Hydroperoxide bicyclase CYP50918A1 of Plasmodiophora brassicae (Rhizaria, SAR): Detection of novel enzyme of oxylipin biosynthesis.

The genome of the cabbage clubroot pathogen Plasmodiophora brassicae Woronin 1877 (Cercozoa, Rhizaria, SAR), possesses two expressed genes encoding the P450s that are phylogenetically related to the enzymes of oxylipin biosynthesis of the CYP74 clan. The cDNA of one of these genes (CYP50918A1) has been expressed in E. coli. The preferred substrate for the recombinant protein, the 13-hydroperoxide of α-linolenic acid (13-HPOT), was converted to the novel heterobicyclic oxylipins, plasmodiophorols A and B (1 and 2) at the ratio ca. 12:1. Compounds 1 and 2 were identified as the substituted 6-oxabicyclo[3.1.0]hexane and 2-oxabicyclo[2.2.1]heptane (respectively) using the MS and NMR spectroscopy, as well as the chemical treatments. The 18O labelling experiments revealed the incorporation of a single 18O atom from [18O2]13-HPOT into the epoxide and ether functions of products 1 and 2 (respectively), but not into their OH groups. In contrast, the 18O from [18O2]water was incorporated only into the hydroxyl functions. One more minor polar product, plasmodiophorol C (3), identified as the cyclopentanediol, was formed through the hydrolysis of compounds 1 and 2. Plasmodiophorols A-C are the congeners of egregiachlorides, hybridalactone, ecklonialactones and related bicyclic oxylipins detected before in some brown and red algae. The mechanism of 13-HPOT conversions to plasmodiophorols A and B involving the epoxyallylic cation intermediate is proposed. The hydroperoxide bicyclase CYP50918A1 is the first enzyme controlling this kind of fatty acid hydroperoxide conversion.

Leishmania mexicana gp63 is the enzyme responsible for cyclooxygenase (COX) activity in this parasitic protozoa.

Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production. To identify the protein responsible for COX activity in Leishmania mexicana, we examined total extracts of promastigotes and samples with COX activity were subjected to ion exchange column purification and precipitation with ammonium sulphate; fractions with activity were analyzed by SDS-PAGE and Western blot using an anti-mouse COX-2 polyclonal antibody. Results showed that in those samples with enzymatic activity, the anti-mouse COX-2 polyclonal antibody recognized a protein with an approximate molecular weight of 66 KDa. Bands recognized by the antibody were subjected to mass spectrometry analysis and the results showed that several peptides from the bands purified by two different methods, and that were recognized by the anti-mouse COX-2 polyclonal antibody corresponded to the Leishmania mexicana gp63 surface protease. L. mexicana gp63 was purified by a Concanavalin A (Con-A) affinity column and subjected to immunoprecipitation with a commercial anti-Leishmania gp63 polyclonal antibody; the immunoprecipitated sample was analyzed for COX activity showing that the anti-gp63 antibody did immunoprecipitate the COX activity. The presence of COX activity was further confirmed in amastigotes extracts of L. mexicana. Moreover, a recombinant gp63 protein was produced and its COX activity tested, confirming that gp63 is the molecule responsible for COX activity.

Evidence for the presence of prostaglandin H synthase like enzyme in female Setaria cervi and its inhibition by diethylcarbamazine.

Experimental evidence has shown that Setaria cervi a bovine filarial parasite contains significant amount of prostaglandin H synthase like activity in the somatic extract of its different life stages. A protein with characteristics of prostaglandin H synthase was purified to homogeneity from female somatic extract using a combination of affinity and gel filtration chromatography. Molecular weight of purified enzyme was 70kDa as determined by SDS-PAGE. Purified enzyme showed high activity with arachidonic acid and TMPD substrates suggests the presence of both cyclooxygenase and peroxidase activity in enzyme. Fluorescence spectroscopy and hemin-associated peroxidase activity confirmed presence of heme in purified enzyme. The K(m) and V(max) values using arachidonic acid were determined to be 79+/-1.5microM and 0.165+/-0.2U/ml, respectively. Further, indomethacin and aspirin, specific inhibitors for PGHS, significantly inhibited the enzyme activity. Diethylcarbamazine, an antifilarial drug inhibited the microfilarial PGHS like activity as well as their motility. Here we are reporting for the first time PGHS like activity in filarial parasite and its inhibition with DEC which provide that this enzyme could be used as a drug target.

Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes.

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.

Modulatory influence of sodium 2-propenyl thiosulfate from garlic on cyclooxygenase activity in canine platelets: possible mechanism for the anti-aggregatory effect.

We previously found that sodium 2-propenyl thiosulfate (2PTS) has an anti-aggregatory effect in vitro on both canine and human platelets at relatively low concentrations, but the extent of aggregation tends to return to the control level at high concentrations. To clarify the mechanism of this modulatory influence of 2PTS on the aggregation of platelets, we investigated the effects of 2PTS on cyclooxygenase (COX) activity and the reduced glutathione (GSH) concentration in canine platelets. Platelet COX activity was inhibited by 2PTS in a dose-dependent manner up to 0.1 mM, but tended to return to the control level at 1 mM. In contrast, the platelet GSH concentration decreased in a dose-dependent manner after treatment with 2PTS and a significant decrease was observed at 0.1 mM (P<0.05) and 1 mM (P<0.001). Furthermore, the activity of purified COX-1 was directly inhibited by addition of GSH in a dose-dependent manner. From these results, we conclude that the 2PTS-induced inhibition of platelet aggregation occurs as a result of inhibition of COX activity. Additionally, 2PTS may have a modulatory effect on platelet aggregation by affecting the platelet GSH concentration.

Cytosolic phospholipase A2-alpha and cyclooxygenase-2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-alpha in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-alpha was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-alpha with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-alpha occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-alpha interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-alpha was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-alpha and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling.

Depletion of iNOS-derived nitric oxide by prostaglandin H synthase-2 in inflammation-activated J774.2 macrophages through lipohydroperoxidase turnover.

PGHS-2 (prostaglandin H synthase-2) is induced in mammalian cells by pro-inflammatory cytokines in tandem with iNOS [high-output ('inducible') nitric oxide synthase], and is co-localized with iNOS and nitrotyrosine in human atheroma macrophages. Herein, murine J774.2 macrophages incubated with lipopolysaccharide and interferon gamma showed induction of PGHS-2 and generated NO using iNOS that could be completely depleted by 12(S)-HPETE [12(S)-hydroperoxyeicosatetraenoic acid; 2.4 muM] or hydrogen peroxide (500 microM) (0.42+/-0.084 and 0.38+/-0.02 nmol x min(-1) x 10(6) cells(-1) for HPETE and H2O2 respectively). COS-7 cells transiently transfected with human PGHS-2 also showed HPETE- or H2O2-dependent NO decay (0.44+/-0.016 and 0.20+/-0.04 nmol x min(-1) x 10(6) cells(-1) for 2.4 microM HPETE and 500 microM H2O2 respectively). Finally, purified PGHS-2 consumed NO in the presence of HPETE or H2O2 (168 and 140 microM x min(-1) x microM enzyme(-1) for HPETE and H2O2 respectively), in a haem-dependent manner, with 20 nM enzyme consuming up to 4 microM NO. K(m) (app) values for NO and 15(S)-HPETE were 1.7+/-0.2 and 0.45+/-0.16 microM respectively. These data indicate that PGHS-2 catalytically consumes NO during peroxidase turnover and that pro-inflammatory cytokines simultaneously upregulate NO synthesis and degradation pathways in murine macrophages. Catalytic NO consumption by PGHS-2 represents a novel interaction between NO and PGHS-2 that may impact on the biological effects of NO in vascular signalling and inflammation.

Cyclooxygenase 2 (COX-2) as a target for therapy and noninvasive imaging.

Prostaglandins modulate a wide range of biologic functions, including wound healing, temperature regulation, reproduction, and many aspects of immune function. Exaggerated production of prostaglandins contributes to a large number pathophysiologies. The critical enzyme in prostaglandin biosynthesis is prostaglandin synthase, also known as cyclooxygenase (COX). The nonsteroidal anti-inflammatory drugs (NSAIDs), one of the largest classes of pharmaceutical agents, exert most of their biologic effects by inhibiting cyclooxygenase production of prostaglandins. The discovery of a second, inducible form of cyclooxygenase, now known as COX-2, responsible for the production of prostaglandins in most pathological states, revived a relatively moribund research area in biochemistry, physiology, and pharmacology, and led to the search for and discovery of a new class of pharmacologic agents. The coxibs have greater efficacy and substantially ameliorated side effects when compared to the classic NSAIDs. Because of the pervasive role of COX-2 in a wide range of human pathologies, the coxibs have been the most successful entry into the pharmaceutical market in history, responsible for 6-10 billion dollars in sales annually. The ability to noninvasively monitor COX-2 expression with molecular imaging probes will provide a corresponding advance in diagnosing COX-2-based disease, monitoring progression of such diseases, and evaluating alternative therapies.

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.

Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol.

The antiinflammatory agent darbufelone, ((Z)-5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl] methylene]-2-imino-4-thiazolidinone, methanesulfonate salt), was discovered as a dual inhibitor of cellular prostaglandin and leukotriene production. To study the mechanism of action of this drug, we expressed human prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2 and purified the recombinant enzymes using buffers that contain octylglucoside. In cyclooxygenase assays following a 15-min incubation of enzyme with inhibitor, darbufelone potently inhibits PGHS-2 (IC(50) = 0.19 microM) but is much less potent with PGHS-1 (IC(50) = 20 microM). Interestingly, when the assay buffer contains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS-2 due to a detergent interaction that is detectable by absorption spectroscopy. We therefore used octylglucoside, which does not affect darbufelone in this way, in place of Tween 20 in our PGHS buffers. Inhibition of PGHS-2 with darbufelone is time dependent: with no preincubation, darbufelone is a weak inhibitor (IC(50) = 14 microM), but after a 30-min incubation it is 20-fold more potent. Plots of PGHS-2 activity vs preincubation time at various darbufelone concentrations reach a plateau. This finding is inconsistent with irreversible or one-step slow-binding inhibition. A two-step slow-binding inhibition model is proposed in which the E.I complex (K(i) = 6.2 +/- 1.9 to 14 +/- 1 microM) slowly transforms (k(5) = 0.015-0.030 s(-)(1)) to a tightly bound E.I form with K(i) = 0.63 +/- 0.07 microM and k(6) = 0.0034 s(-)(1). In steady-state kinetics inhibition experiments performed with no preincubation, we find that darbufelone is a noncompetitive inhibitor of PGHS-2 (K(i) = 10 +/- 5 microM). Darbufelone quenches the fluorescence of PGHS-2 at 325 nm (lambda(ex) = 280 nm) with K(d) = 0.98 +/- 0.03 microM. The PGHS substrate, arachidonate, and various cyclooxygenase inhibitors do not alter this binding affinity of darbufelone but a structural analogue of darbufelone competes directly for binding to PGHS-2. Di-tert-butyl phenols such as darbufelone may inhibit PGHS-2 by exploiting a previously unrecognized binding site on the enzyme.

NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps.

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-kappaB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-kappaB is seen in stromal macrophages that also express COX-2 and TNF-alpha, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-kappaB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

Identification of a novel prostaglandin f(2alpha) synthase in Trypanosoma brucei.

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH(2). Among all PGs synthesized, PGF(2alpha) was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF(2alpha) synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF(2alpha) in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

The formation of stable fatty acid substrate complexes in prostaglandin H(2) synthase-1.

We have developed a protocol to purify apo-ovine (o) prostaglandin endoperoxide H(2) synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The resulting apo enzyme can then be reconstituted with Co(3+)-protoporphyrin IX instead of Fe(3+)-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate complexes for biophysical characterization. Co(3+)-protoporphyrin IX reconstituted oPGHS-1 (Co(3+)-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to that of cobalt-reconstituted horseradish peroxidase and myoglobin and suggests, along with resonance Raman spectroscopy, that the Co(3+)-protoporphyrin IX group is one in a six-coordinate, cobalt(III) state. However, Co(3+)-oPGHS-1 does not display cyclooxygenase or peroxidase activity, nor does the enzyme produce prostaglandin products when incubated with [1-(14)C]arachidonic acid. The cocrystallization of Co(3+)-oPGHS-1 and the substrate arachidonic acid (AA) has been achieved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-beta-d-glucopyranoside. Crystals are hexagonal, belonging to the space group P6(5)22, with cell dimensions of a = b = 181.69 A and c = 103.74 A, and a monomer in the asymmetric unit. GC-MS analysis of dissolved crystals indicates that unoxidized AA is bound within the crystals.

Purification and characterization of the human recombinant histidine-tagged prostaglandin endoperoxide H synthases-1 and -2.

We have used in vitro mutagenesis to introduce a six residue histidine sequence (His-tag) near the amino terminal end of the human PGHS-1 and -2 and have expressed these proteins using the baculovirus system. The His-tags are located one and two amino acids beyond the signal peptide cleavage sites of PGHS-1 and PGHS-2, respectively, positions that do not affect their activities or sensitivities to nonsteroidal anti-inflammatory drugs. When expressed in sf-21 cells, the His-tagged enzymes have K(m) values for arachidonate, and IC(50) values for inhibition by nonsteroidal anti-inflammatory drugs that are similar to values reported for the nontagged enzymes. The His-tags allowed for purification of the PGHSs by a simplified protocol involving nickel-affinity and anion exchange FPLC chromatography. The specific activities and recoveries for the purified enzymes were as good or better than those reported previously for purification of the non-tagged PGHS. These baculovirus constructs should provide a convenient source for pharmacologic and biophysical studies that require large scale preparation of human PGHSs.

Regulation of prostaglandin H2 synthase activity by nitrogen oxides.

Nitric oxide and its derivatives have been shown to both activate and inhibit prostaglandin H(2) synthase 1 (PGHS-1). We set out to determine the mechanisms by which different nitrogen oxide derivatives modulate PGHS-1 activity. To this end, we show that 3-morpholinosydnonimine hydrochloride (SIN-1), a compound capable of generating peroxynitrite, activates purified PGHS-1 and also stimulates PGE(2) production in arterial smooth muscle cells in the presence of exogenous arachidonic acid. The effect of SIN-1 in smooth muscle cells was abrogated by superoxide and peroxynitrite inhibitors, which supports the hypothesis that peroxynitrite is an activating species of PGHS-1. Indeed, authentic peroxynitrite also induced PGE(2) production in arachidonic acid-stimulated cells. In contrast, when cells were exposed to the nitric oxide-releasing compound 1-hydroxy-2-oxo-3-[(methylamino)propyl]-3-methyl-1-triazene (NOC-7), PGHS-1 enzyme activity was inhibited in the presence of exogenous arachidonic acid. Finally, in lipid-loaded smooth muscle cells, we demonstrate that SIN-1 stimulates arachidonic acid-induced PGE(2) production; albeit, the extent of activation is reduced compared to that under normal conditions. These results indicate that formation of peroxynitrite is a key intermediary step in PGHS-1 activation. However, other forms of NO(x)() inhibit PGHS-1. These results may have implications in the regulation of vascular function and tone in normal and atherosclerotic arteries.

A novel mechanism of action of chemically modified tetracyclines: inhibition of COX-2-mediated prostaglandin E2 production.

Tetracyclines (doxycycline and minocycline) inhibit inducible NO synthase expression and augment cyclooxygenase (COX)-2 expression and PGE2 production. In contrast, chemically modified tetracyclines (CMTs), such as CMT-3 and -8 (but not CMT-1, -2, and -5), that lack antimicrobial activity, inhibit both NO and PGE2 production in LPS-stimulated murine macrophages, bovine chondrocytes, and human osteoarthritis-affected cartilage, which spontaneously produces NO and PGE2 in ex vivo conditions. Furthermore, CMT-3 augments COX-2 protein expression but inhibits net PGE2 accumulation. This coincides with the ability of CMT-3 and -8 to inhibit COX-2 enzyme activity in vitro. The action of CMTs is distinct from that observed with tetracyclines because 1) CMT-3-mediated inhibition of PGE2 production coincides with modification of COX-2 protein, which is distinct from the nonglycosylated COX-2 protein generated in the presence of tunicamycin, as observed by Western blot analysis and 2) CMT-3 and -8 have no significant effect on COX-2 mRNA accumulation. In contrast, CMT-3 and -8 do not inhibit COX-1 expression in A549 human epithelial cells at the level of protein and mRNA accumulation or modification of COX-1 protein. CMT-3 and -8 inhibit the sp. act. of COX-2 (but not COX-1) in cell-free extracts. These results demonstrate differential action of CMT-3 (Metastat) on COX-1 and -2 expression, which is distinct from other tetracyclines.

Altered gene expression of prostacyclin synthase and prostacyclin receptor in the thoracic aorta of spontaneously hypertensive rats.

OBJECTIVE: The aim of this study was to evaluate the possible role of prostacyclin (PGI2) in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). METHODS: Measurement of mRNA and protein levels of PGH synthase (PGHS)-1, PGI2 synthase and the PGI2 receptor, in the thoracic aorta was performed in SHR aged 5, 10, 20, and 40 weeks old and in age-matched normotensive Wistar-Kyoto (WKY) rats with a competitive polymerase chain reaction method and immunoblotting. Aortic production of 6-keto-PGF1 alpha, the main metabolite of PGI2, was also measured. RESULTS: Compared with age-matched WKY rats, PGHS-1 mRNA and protein levels in the thoracic aorta of SHR increased with age, reaching three- and twofold higher than WKY rats at 40 weeks old, respectively. PGI2 synthase mRNA and protein levels in SHR were significantly higher than in WKY rats at 20 and 40 weeks old. In contrast, PGI2 receptor mRNA levels in SHR were consistently lower than in WKY rats at all ages. CONCLUSIONS: These results provide evidence that hypertension elicits alterations in levels of arachidonic acid metabolites, including PGH2 and PGI2. They also suggest that the decreased expression of PGI2 receptor mRNA in prehypertensive SHR could be one of the causes of hypertension in SHR.