 0.01). CONCLUSIONS: These results are consistent with previous findings of an abnormal fatty acid status in atopic subjects and may account for some of the inconsistent results from studies of the effect of breastfeeding on the subsequent development of atopic dermatitis. We conclude that further studies to examine the effects of supplementation of the diet of breastfeeding mothers with long chain polyunsaturates should be done.

Dietary (n-3) fatty acid and vitamin E interactions in rats: effects on vitamin E status, immune cell prostaglandin E production and primary antibody response.

To examine the interaction between dietary fat and vitamin E at the level of the rat immune system, a 2 x 3 factorial study was designed. Weanling female Sprague Dawley rats were fed for 8-9 wk diets that contained either corn oil (CO diet) or fish oil (FO diet) and one of three levels (30, 300, 900 mg/kg) of all-rac-alpha-tocopheryl acetate. At the lowest level of dietary vitamin E, alpha-tocopherol content of splenocytes from FO-fed rats was approximately 40% lower (P less than 0.05) than in those from CO-fed rats. Supplementation with all-rac-alpha-tocopheryl acetate elevated alpha-tocopherol in splenocytes from FO-fed rats but not in those from CO-fed rats, and reduced the relative proportion of arachidonic acid and eicosapentaenoic acid in the serum of CO-fed and FO-fed rats, respectively. Prostaglandin E production by isolated immune cells was not affected by all-rac-alpha-tocopheryl acetate supplementation. However, feeding the FO diet consistently reduced prostaglandin E synthesis by 70-80% as compared with the CO diet. Antibody production against sheep RBC was highest in rats fed the FO diet supplemented with 900 mg all-rac-alpha-tocopheryl acetate/kg of diet. However, antibody response was not directly correlated to diet-induced changes in immune cell prostaglandin E production or alpha-tocopherol content. Our data suggest that there are significant interactions between vitamin E and (n-3) fatty acids that affect the immune system and that further research in this area is warranted.

Evaluation of assay procedures measuring macrophage stimulation by immunomodulators in vitro.

Several assay procedures for measuring the stimulatory effect on macrophages (møs) of bacterial-derived immunomodulators (OM-85, OM-89, OM-163, Laboratoires OM, Meyrin/Geneva, Switzerland) were evaluated with regard to their complexity, speed, and general convenience. To this effect, bone marrow-derived or peritoneal exudate macrophages were exposed to the immunomodulators in vitro, then tested for metabolic stimulation (glucose oxidation through the hexosemonophosphate shunt pathway, synthesis of type E prostaglandins, release of superoxide, and production of L-arginine-derived nitrogen oxidation products), as well as for enhancement of functional activities (production of tumor necrosis factor-alpha, extracellular cytolysis of P815 target cells, and intracellular parasite destruction). All these tests were found to provide adequate measurements of the mø response to the immunomodulators, with significant effects detectable using the compounds in the ng/ml to microgram/ml range. Concomitant incubation with crude macrophage activating factor or with recombinant murine interferon-gamma (IFN-gamma) dramatically increased the sensitivity of møs to the immunomodulators, and was an absolute requirement for induction of mø cytotoxic activities by the bacterial extracts. The measurement of nitrite production by møs exposed to the immunomodulators with or without treatment with 10 U/ml of IFN-gamma was found to be a highly convenient procedure, which correlated well with functional assays.

Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids.

Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.

Effect of fish oil supplementation on the excretion of the major metabolite of prostaglandin E in healthy male subjects.

We investigated the effect of fish oil supplementation on the synthesis of prostaglandin E (PGE) in vivo by measuring the excretion of its catabolite, PGE-M, in 24-hr urine by gas chromatography/mass spectrometry. Forty healthy male volunteers (24-57 years of age) consumed a controlled basal diet providing 40% of energy from fat (P/S ratio about 0.8:1), 130 mg/1000 kcal cholesterol, and a minimum of 22 mg/day of alpha-tocopherol (alpha-T), for three experimental periods lasting a total of 28 weeks. During period 1 (10 weeks) the diet was supplemented with placebo (PO) capsules (15 X 1 g/day) consisting of a blend of fats approaching the fatty acid profile of the basal diet. This was followed by a second 10-week period during which the subjects received 15 X 1 g/day capsules of fish oil concentrate (FOC). During period 3 (8 weeks) they continued the 15 g/day intake of FOC but received an additional 200 mg/day of alpha-T. PO and FOC capsules contained 1 mg alpha-T/g fat as antioxidant. A 14% reduction of PGE-M excretion was observed after 10 weeks of FOC supplementation (period 2), compared to an identical period of placebo supplementation (period 1), P = 0.009. PGE-M excretion during the last week of period 3 was not significantly different from that at the end of period 2. The reduction in PGE synthesis in response to the relatively high marine oil supplementation was large in many subjects participating in this study.

Effect of dietary alpha-linolenic acid on growth, metastasis, fatty acid profile and prostaglandin production of two murine mammary adenocarcinomas.

The purpose of this study was to determine whether dietary (n-3) fatty acids would affect mammary tumor growth and metastasis. Weanling female BALB/c mice were fed diets that contained 10% corn oil (CO), linseed oil (LO) or a fish oil-corn oil mix (FO) for 3-8 wk prior to receiving subcutaneous injections of one of two syngeneic mammary tumor cell types (410 and 410.4). Tumor growth was assessed by monitoring mean tumor diameter and tumor weight upon removal. Feeding LO, but not FO, reduced the growth (p less than 0.05) of 410.4 mammary tumors compared with growth in those fed CO. Metastasis data paralleled the tumor growth rate. Feeding LO and FO enhanced (p less than 0.005) incorporation of (n-3) fatty acids into tumors. Tumor prostaglandin E (PGE) production was reduced (p less than 0.005) by LO and FO, compared with CO. FO feeding reduced 410.4 tumor PGE synthesis more (p less than 0.05) than LO feeding, yet tumor growth was only inhibited by LO. These data suggest an inhibitory effect of dietary linolenic acid [i.e., 18:3 (n-3)] on mammary tumor growth and metastasis. However, this effect did not directly correlate with diet-induced changes in PGE synthesis.

Effect of dietary iron overload on lipid peroxidation, prostaglandin synthesis and lymphocyte proliferation in young and old rats.

The effect of dietary iron overload on lipid peroxidation (LP), prostaglandin (PG) synthesis and lymphocyte proliferation was examined in young and old F344 rats. Rats 4 and 19-22 mo old were fed AIN-76 diet for 11-12 wk supplemented with 2.5% carbonyl iron obtained from two sources (Type A and B). Animals supplemented with Type A iron showed reduced food intake and weight gain associated with marked increases in extrahepatic and hepatic iron concentration. Rats receiving Type B iron had food intakes and body weights similar to those of controls but exhibited small increases in tissue iron concentration. Old control rats compared to young had significantly higher conjugated dienes (CD) in hepatic microsomes. Feeding Type A iron diets induced a significantly higher level of CD in hepatic microsomes from old rats compared to young rats. Iron overloaded rats also showed highly correlated (r = 0.94) increases in the urinary excretion of thiobarbituric acid-reactive substances and PG metabolites indicating increased in vivo LP and PG synthesis. Mitogen-stimulated PGE2 synthesis in young rats was increased at 4 wk in association with enhanced T-cell proliferation stimulated by Concanavalin A. Lymphocyte proliferation was significantly lower in old than in young control or iron-treated rats. The lack of efficacy of Type B vs. Type A iron appears due to a larger particle size and lower bioavailability. In conclusion, iron overloading increases in vivo LP and PG metabolism. Furthermore, the mitogenic response to Concanavalin A in young rats is enhanced after 4 wk of iron overloading.

Suppression of human synovial cell proliferation by dihomo-gamma-linolenic acid.

Prostaglandin E1 (PGE1) and oils enriched in its precursor fatty acids suppress inflammation and joint tissue injury in several animal models. Since synovial cell proliferation is a hallmark of rheumatoid arthritis, we studied the effect of dihomo-gamma-linolenic acid (DGLA), an immediate precursor of PGE1, on the growth of human adherent synovial cells (ASC) in tissue culture. When stimulated by appropriate concentrations of recombinant interleukin-1 beta (rIL-1 beta), ASC proliferate and produce PGE. DGLA-enriched medium suppressed both baseline and rIL-1 beta-stimulated ASC growth fivefold, compared with medium supplemented with arachidonic acid. Indomethacin reduced the effect of the DGLA. Synovial cells incorporated the DGLA, and rIL-1 beta-stimulated cells that were incubated with DGLA exhibited a 14-fold increase in PGE1 (to 25.2 +/- 6.0 ng/ml, mean +/- SD) and a 70% decrease in PGE2 (to 25.2 +/- 4.2 ng/ml) compared with cells in control medium. At equivalent concentrations (5 x 10(-7) M), PGE1 increased the level of cellular cAMP to a greater extent than did PGE2 (16.8 +/- 2.0 pmoles versus 4.3 +/- 1.9 pmoles, mean +/- SEM). Exogenous PGE1 was also a more effective inhibitor of cell growth. Similarly, cAMP concentrations in cells exposed to DGLA for 6 hours were greater than concentrations in arachidonic acid-enriched cultures (17.8 +/- 3.3 pmoles versus 2.1 +/- 2.0 pmoles). These observations suggest that DGLA can restrain ASC growth, an effect which may be due to its capacity to increase PGE1 production and subsequent cellular cAMP concentration.

The antihypertensive effects of fish oil. A controlled study of polyunsaturated fatty acid supplements in essential hypertension.

Both n-3 and n-6 polyunsaturated fats have been suggested to lower blood pressure, an effect ascribed to altered biosynthesis of eicosanoids. To test these hypotheses, we studied blood pressure and eicosanoid production during supplementation of dietary fat for four weeks in 32 men with mild essential hypertension. Supplementation was preceded and followed by four-week run-in and recovery periods. Groups of eight subjects received either 10 ml or 50 ml of fish oil (3 or 15 g of n-3 fatty acids) daily, 50 ml of safflower oil (39 g of n-6 fatty acids), or 50 ml of a mixture of oils that approximated the types of fat present in the American diet. The biosynthesis of eicosanoids was assessed by the measurement of urinary metabolites. Blood pressure decreased in the men who received the high dose of fish oil (systolic pressure by a mean of 6.5 mm Hg [P less than 0.03] and diastolic pressure by 4.4 mm Hg [P less than 0.015]), but not in the other groups. Although the formation of vasodilatory prostacyclins (prostaglandins I2 and I3) increased initially, this increase was not maintained as blood pressure fell. The level of thromboxane A2 metabolites fell; metabolites of thromboxane A3 were detected in the groups receiving fish oil. The formation of prostaglandin E2 increased during supplementation with safflower oil and tended to decrease with fish oil; no prostaglandin E3 metabolite was detected. Our data indicate that high doses of fish oil can reduce blood pressure in men with essential hypertension. However, the clinical usefulness and safety of fish oil in the treatment of hypertension will require further study.

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages.

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.

Suppression of foam cell lesions in hypercholesterolemic rabbits by inhibition of thromboxane A2 synthesis.

Hypercholesterolemia induces adhesion of blood-borne monocytes to vascular endothelium and their subsequent migration into the intima, where foam cell lesions subsequently develop. The regulating mechanisms for the adhesion and migration are unclear. In this study, a specific thromboxane A2 (TXA2) synthetase inhibitor, UK-38485, was used to treat rabbits fed an atherogenic diet to determine whether inhibition of TXA2, the major metabolite of arachidonic acid in monocytes, affects lesion development. Rabbits were fed a diet supplemented with 2% cholesterol and 8% peanut oil for 12 weeks with or without UK-38485 at a dosage that maintained 80% to 90% inhibition of TXA2 formation in serum. The treatment with UK-38485 had no effect on total serum cholesterol. Both the treated and untreated groups developed subpopulations of high and low responders with respect to the extent of lesion coverage, forming a bimodal distribution. The treatment with UK-38485 significantly (p less than 0.001) reduced the percentage of the thoracic aorta covered by lesions when treated low responders (5.3 +/- 1.0%, n = 12) were compared to untreated low responders (23.6 +/- 2.9%, n = 12). However, UK-38485 had no effect when treated high responders (76.3%, n = 3) were compared to untreated high responders (72.0%, n = 12). Lesion coverage was not correlated with serum cholesterol levels. Stimulation of isolated rabbit monocytes in autologous plasma with 0.66 mM arachidonic acid in the presence of increasing concentrations of UK-38485 caused a dose-dependent inhibition of TXA2 and a concurrent increase in prostaglandin E2 (PGE2).(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin and prostaglandin E2 secretions by bovine pulmonary microvessel endothelial cells are altered by changes in culture conditions.

The isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established. Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma-derived serum, bovine retinal growth extract (1%), and heparin (90 micrograms/ml) on gelatin coated plates. A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma-derived serum. Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages. MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII-related antigen and uptake of 1,1'-dioctacecyl-1,3,3,3'3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (Dil-Ac-LDL). MVE cells were also positive for angiotensin-converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence. MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 +/- 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGI2) and thromboxane (Tx) A2 (316 +/- 43 and 588 +/- 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay. However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187. In contrast, MVE cells cultured without heparin and growth factor secreted more PGI2 than PGE2 (862 +/- 84 and 89 +/- 12 respectively). Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGI2 and PGE2 production (P less than 0.01). Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGI2. These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.