Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells.

There are two established depigmenting agent assays currently in use. However, these methods are unreliable and time-consuming. Therefore, it will be valuable to establish a better assay system for depigmenting agent analysis. In this study, we established a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the microphthalmia-associated transcription factor (MITF), tyrosinase (Tyr) and dopachrome tautomerase (Dct) genes in MeWo human melanoma cells. We used several melanogenesis regulators, including theophylline, hesperetin, arbutin and rottlerin, to confirm the function of this assay system. The established MeWo/pMITF-EGFP, MeWo/pTyr-EGFP and MeWo/pDct-EGFP stable cells integrated the pMITF-EGFP, pTyr-EGFP and pDct-EGFP plasmids into their genomic DNA. These stably transfected cells were used to examine alterations in the expression of the MITF, Tyr and Dct genes. All of the tested compounds, including theophylline, hesperetin, arbutin and rottlerin, could be analyzed in the stable cells, producing reliable results. Therefore, we believe that this melanogenesis regulation assay system can be used as a rapid and reliable assay system to analyze the regulation of melanogenesis by many known or unknown compounds.

Inhibitory effects of adlay extract on melanin production and cellular oxygen stress in B16F10 melanoma cells.

The aim of this study was to determine the effects of adlay extract on melanin production and the antioxidant characteristics of the extract. The seeds were extracted by the supercritical fluid CO2 extraction (SFE) method. The effect of adlay extract on melanin production was evaluated using mushroom tyrosinase activity assay, intracellular tyrosinase activity, antioxidant properties and melanin content. Those assays were performed spectrophotometrically. In addition, the expression of melanogenesis-related proteins was determined by western blotting. The results revealed that the adlay extract suppressed intracellular tyrosinase activity and decreased the amount of melanin in B16F10 cells. The adlay extract decreased the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2). The extract also exhibited antioxidant characteristics such as free radical scavenging capacity and reducing power. It effectively decreased intracellular reactive oxygen species (ROS) levels in B16F10 cells. We concluded that the adlay extract inhibits melanin production by down-regulation of MITF, tyrosinase, TRP-1 and TRP-2. The antioxidant properties of the extract may also contribute to the inhibition of melanogenesis. The adlay extract can therefore be applied as an inhibitor of melanogenesis and could also act as a natural antioxidant in skin care products.

Kaliziri extract upregulates tyrosinase, TRP-1, TRP-2 and MITF expression in murine B16 melanoma cells.

BACKGROUND: Kaliziri extract (KZE) is a traditional Uyghur medicine (TUM), used by traditional hospitals in China as an injection for treatment of vitiligo for more than 30 years. Clinical application has shown that this medicine has obvious therapeutic effects. However, its phytochemical analysis and mechanism have not been examined. METHODS: KZE was extracted from seeds of Kaliziri [Vernonia anthelmintica (L.) Willd.] in ethanol-water (80:20, v/v), its components were identified by LC-MS/MS, and the signaling pathway of melanin synthesis in KZE treated murine B16 melanoma cells was examined by western blotting. RESULTS: Liquid chromatography-mass spectrometry analysis confirmed that the main components of KZE are flavonoids. KZE increased the tyrosinase activity and melanin content in a dose-dependent manner at concentrations of 5-40 µg/ml, and treatment with 20 µg/ml of KZE enhanced the expression of tyrosinase in B16 cells in a time-dependent manner. CONCLUSIONS: KZE induced melanogenesis by increasing the expression of TYR, TRP-1, TRP-2 and MITF in B16 cells.

Cancer-induced anorexia in tumor-bearing mice is dependent on cyclooxygenase-1.

It is well-established that prostaglandins (PGs) affect tumorigenesis, and evidence indicates that PGs also are important for the reduced food intake and body weight loss, the anorexia-cachexia syndrome, in malignant cancer. However, the identity of the PGs and the PG producing cyclooxygenase (COX) species responsible for cancer anorexia-cachexia is unknown. Here, we addressed this issue by transplanting mice with a tumor that elicits anorexia. Meal pattern analysis revealed that the anorexia in the tumor-bearing mice was due to decreased meal frequency. Treatment with a non-selective COX inhibitor attenuated the anorexia, and also tumor growth. When given at manifest anorexia, non-selective COX-inhibitors restored appetite and prevented body weight loss without affecting tumor size. Despite COX-2 induction in the cerebral blood vessels of tumor-bearing mice, a selective COX-2 inhibitor had no effect on the anorexia, whereas selective COX-1 inhibition delayed its onset. Tumor growth was associated with robust increase of PGE(2) levels in plasma - a response blocked both by non-selective COX-inhibition and by selective COX-1 inhibition, but not by COX-2 inhibition. However, there was no increase in PGE(2)-levels in the cerebrospinal fluid. Neutralization of plasma PGE(2) with specific antibodies did not ameliorate the anorexia, and genetic deletion of microsomal PGE synthase-1 (mPGES-1) affected neither anorexia nor tumor growth. Furthermore, tumor-bearing mice lacking EP(4) receptors selectively in the nervous system developed anorexia. These observations suggest that COX-enzymes, most likely COX-1, are involved in cancer-elicited anorexia and weight loss, but that these phenomena occur independently of host mPGES-1, PGE(2) and neuronal EP(4) signaling.

Thirst deficits in aged rats are reversed by dietary omega-3 fatty acid supplementation.

During heat waves many elderly individuals die as a consequence of dehydration. This is partially due to deficits in mechanisms controlling thirst. Reduced thirst following dipsogenic stimuli is well documented in aged humans and rodents. Low in vivo long-chain omega-3 fatty acid levels, as can occur in aging, have been shown to alter body fluid and sodium homeostasis. Therefore, the effect of dietary omega-3 fatty acid supplementation on drinking responses in aged rats was examined. Omega-3 fatty acids reversed thirst deficits in aged rats following dehydration and hypertonic stimuli; angiotensin (ANG) II induced drinking was unaffected in aged rats. Plasma atrial natriuretic peptide (ANP) and arginine vasopressin (AVP) were altered with age, but not affected by diet. Aged omega-3 fatty acid deficient animals displayed increased hypothalamic cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase (COX)-2, and prostaglandin E (PGE) synthase messenger (m)RNA expression, compared with animals that received omega-3 fatty acids. The aged low omega-3 fatty acid fed animals had significantly elevated hypothalamic PGE(2) compared with all other groups. Hypothalamic PGE(2) was negatively correlated with drinking induced by both dehydration and hypertonicity. The results indicate that PGE(2) may be the underlying mechanism of the reduced thirst observed in aging.

Quercetin inhibits α-MSH-stimulated melanogenesis in B16F10 melanoma cells.

Quercetin is known to inhibit tyrosinase activity and melanin production in melanocytes. However, several reports suggest that quercetin has different and opposite effects on melanogenesis. This study examined the precise effects of quercetin on melanogenesis using cell-free assay systems and melanocytes. Quercetin inhibited the monophenolase and diphenolase activities of tyrosinase, and melanin synthesis in cell-free assay systems. Quercetin induced mild stimulation of the tyrosinase activity and dihydroxyphenylalaminechrome tautomerase (TRP-2) expression but only at low concentrations (<20 µm) in B16F10 melanoma cells. In contrast, the addition of 50 µm quercetin to the cells led to a significant decrease in the activity and synthesis of tyrosinase, as well as a decrease in the expression of tyrosinase-related protein-1 and TRP-2 proteins, regardless of the presence or absence of α-melanocyte stimulating hormone (α-MSH). Quercetin also reduced the intracellular cAMP and the phosphorylated protein kinase A levels in α-MSH-stimulated B16F10 cells. Moreover, quercetin (20 µm) diminished the expression and activity of tyrosinase, and melanin content in cultured normal human epidermal melanocytes. These effects were not related to its cytotoxic action. Although the in vivo effects of quercetin are still unclear, these results suggest that quercetin could play important roles in controlling melanogenesis.

Inhibition of hypoxia inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis.

PURPOSE: 2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. EXPERIMENTAL DESIGN: To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE-2). RESULTS: DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 mumol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1beta-mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1alpha. Moreover, DPE blocked mPGEs-1-dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1alpha, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal-related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1alpha expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1alpha translation. CONCLUSIONS: We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1alpha/mPGEs-1/VEGF axis.

Molecular cloning and mRNA expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from Hyoscyamus niger.

In the endeavor to enhance the production of pharmaceutically valuable tropane alkaloids including hyoscyamine and scopolamine in Hyoscyamus niger, methyl jasmonate (MeJA) showed significant stimulation both in tropane biosynthetic pathway enzymes activities and tropane alkaloids yields. Therefore it was speculated that genetic engineering of jasmonate biosynthetic pathway might enhance the endogenous jasmonate concentration, followed by stimulating the production of tropane alkaloids. Herein a full-length cDNA encoding allene oxide synthase (AOS, EC 4.2.1.92), the first committed step enzyme in jasmonate biosynthetic pathway was reported (named HnAOS, GenBank accession: EF532599). HnAOS was a novel member of the cytochrome P450 (CYP74A) subfamily. Real-time quantitative PCR analysis showed that HnAOS mRNA accumulated mainly in stems, and responded significantly to wounding or methyl jasmonate.

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs.

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I(2), thromboxane A(2) and prostaglandin F(2alpha). The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H(2) production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.

Molecular cloning, characterization and expression of a jasmonate biosynthetic pathway gene encoding allene oxide cyclase from Camptotheca acuminata.

AOC (allene oxide cyclase; EC 5.3.99.6), an essential enzyme in jasmonic acid and its methyl ester biosynthesis, was cloned from Camptotheca acuminata (named as CaAOC), a native medicinal plant species in China. CaAOC had significant similarity at the amino-acid level with AOCs from other plant species. Comparison between the sequences of the full-length cDNA and genomic DNA of CaAOC revealed that the genomic DNA of CaAOC contained an 89-bp intron and a 240-bp intron. Southern-blot analysis indicated that CaAOC was a multiple-copy gene, and real-time quantitative PCR analysis showed that CaAOC was expressed constitutively in all organs tested, with the highest expression level in leaves. The results from treatment experiments using different signalling components, including methyl jasmonate, abscisic acid, salicylic acid and H(2)O(2), revealed that expression of CaAOC had a prominent diversity. Heavy metal (copper) significantly enhanced CaAOC expression, whereas wounding (induced by UV-B) was not so effective.

[Molecular cloning and expression profile of a jasmonate biosynthetic pathway gene allene oxide cyclase from Hyoscyamus niger].

Hyoscyamus niger L. is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary metabolites named as tropane alkaloids. As a family of signaling phytohormones, jasmonates play significant roles in the biosynthesis of many plant secondary metabolites. In jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, [...] EC 5.3.99.6 [...]) catalyzes the most important step. Here we cloned a cDNA from H. niger, named HnAOC (GenBank accession: AY708383), which was 1044 bp long, with a 747 bp open reading frame (ORF) encoding a polypeptide of 248 amino acid residues. Southern blot analysis indicated that it was a multi-copy gene. RT-PCR analysis revealed that the expression of HnAOC was regulated by various stresses and elicitors, with methyl-jasmonate showing the most prominent inducement. The characterization of HnAOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis ofjasmonates.

MF63 [2-(6-chloro-1H-phenanthro[9,10-d]imidazol-2-yl)-isophthalonitrile], a selective microsomal prostaglandin E synthase-1 inhibitor, relieves pyresis and pain in preclinical models of inflammation.

Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal prostaglandin E(2) (PGE(2)) synthase in the cyclooxygenase pathway. Inhibitors of mPGES-1 may block PGE(2) production and relieve inflammatory symptoms. To test the hypothesis, we evaluated the antipyretic and analgesic properties of a novel and selective mPGES-1 inhibitor, MF63 [2-(6-chloro-1H-phenanthro-[9,10-d]imidazol-2-yl)isophthalonitrile], in animal models of inflammation. MF63 potently inhibited the human mPGES-1 enzyme (IC(50) = 1.3 nM), with a high degree (>1000-fold) of selectivity over other prostanoid synthases. In rodent species, MF63 strongly inhibited guinea pig mPGES-1 (IC(50) = 0.9 nM) but not the mouse or rat enzyme. When tested in the guinea pig and a knock-in (KI) mouse expressing human mPGES-1, the compound selectively suppressed the synthesis of PGE(2), but not other prostaglandins inhibitable by nonsteroidal anti-inflammatory drugs (NSAIDs), yet retained NSAID-like efficacy at inhibiting lipopolysaccharide-induced pyresis, hyperalgesia, and iodoacetate-induced osteoarthritic pain. In addition, MF63 did not cause NSAID-like gastrointestinal toxic effects, such as mucosal erosions or leakage in the KI mice or nonhuman primates, although it markedly inhibited PGE(2) synthesis in the KI mouse stomach. Our data demonstrate that mPGES-1 inhibition leads to effective relief of both pyresis and inflammatory pain in preclinical models of inflammation and may be a useful approach for treating inflammatory diseases.

Activation of peroxisome proliferator-activated receptor gamma inhibits interleukin-1beta-induced membrane-associated prostaglandin E2 synthase-1 expression in human synovial fibroblasts by interfering with Egr-1.

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial fibroblasts. PPARgamma ligands 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model.

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.

Cytoplasmic prostaglandin E2 synthase is dominantly expressed in cultured KAT-50 thyrocytes, cells that express constitutive prostaglandin-endoperoxide H synthase-2. Basis for low protaglandin E2 production.

The recent identification and cloning of two glutathione-dependent prostaglandin E(2) synthase (PGES) genes has yielded important insights into the terminal step of PGE(2) synthesis. These enzymes form efficient functional pairs with specific members of the prostaglandin-endoperoxide H synthase (PGHS) family. Microsomal PGES (mPGES) is inducible and works more efficiently with PGHS-2, the inflammatory cyclooxygenase, while the cytoplasmic isoform (cPGES) pairs functionally with PGHS-1, the cyclooxygenase that ordinarily exhibits constitutive expression. KAT-50, a well differentiated thyroid epithelial cell line, expresses high levels of PGHS-2 but surprisingly low levels of PGE(2) when compared with human orbital fibroblasts. Moreover, PGHS-1 protein cannot be detected in KAT-50. We report here that KAT-50 cells express high basal levels of cPGES but mPGES mRNA and protein are undetectable. Thus, KAT-50 cells express the inefficient PGHS-2/cPGES pair, and this results in modest PGE(2) production. The high levels of cPGES and the absence of mPGES expression result from dramatic differences in the activities of their respective gene promoters. When mPGES is expressed in KAT-50 by transiently transfecting the cells, PGE(2) production is up-regulated substantially. These observations indicate that naturally occurring cells can express a suboptimal profile of PGHS and PGES isoforms, resulting in diminished levels of PGE(2) generation.

Functional expression and characterization of Aedes aegypti dopachrome conversion enzyme.

A full-length mosquito dopachrome conversion enzyme (DCE) and its truncated form lacking the last 54 carboxyl-terminal amino acid residues are expressed using a baculovirus/insect cell expression system. The full-length recombinant DCE displayed multiple bands during native PAGE with substrate staining, but only one active band was detected when the truncated recombinant DCE was analyzed under identical analysis conditions. Our data suggest that the last 50 some carboxyl-terminal residues are involved in the polymerization of the DCE molecules and that the proposed DCE isozymes likely reflect the presence of multimers of the same DCE molecules. The significance of the recombinant DCE in accelerating the melanization pathway is demonstrated by a rapid production of melanin in a dopa and tyrosinase reaction mixture in the presence of recombinant DCE. The DCE sequence data obtained in our previous study, together with results of functional expression and biochemical characterization achieved in this study, provide a necessary reference for the study of other insect DCEs.

Melanosomal defects in melanocytes from mice lacking expression of the pink-eyed dilution gene: correction by culture in the presence of excess tyrosine.

Mutations in the murine pink-eyed dilution (p) gene, or its human homologue P, result in oculocutaneous albinism. Melanocytes cultured from mice lacking p gene expression exhibit defective melanogenesis, but following culture in the presence of high concentrations of L-tyrosine, increased melanin deposition is observed. Electron microscopy and image analysis demonstrated that untreated p mutant melanocytes exhibited small melanosomes, largely of stages I-II. Following tyrosine treatment, increased proportions of stage III-IV melanosomes, almost normal in size, were observed. Levels of tyrosinase protein and to a lesser extent of tyrosinase-related protein-1 (TRP-1) were subnormal but rose dramatically following stimulation by tyrosine. Levels of TRP-2 and Pmel17/silver gene product were not altered, nor were the levels of mRNA for tyrosinase, TRP-1, TRP-2, or the Pmel17/silver gene product. As expected, the 110-kDa product of the p gene was absent from both stimulated and unstimulated p mutant cells. In a melanoblast line derived from the same mice, excess tyrosine failed to stimulate visible melanogenesis or increase the low levels of tyrosinase. The melanosomes in these cells were smaller still than those in the mutant melanocytes even when cultured in the presence of excess tyrosine. Thus, absence of the p gene product affects melanosomal structure and protein composition at the posttranscriptional level. These defects are correctable at least in part by supplementation with L-tyrosine.

Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells.

To enlighten the mechanism of inhibition of prostacyclin (PGI2) production by n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, cultured endothelial cells were incubated with albumin bound-EPA or -DHA for 22 h. Under these conditions, PGI2 formation in response to bradykinin, calcium ionophore or exogenous arachidonic acid was equally inhibited by 50%, suggesting that the inhibition might occur downstream the phospholipase step, likely at the level of PGH synthase and/or PGI2 synthase activities. Western blot analysis indicated that the mass of the constitutive isoform of PGH synthase (PGH synthase 1), but not PGI2 synthase, was significantly reduced in n-3 fatty acid-enriched cells. In subsequent experiments, PGH synthase 1 mRNA level, measured by northern blotting, was also decreased in n-3 supplemented cells. This reduction was not due to mRNA destabilization. None of these parameters were altered by similar enrichment with oleic acid (OA). These results suggest that EPA and DHA may affect PGH synthase 1 expression, presumably at the transcriptional level.