Formation and antiproliferative effect of prostaglandin E(3) from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E(3) (PGE(3)) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE(3.) The extracellular ratio of PGE(3) to PGE(2) increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE(3), respectively, than that produced by COX-1. Exposure of A549 cells to 1 microM PGE(3) inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE(3), however, had no effect. When A549 cells were exposed to EPA (25 microM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 microM). This effect appears to be associated with a 50% reduction of PGE(3) formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE(2), an increase in the level of PGE(3), and PGE(3)-mediated inhibition of tumor cell proliferation.     

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ω3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10 μM of these two ω3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of ω3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.     

In vitro prostaglandin synthesis by human glomeruli and papillae

We have investigated in vitro prostaglandin synthesis by human isolated glomeruli and papillary homogenates and compared the results with those obtained in parallel studies using rat material. Prostaglandins were measured by two methods, namely radiometric high performance liquid chromatography after incubation with ¹⁴C arachidonic acid and radioimmunoassay. The relative abundance of various prostaglandins synthesized by glomeruli was different in man (6 keto PGF_1α > TXB₂ > PGF_2α > PGE₂) and in the rat (PGE₂ TXB₂ > 6 keto PGF_1α). Unidentified peaks eluting between 6 keto PGF_1α and TXB₂ were observed only in rat glomeruli. These peaks were suppressed by indomethacin. Direct radioimmunoassay of prostaglandins in the incubation medium of human glomeruli confirmed the predominance of 6 keto PGF_1α synthesis and showed its stimulation by arachidonic acid, its progressive decrease with time and its linear relationship with glomerular protein at low concentrations. On the contrary, the profile of prostaglandin synthesis by the papilla was similar in man and in the rat, PGE₂ and PGF_2α being the major products in both species. However, related to one mg of protein, papillary synthesis of these two prostaglandins was greater in the rat. These results show that PGI₂ is the major prostaglandin synthesized in human glomeruli and suggest a role for this prostaglandin in glomerular physiology in man.     

Selective modulation of the human platelet thromboxane A2/prostaglandin H2 receptor by eicosapentaenoic and docosahexaenoic acids in intact platelets and solubilized platelet membranes.

We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.     

Lipoxygenase in trout gill tissue acting on arachidonic, eicosapentaenoic and docosahexaenoic acids

Lipoxygenase activity was characterized in the gill tissue of fresh-water trout. Incubation of arachidonic acid with gill preparations yielded 12-hydroxyeicosatetraenoic acid as the major product, suggesting a 12-lipoxygenase. Eicosapentaenoic acid was similarly converted to the 12-hydroxyeicosapentaenoic acid. Both arachidonic acid and docosahexaenoic acid were converted with equal apparent velocities and affinities into single monohydroxy derivatives. Analyses of the hydroxy product of docosahexaenoic acid were consistent with 14-hydroxydocosahexaenoic acid. This enzyme activity was localized to the cytosolic fraction and displayed a broad pH optimum around pH 7. The enzyme was insensitive to the cyclooxygenase inhibitors indomethacin and aspirin but activity was strongly inhibited in the presence of the lipoxygenase inhibitors, SnCl2 (5 mM), esculetin (10 microM) and eicosatetraynoic acid (100 microM).     

Effects of eicosapentaenoic acid, gamma-linolenic acid and prostaglandin E1 on three human colon carcinoma cell lines.

Several studies have demonstrated that certain essential fatty acids present a specific cytotoxicity for tumor cells. However, no investigation of this type has been performed on human colon cancer cells to date. This study investigated the effect of gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and prostaglandin (PG) E1 on the proliferation and metabolism of three human colon cancer cell lines: HT 29, HRT 18, and CACO 2. GLA, EPA and PGE1 all inhibited the proliferation of the three cell lines, but with a decreasing gradient of sensitivity: HRT 18 > HT 29 > CACO 2, and with different IC50 values. PGE1 was markedly less effective than the other two. GLA and EPA increased lipid peroxidation and membrane fluidity in a dose-dependent manner. The presence of indomethacin did not modify the effects of GLA and EPA. In addition, PGE1 had little effect on membrane fluidity and lipid peroxidation. The antitumoral effect thus does not appear to be mediated by PGE1. Addition of vitamin E decreased the effects of GLA and EPA, which supports the hypothesis of direct action by these fatty acids. In conclusion, while EPA and GLA have an antitumoral effect in vitro, their effect on primary cultures of normal human colon cells must be investigated to determine whether this effect is specific to tumoral cells, as has been observed for other cell types.     

Effects of estradiol-17 beta and progesterone on the synthesis of prostaglandin F2 alpha, prostaglandin E2 and prostaglandin I2 by fibroblasts from human endometrium in vitro

Estradiol-17 beta increases the production of prostaglandin F2 alpha (PGF2 alpha) in long term monolayer cell cultures of the human endometrium in a dose dependent manner. Progesterone in pharmacological dosage stimulates the syntheses of PGF2 alpha and of prostaglandin E2 (PGE2). The synthesis of prostaglandin I2 (PGI2) is not influenced by sex steroids in long term monolayer cell cultures of the human endometrium.     

Incorporation of eicosapentaenoic and docosahexaenoic acids by a yeast (FO726A)

An eicosapentaenoic acid (EPA)- and docosahexaenoic acid (DHA)-incorporating yeast, FO726A, was putatively identified as Candida guilliermondii on the basis of morphological, physiological and biochemical characteristics. Culture conditions for FO726A were investigated with respect to cell mass productivity, cellular accumulation of total lipid, triglyceride (TG), EPA and DHA. When grown at 20 degrees C for 24 h in an optimal medium containing 1 g scrap fish oil, the yeast yielded 820 mg dry cells which consisted of 40.7% lipid, 40.2% protein and 14.1% carbohydrate. The lipid (334mg) consisted of 300 mg TG (36.6% of dry cells), 23.2 mg EPA (2.8%) and 54.8 mg DHA (6.7%), and the recovery rates of EPA and DHA from the fish oil were 27.1 and 43.6%, respectively. The positional distributions of fatty acids in the TG from the yeast were then investigated and compared with those in the TG from the fish oil. The EPA and DHA in the fish oil were concentrated more in the sn-1,3 positions (8.8 and 13.7%, respectively) than in the sn-2 position (3.7 and 10.8%, respectively). In the case of the TG from the yeast, EPA was present to a greater extent in the sn-1,3 positions than in the sn-2 position. In contrast, DHA was preferentially present in the sn-2 position, approximately twice that in the sn-1,3 positions.     

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors

The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).     

Suppression of prostaglandin E2 receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth

Prostaglandin E(2) (PGE(2)), a major cyclooxygenase (COX-2) metabolite, plays important roles in tumor biology and its functions are mediated through one or more of its receptors EP1, EP2, EP3, and EP4. We have shown that the matrix glycoprotein fibronectin stimulates lung carcinoma cell proliferation via induction of COX-2 expression with subsequent PGE(2) protein biosynthesis. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibited this effect and induced cellular apoptosis. Here, we explore the role of the PGE(2) receptor EP2 in this process and whether the inhibition observed with PPARgamma ligands is related to effects on this receptor. We found that human non-small cell lung carcinoma cell lines (H1838 and H2106) express EP2 receptors, and that the inhibition of cell growth by PPARgamma ligands (GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone [also known as BRL49653]) was associated with a significant decrease in EP2 mRNA and protein levels. The inhibitory effects of BRL49653 and ciglitazone, but not PGJ2, were reversed by a specific PPARgamma antagonist GW9662, suggesting the involvement of PPARgamma-dependent and -independent mechanisms. PPARgamma ligand treatment was associated with phosphorylation of extracellular regulated kinase (Erk), and inhibition of EP2 receptor expression by PPARgamma ligands was prevented by PD98095, an inhibitor of the MEK-1/Erk pathway. Butaprost, an EP2 agonist, like exogenous PGE(2) (dmPGE(2)), increased lung carcinoma cell growth, however, GW1929 and troglitazone blocked their effects. Our studies reveal a novel role for EP2 in mediating the proliferative effects of PGE(2) on lung carcinoma cells. PPARgamma ligands inhibit human lung carcinoma cell growth by decreasing the expression of EP2 receptors through Erk signaling and PPARgamma-dependent and -independent pathways.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

Macrolide antibiotics inhibit prostaglandin E2 synthesis and mRNA expression of prostaglandin synthetic enzymes in human leukocytes

We investigated the action of macrolide antibiotics, which are considered to have anti-inflammatory activity, on lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 synthesis and the expression of mRNAs for cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX)-1, and COX-2 in human leukocytes. The production of LPS-stimulated PGE2 was significantly increased in peripheral polymorphonuclear leukocytes (PMNLs) and in mononuclear leukocytes (MNLs). Amounts of mRNAs for COX-2 and cPLA2, but not for COX-1, were enhanced by LPS in PMNLs and MNLs. The LPS-enhanced PGE2 synthesis and the expression of cPLA2 and COX-2 mRNAs were inhibited by clarithromycin, azithromycin and dexamethasone in PMNLs and MNLs. The mRNA expression of COX-1 in PMNLs was decreased by clarithromycin and azithromycin. Macrolide antibiotics inhibited PGE2 synthesis in human leukocytes by suppressing cPLA2, COX-1, and COX-2 mRNA expression. These data indicate one mechanism of macrolide anti-inflammatory activity.     

Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexaenoic acids in glycerides

Lipoprotein lipase from Pseudomonas sp. was the best enzyme to concentrate eicosapentaenoic and docosahexaenoic acids (EPA and DHA) in sardine oil by acidolysis reaction, and acetone was more effective than n-hexane as a solvent for dissolving the reactants and concentrating the two fatty acids. The water concentration in the reaction mixture was a decisive factor governing the enrichment of EPA and DHA and the yield of glycerides. EPA and DHA were more concentrated, but the yield of glycerides decreased, when the water concentration was increased gradually. Thus, the concentration rates of both the fatty acids were low with 0.25% water, although a considerable amount of diglyceride was detectable in the reaction products. The effect of reaction temperature was very slight with the use of acetone; however, the ratio DHA/EPA increased when the temperature was lowered in the presence of n-hexane. When acidolysis was performed at 25°C for 1 h, using 10,000 units of lipase per g of the reactants, the total percentage of EPA and DHA reached 65% in the glycerides and the recoveries of the two acids were 87.4 and 81.3%, respectively, based on the contents in the original sardine oil. The relationship of the enzyme substrate specificity to the reaction results was also investigated.     

Alteration of in vitro murine peritoneal macrophage function by dietary enrichment with eicosapentaenoic and docosahexaenoic acids in menhaden fish oil

The effects of diets containing menhaden fish oil (MFO), compared with those of diets containing safflower oil (SAF) or an essential fatty acid deficient hydrogenated coconut oil (HCO), on in vitro activation of tumoricidal capacity by murine macrophages were assessed. Mice fed the experimental diets for 4 weeks were injected intraperitoneally with sterile thioglycollate broth 3 days before use. There was no difference between any of the groups with respect to total peritoneal exudate cells or the percentage of macrophages, although the fatty acid profile of purified adherent macrophages closely paralleled that of the diets. Macrophages from mice fed MFO killed fewer P815 mastocytoma cells upon activation with recombinant interferon gamma (IFN gamma) and lipopolysaccharide. Macrophages from all diets were equally competent for tumoricidal capacity when activated pharmacologically with calcium ionophore, phorbol 12-myristate 13-acetate, and lipopolysaccharide (LPS), suggesting that MFO diet macrophages were hyporesponsive to IFN gamma. Priming with higher concentrations of IFN gamma restored the partial defect in activation of MFO macrophages. When activated for 24 hr with high levels of LPS, macrophages from mice fed SAF displayed little cytolytic capacity; addition of indomethacin. (1 microM) resulted in enhanced levels of P815 kill. In contrast, MFO and HCO diet macrophages were highly cytolytic with similar LPS treatment with or without indomethacin. Macrophages from mice fed SAF produced threefold more prostaglandin E in response to LPS than did MFO and HCO diet macrophages. These results suggest that dietary manipulation of fatty acids can alter activation of tumoricidal capacity of macrophages, possibly both dependent and independent of changes in eicosanoid synthesis.     

Primary open-angle glaucoma patients have reduced levels of blood docosahexaenoic and eicosapentaenoic acids

The aetiology of primary open-angle glaucoma (POAG), which is the commonest cause of non-remediable blindness and visual impairment, is not well understood. Nevertheless, increased intraocular pressure, and vascular factors such as ocular blood flow deficits are thought to be risk factors. There is evidence of decreased optic nerve blood velocity and increased red blood cell aggregability in POAG. These factors are influenced by fatty acids. We have investigated if glaucoma patients have abnormal blood fatty acid composition. Patients with POAG (n=10) and their healthy siblings (n=8) were enrolled. Compared with their healthy siblings, the glaucoma patients had reduced eicosapentaenoic (EPA, P<0.01), and docosahexaenoic (DHA, P<0.05) fatty acids and total omega3 long-chain polyunsaturated fatty acids (LCPUFA) (P<0.05) in red cell choline phosphoglycerides (CPG); decreased EPA (P<0.05) in ethanolamine phosphoglycerides (EPG); lower EPA (P<0.05) and total omega3 LCPUFA (P<0.05) in serine phosphoglycerides (SPG). Similarly, they had reduced EPA, DHA and total omega3 LCPUFA in plasma CPG (P<0.005) and triglycerides (P<0.05). These findings may be significant, since EPA and DHA could modulate impaired systemic microcirculation and ocular blood flow and optic neuropathy, which are the main physiological changes associated with glaucoma.     

Non-small-cell lung carcinoma cells invade human bronchial mucosa In vitro

Summary To study invasion of lung cancer in vitro a novel three-dimensional coculture assay consisting of living human tissues has been developed. Multicellular spheroids initiated from a new large-cell lung carcinoma cell line (GaL23), found to be invasive in immunodeficient mice, were confronted with precultured bronchial fragments derived from mucosal biopsies obtained during routine fiberoptic bronchoscopy. The bronchial fragments consist of a stromal core with scattered fibroblasts covered by a continuous surface epithelium resting on a basal lamina. During the first 2 wk of confrontation, a gradual retraction of the bronchial epithelium with subsequent adhesion of the tumor cells to the underlying basal lamina occurred. The following week, a limited invasion of tumor cells into the bronchial stroma was seen. To facilitate the entrance of tumor cells through the mucosal surface, the surface epithelium was removed prior to coculture by ethylenediaminetetraacetic acid (EDTA) buffer treatment. Upon confrontation, GaL23 cells then rapidly attached to and migrated on the exposed basal lamina and an increasing number of tumor cells was seen in the stroma during the first week of culture. This model offers opportunities for studying mechanisms of lung cancer adhesion, migration, and invasion using human bronchial mucosa as the natural target tissue.     

Prostaglandin E precursor fatty acids inhibit human IL-2 production by a prostaglandin E-independent mechanism

Essential fatty acids, from which PG derive, can participate in development and regulation of immune responses and have been shown to suppress inflammation and tissue injury in animal models. In this report, we investigate the effects of the immediate (DGLA, precursor to PGE1), arachidonic acid (AA, PGE precursors, dihomogamma linolenic acid (DGLA, precursor to PGE1), arachidonic acid (AA, precursor to PGE2), and eicosapentaenoic acid (EPA, precursor to PGE3) on IL-2 production by PHA-stimulated human PBMC. DGLA and AA inhibited IL-2 production in a dose-dependent manner: half-maximal inhibition was obtained by using the fatty acids at the dose of 10 micrograms/ml without significant effects on cell viability. EPA inhibited IL-2 production by PBMC of only some donors. Incubation of cells in the presence of oleic, stearic, and palmitic acids, which are not PG precursors, did not affect mitogen-induced IL-2 production. A progressive increase in incorporation of DGLA into cellular lipids was observed over a 48-h incubation period. IL-2 production was reduced also when PBMC were pretreated overnight with DGLA or AA and washed before exposure to PHA. Whereas addition of the cyclo-oxygenase inhibitor, indomethacin, at the time of mitogenic stimulation led to increased IL-2 production and prevented mitogen- and fatty acid-induced increases in PGE release, it had no significant effect on the capacity of the fatty acids to suppress IL-2 production. Time course experiments showed that DGLA and AA inhibited IL-2 production even at times of minimal or no PGE release by the treated cultures. Moreover, DGLA and AA inhibited IL-2 production by the human leukemia T cell line Jurkat which, when appropriately induced, is able to release high levels of IL-2 in the absence of accessory cells and measurable PGE production. Taken together, these data indicate that essential fatty acids inhibit IL-2 production directly without conversion into their cyclo-oxygenase pathway products, and suggest that human lymphocyte function may be altered profoundly by small changes in their fatty acid profile.     

Effect of omega-3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine-induced contractions.

The relaxant responses of the rat thoracic aorta to omega-3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine- and potassium-induced contractions were investigated. Relaxation was enhanced in vessels contracted with norepinephrine. Docosahexaenoic acid at concentrations as low as 1, 3, and 10 microM evoked significant relaxant responses (15, 23, 30%) in norepinephrine-contracted vessels as compared with responses (5, 9, 12%) in potassium-contracted vessels. Results for eicosapentaenoic acid under similar conditions were 3, 8, and 19% in norepinephrine-contracted vessels and 3, 3, and 8% in potassium-contracted vessels. Pretreatment with eicosapentaenoic (10 microM) or docosahexaenoic acids (1-10 microM) decreased the contractile response to physiologic concentrations of norepinephrine. In the presence of calcium-free medium, the omega-3 fatty acids (1-30 microM) significantly abolished sustained norepinephrine contractions but did not reduce the phasic contractions when incubated prior to norepinephrine contraction. Comparatively, the effects of docosahexaenoic acid were greater than eicosapentaenoic acid. These findings suggest that the relaxant effects of the omega-3 fatty acids are specific to the mode of contraction, i.e., alpha-adrenoceptor stimuli. This effect may be related to intracellular calcium mechanisms, since both fatty acids reversed norepinephrine-induced sustained contractions in the absence of extracellular calcium.