PGH2 Research Articles

Early Growth Response Factor-1 Mediates Prostaglandin E2-dependent Transcriptional Suppression of Cytokine-induced Tumor Necrosis Factor-α Gene Expression in Human Macrophages and Rheumatoid Arthritis-affected Synovial Fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic proinflammatory cytokine that modulates a broad range of inflammatory and immunological processes. We have investigated the potential immunomodulatory properties of prostaglandin E2 (PGE2) by examining the molecular mechanism by which the eicosanoid suppresses T-cell-derived interleukin-17 (IL-17)-induced TNF-alpha mRNA expression and protein synthesis in human macrophages and rheumatoid arthritis-affected synovial fibroblasts. Initial studies confirmed that PGE2 induces egr-1 mRNA expression and protein synthesis by restricted SAPK2/p38 MAPK-dependent activating transcription factor-2 (ATF-2) dimer transactivation of the egr-1 promoter as judged by studies using wild-type (WT) and deletion mutant egr-1 promoter constructs, Northern and Western blotting, and standard and supershift electrophoretic mobility shift analyses. Using human leukemic monocytic THP-1 cells stably transfected with WT and dominant-negative mutant expression constructs of Egr-1, cotransfected or not with a WT pTNF-615SVOCAT construct, we observed that PGE2 inhibition of IL-17-stimulated TNF-alpha mRNA expression and promoter activity was dependent on Egr-1 expression, as mutants of Egr-1, alone or in combination, markedly abrogated any inhibitory effect of PGE2. Standard and supershift electrophoretic mobility shift analysis, signaling "decoy" overexpression studies, and pTNF-615SVOCAT promoter assays using WT and mutant promoter constructs revealed that IL-17-up-regulated promoter activity was largely dependent on ATF-2/c-Jun transactivation. PGE2 suppression of IL-17-induced ATF-2/c-Jun transactivation and DNA binding was dependent on Egr-1-mediated inhibition of induced c-Jun expression. We suggest that egr-1 is an immediate-early PGE2 target gene that may be a key regulatory factor in mediating eicosanoid control of genes involved in the immune and inflammatory responses.

Prostaglandin E2 Induces MUC8 Gene Expression via a Mechanism Involving ERK MAPK/RSK1/cAMP Response Element Binding Protein Activation in Human Airway Epithelial Cells

MUC8 gene expression is overexpressed in nasal polyp epithelium and is also increased by treatment with inflammatory mediators in nasal epithelial cells. These data suggest that MUC8 may be one of important mucin genes expressed in human airway. However, the mechanisms of various inflammatory mediator-induced MUC8 gene expression in normal nasal epithelial cells remain unclear. We examined the mechanism by which prostaglandin E(2) (PGE2), an arachidonic acid metabolite, increases MUC8 gene expression levels. Here, we show that ERK mitogen-activated protein kinase is essential for PGE2-induced MUC8 gene expression in normal human nasal epithelial cells and that p90 ribosomal S 6 protein kinase 1 (RSK1) mediates the PGE2-induced phosphorylation of cAMP-response element binding protein. Our results also indicate that cAMP-response element at the -803 region of the MUC8 promoter is an important site of PGE2-induced MUC8 gene expression. In conclusion, this study gives insights into the molecular mechanism of PGE2-induced MUC8 gene expression in human airway epithelial cells.

Determination of the Structural Environment of the Tyrosyl Radical in Prostaglandin H2 Synthase-1: A High Frequency ENDOR/EPR Study

The catalytically active tyrosyl radical which gives rise to the "wide doublet" (WD1) signal in ovine Prostaglandin H2 Synthase-1 has been studied using high frequency (HF) pulsed ENDOR and EPR. A hydrogen-bonded deuteron was directly detected in HFENDOR (130 GHz) spectra of 1H2O/2H2O-exchanged samples. The HFENDOR spectral simulations required a distribution in hydrogen bond distances to achieve proper fits. This range of distances was consistent with that used to model the distribution in gX values detected in pulsed HFEPR spectra. Possible hydrogen-bonding partners, as well as implications regarding the mechanism of self-inactivation for PGHS, are discussed.

The Janus Face of Cyclooxygenase-2 in Ischemic Stroke

The rate-limiting enzyme for prostanoid synthesis cyclooxygenase-2 (COX-2) has been implicated in the basic mechanisms of several brain diseases, including stroke, multiple sclerosis and neurodegenerative diseases.1 The approval by the Food and Drug Administration (FDA) of highly selective COX-2 inhibitors for the treatment of pain and rheumatoid arthritis (RA) raised the possibility that these agents could also be used in the treatment of neurological diseases including stroke. However, the occurrence of serious cardiovascular complications in patients receiving COX-2 inhibitors has led to the recent withdrawal from the market of a popular COX-2 inhibitor and has called for a re-evaluation of the therapeutic potential of these drugs. In this article, we briefly review the role of COX-2 in ischemic brain injury and re-examine the validity of the COX-2 pathway as a therapeutic target for ischemic stroke. COX enzymes catalyze the conversion of arachidonic acid into prostaglandin H2 (PGH2; Figure).2 Arachidonic acid, produced by the breakdown of membrane phospholipids, is metabolized by COX into PGH2 in a 2-step reaction in which the free radical superoxide is also produced.2 Three isoforms of COX have been described. COX-1 is present in most cells and is involved in normal cellular physiology, such as gastric secretion and platelet function.2 COX-2 is expressed constitutively in some organs, such as brain, but is markedly upregulated by a wide variety of stimuli, most notably inflammatory mediators.2 COX-3, a splice variant of COX-1, is highly sensitive to inhibition by acetaminophen and is most abundant in heart and brain.3 The COX reaction product PGH2 is converted into 5 different prostanoids by a corresponding number of isomerases (Figure), the distribution of which is cell and organ specific.2 Thus, PGH2 can give rise to different prostanoids depending on the …

Cyclooxygenase-2 and Thromboxane Synthase in Non-Endocrine and Endocrine Tumors: A Review

Prostaglandins (PG) are members of a large group of hormonally active fatty acids derived from free fatty acids. They are formed from arachidonic acid-the major PG precursor. Cyclooxygenase (COX)-1 and -2 are the rate-limiting steps in PG synthesis. COX-2 is overexpressed in many human non-endocrine and endocrine tumors including colon, breast, prostate, brain, thyroid, and pituitary. COX-2 has an important role in angiogenesis and tumor growth. Thromboxane synthase (TS) catalyzes the synthesis of thromboxane A2 (TXA2), which is derived from arachidonic acid and prostaglandin H2 and is a vasoconstrictor and inducer of platelet aggregation. TXA2 stimulates tumor growth and spread of some tumors and TS appears to have a critical role in tumorigenesis in some organ systems. In this review, we examine the role of COX-2 and TS in various non-endocrine tumors, especially colon, breast, prostate, and brain as well as in endocrine tumors. The accumulating evidence points to an increasingly important role of COX-2 and TS in tumor progression and metastasis.

Lipid Signaling in Neural Plasticity, Brain Repair, and Neuroprotection

The extensive networking of the cells of the nervous system results in large cell membrane surface areas. We now know that neuronal membranes contain phospholipid pools that are the reservoirs for the synthesis of specific lipid messengers on neuronal stimulation or injury. These messengers in turn participate in signaling cascades that can either promote neuronal injury or neuroprotection. Prostaglandins are synthesized as a result of cyclooxygenase activity. In the first step of the arachidonic acid cascade, the short-lived precursor, prostaglandin H2, is synthesized. Additional steps in the cascade result in the synthesis of an array of prostaglandins, which participate in numerous physiological and neurological processes. Our laboratory recently reported that the membrane polyunsaturated fatty acid, docosahexaenoic acid, is the precursor of oxygenation products now known as the docosanoids, some of which are powerful counter-proinflammatory mediators. The mediator 10,17S-docosatriene (neuroprotectin D1, NPD1) counteracts leukocyte infiltration, NF-kappa activation, and proinflammatory gene expression in brain ischemia-reperfusion and is an apoptostatic mediator, potently counteracting oxidative stress-triggered apoptotic DNA damage in retinal pigment epithelial cells. NPD1 also upregulates the anti-apoptotic proteins Bcl-2 and Bcl-xL and decreases pro-apoptotic Bax and Bad expression. Another biologically active messenger derived from membrane phospholipids in response to synaptic activity is platelet-activating factor (PAF). The tight regulation of the balance between synthesis (via phospholipases) and degradation (via acetylhydrolases) of PAF modulates the functions of this lipid messenger. Under pathological conditions, this balance is tipped, and PAF becomes a proinflammatory mediator and neurotoxic agent. The newly discovered docosahexaenoic acid signaling pathways, as well as other lipid messengers related to synaptic activation, may lead to the clarification of clinical issues relevant to stroke, age-related macular degeneration, spinal cord injury, Alzheimer's disease, and other diseases that include neuroinflammatory components.

Adverse Effects of COX-2 Inhibitors

Cyclooxygenase-2 selective inhibitors (COXIBs) were developed with the prime object of minimizing gastrointestinal adverse effects, which are seen with the use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs). Their long-term use is limited by the development of hypertension, edema, and congestive heart failure in a significant proportion of patients. NSAIDs block the activity of both COX isozymes, COX-1 and COX-2, which mediate the enzymatic conversion of arachidonate to prostaglandin H2 (PGH2) and other prostaglandin (PG) metabolites. It is well established that the cardiovascular profile of COX-2 inhibitors can be accounted for by inhibition of COX-dependent PG synthesis. Following the COX-mediated synthesis of PGH2 from arachidonate, PGH2 is metabolized to one of at least five bioactive PGs, including PGE2, PGI2, PGF2, PGD2, or thromboxane A2 (TXA2). These prostanoids have pleiotropic cardiovascular effects, altering platelet function and renal function, and they are acting either as vasodilators or vasoconstrictors. Although COX-1 and COX-2 exhibit similar biochemical activity in converting arachidonate to PGH2in vitro, the ultimate prostanoids they produce in vivo may be different due to differential regulation of COX-1 and COX-2, tissue distribution, and availability of the prostanoid synthases. PGs have been established as being critically involved in mitigating hypertension, helping to maintain medullary blood flow (MBF), promoting urinary salt excretion, and preserving the normal homeostasis of thrombosis, and the researchers found that the use of COX-2 inhibitors caused many serious complications in altering the normal body homeostasis. The purpose of the present research is to explain briefly the side effects of COX-2 inhibitors on the renal and cardiovascular system.

Microsomal prostaglandin E synthase-1: the inducible synthase for prostaglandin E2

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the conversion of prostaglandin (PG)H2 to PGE2. Proinflammatory stimuli markedly increase levels of mPGES-1 expression both in vivo and in vitro. mPGES-1 knockout studies and animal models of inflammatory arthritis also provide a strong basis for the contribution of mPGES-1 in the increased local production of PGE2 observed in inflammatory arthritis. The focus of this article is to review some recent advances in our understanding of mechanisms specific to the regulation of inducible mPGES-1 in inflammatory arthritis.

Thromboxane synthase (TBXAS1) polymorphisms in African-American and Caucasian populations: evidence for selective pressure

Thromboxane synthase (TBXAS1), a cytochrome P450 enzyme, converts prostaglandin H2 into thromboxane A2, a potent vasoconstrictor and inducer of platelet aggregation. Thromboxane A2 has been implicated in modulating cell cytotoxicity and in tumor growth and metastasis. Twelve coding-region variants were identified in the human TBXAS1 gene in 48 African-American and 46 Caucasian individuals, of which eight were amino-acid substitutions. The latter were confirmed in an independent Caucasian population (n=94 unrelated individuals). We performed an evolutionary analysis of patterns of nucleotide diversity and identified patterns of amino acid replacement in human-mouse comparisons consistent with purifying selection on an inter-species time scale using the McDonald-Kreitman test. We also observed patterns of nucleotide diversity within humans consistent with purifying selection acting on existing polymorphism using Tajima's D within coding regions. These evolutionary tests suggest that some of the rare coding variations observed in the human population are deleterious. We used two sequence-homology-based software programs and molecular modeling to predict the potential impact of these polymorphisms on TBXAS1 function. The c.772C>T (p.Lys258Glu), c.1249C>G (p.Gln417Glu), and c.1348G>A (p.Glu450Lys) substitutions are predicted as most likely to alter protein function; another, c.1352C>A (p.Thr451Asn), may also affect function. Given the evolutionary evidence, these variants may be functional and therefore of relevance for disease endpoints related to inflammation and angiogenesis, as well as for the pharmacogenetics of non-steroidal anti-inflammatory drugs.

Parallel synthesis and in vitro activity of novel anthranilic hydroxamate-based inhibitors of the prostaglandin H2 synthase peroxidase activity

Currently available non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are directed at the cyclooxygenase (COX) site, but not the peroxidase (POX) activity of prostaglandin H2 synthase (PGHS). They are thus unable to inhibit the free-radical induced tissue injury associated with PGHS peroxidase activity, which can occur independently of the COX site. A lead compound, anthranilic hydroxamic acid (AHA) was found to have significant PGHS-POX inhibitory activity (IC50= 72 microM). To define the critical parameters for PGHS-POX inhibition, we investigated 29 AHA derivatives, synthesised from their acid precursors, using solid phase synthesis. In vitro analysis demonstrated a ten-fold improvement in inhibition with 3,5-diiodoanthranilic hydroxamic acid (IC50= 7 microM).

Higher viscosity participates in the regulation of coronary flow via nitric oxide and indomethacin-sensitive contracting factor

Few studies have reported on the association of viscosity with coronary circulation. We evaluated the change in coronary flow after dextran was added to a perfusion solution to increase viscosity in isolated rat hearts. We also measured NOx- production induced by the change in shear stress in the coronary effluent, as a marker of NO synthesis. The baseline coronary flow was not influenced by the presence of either the cyclooxygenase inhibitor indomethacin, the thromboxane A2 (TXA2)-prostaglandin H2 (PGH2) receptor antagonist ONO-3708, or the TXA2 synthase inhibitor OKY-046. After exposure to solution containing 0.5% dextran, the coronary flow first decreased and then gradually increased until 10 min. The initial decrease in coronary flow was inhibited by indomethacin, ONO-3708, and OKY-046 individually. The gradual increase was completely inhibited by the NO inhibitor L-NAME, but not by indomethacin or ONO-3708. OKY-046 partially inhibited the increase. NOx- levels in the effluent were higher after the dextran solution was administered, and the increased NOx- levels were inhibited by L-NAME. The increased NOx- levels were not inhibited by inhibitors of the cyclooxygenase pathway. It appears that a higher viscosity of perfusion solution induced a gradual increase in NO production and was associated with increased production of indomethacin-sensitive contracting factor.

Argan (Argania spinosa) oil lowers blood pressure and improves endothelial dysfunction in spontaneously hypertensive rats

Traditionally hand-pressed argan oil, obtained from Argania spinosa seeds, is eaten raw in south-west Morocco; its rich composition of tocopherols, MUFA and PUFA make a study of its actions on risk factors for CVD, such as hypertension, interesting. The effects of 7 weeks of treatment with argan oil (10 ml/kg) on the blood pressure and endothelial function of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats were investigated. Systolic blood pressure and heart rate were measured every week by the tail-cuff method and endothelial function was assessed by carbachol (10(-8) to 10(-4) M)-induced relaxations of aortic rings and small mesenteric arteries pre-contracted with phenylephrine. Argan-oil administration reduced the mean blood pressure of SHR after the fifth week of treatment (P<0.05) and increased (P<0.01) the endothelial responses of arteries from SHR. The NO synthase inhibitor, L-N-omega-nitroarginine (3 x 10(-5) M) revealed a greater participation of NO in the relaxant effect after the treatment. When cyclooxygenase (COX) was blocked with indomethacin (10(-5) M), an involvement of COX products in the endothelium-dependent response was characterized. Enzyme immunoassay of thromboxane B2 showed a significant decrease (P<0.05) in the release of thromboxane A2 in both aorta and small mesenteric artery after argan-oil treatment of SHR. Experiments in the presence of the thromboxane A2-prostaglandin H2 receptor antagonist ICI 192,605 (10(-5) M) confirmed this result. Results after incubation with the antioxidants superoxide dismutase and catalase suggested that a decreased oxidative stress might contribute to explain the beneficial effects of argan-oil treatment.

On the mechanism of biosynthesis of 19-hydroxyprostaglandins of human seminal fluid and expression of cyclooxygenase-2, PGH 19-hydroxylase (CYP4F8) and microsomal PGE synthase-1 in seminal vesicles and vas deferens

The predominating prostaglandins of human seminal fluid are 19 R-hydroxyprostaglandins E 1 and E 2, conceivably formed sequentially by prostaglandin H (PGH) synthase-2, PGH 19-hydroxylase (CYP4F8), and microsomal PGE synthase-1 of seminal vesicles. Our aim was to study this enzyme system. Quantification by real-time PCR suggested that the transcripts of PGH synthase-2, CYP4F8, and microsomal PGE synthase-1 were abundant and correlated in seminal vesicles of seven patients ( p < 0.05). The three enzymes were detected in seminal vesicles by Western blot analysis, and immunohistological analysis confirmed the localization to the epithelia of seminal vesicles and distal vas deferens. Immunofluorescence analysis showed co-localization of the three enzymes in epithelial cells of seminal vesicles and vas deferens. 19-Hydroxy-PGE compounds were detected by mass spectrometry in the mucosa of distal vas deferens. Recombinant CYP4F8 catalyzes n-2 hydroxylation of PGH 1 and PGH 2 and n-3 hydroxylation of arachidonic acid. Arachidonic acid was oxidized to 18-hydroxyarachidonic acid and to PGE 2 and by microsomes of seminal vesicles in the presence of NADPH and GSH, and to relatively small amounts of 19-hydroxy-PGE 2. We conclude that PGH synthase-2, CYP4F8, and PGE synthase-1 likely forms 19-hydroxy-PGE compounds in seminal vesicles and vas deferens, but the catalytic properties of CYP4F8 suggest additional biological functions. Recombinant CYP4F8 was also found to catalyze n-2 hydroxylation of PGI 2 and carbaprostacyclin ( K m ∼ 40 μM), and n-2 and n-3 hydroxylation of carbocyclic TXA 2.

110 Reduced Expression of Microsomal Prostaglandin Synthase 1 Attenuates Ventilatory Effects of Interleukin-1â in Neonatal DBA/1LACJ Mice

Background: Infection and apnea represent major medical concerns in the neonatal population, and the pro-inflammatory cytokine interleukin-1Â (IL-1Â ) may serve as a critical link between these events. We recently showed that IL-1Â depresses respiration and anoxic survival via a prostaglandin-dependent mechanism (Olsson et al, 2003). In brain, microsomal prostaglandin E synthase 1 (mPGES-1) has been demonstrated crucial for the development of endotoxin-induced fever and is widely expressed in endothelial cells of the blood-brain-barrier after IL-1Â provocation. These same cells also express the IL-1 receptor as well as cyclooxygenase-2, which catalyzes the formation of prostaglandin H2 (PGH2) from arachidonic acid. mPGES-1 subsequently synthesizes prostaglandin E2 (PGE2) from PGH2. This study tested the hypothesis that IL-1Â alters respiratory control in newborn mice via a PGE2-mediated pathway.Methods: Respiratory behavior was examined using flow plethysmography in 9 day-old DBA/1lacJ wildtype mice and heterozygote knockout mice for mPGES-1. At 70 min after an intraperitoneal injection of either IL-1Â or saline, mice were placed unrestrained in the plethysmograph chamber. Basal respiration and the ventilatory response to anoxia (100% N2) and hyperoxia (100% O2) were then examined. Genotyping was performed after experimentation in all animals.Results: During normoxia, wildtype mice given IL-1Â exhibited a reduced respiratory frequency and tidal volume compared to control animals. Heterozygote knockout mice for mPGES-1 experienced less depression of basal respiration by IL-1Â. In response to anoxia, the IL-1Â -treated wildtype mice displayed fewer gasps and were unable to sustain gasping efforts for as long as control animals. They also were less able to autoresuscitate and survive following severe hypoxic apnea compared to control animals. These deleterious effects of IL-1Â were prevented in mice with a reduced expression of mPGES-1. Skin temperature was similar between groups prior to and following experimentation, suggesting that the results observed here are not due to confounding systemic changes induced by IL-1Â.Conclusion: This study indicates that IL-1Â adversely affects respiration and autoresuscitation in newborn mice and that these changes are mediated by PGE2. These findings may have clinical implications for the screening and treatment of neonatal apnea associated with infection.

Crystal structure of Y10F mutant of Sh28GST: insight into GSH activation mechanism and isomerisation of prostaglandin H2 to prostaglandin D2

Crystal structure of Y10F mutant of Sh28GST: insight into GSH activation mechanism and isomerisation of prostaglandin H2 to prostaglandin D2

Role of Ca2+-independent phospholipase A2 and n−3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A2 and cyclooxygenases. These enzymes release the prostaglandin precursor, the n−6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H2. Docosahexaenoic acid, which belongs to the n−3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca2+ signaling, which results in changes of activity of Ca2+-dependent phospholipase A2, hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A2, i.e. Ca2+-dependent phospholipase A2 and Ca2+-independent phospholipase A2, respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca2+- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca2+-independent phospholipase A2 and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca2+-independent phospholipase A2 and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.

Modification of prostanoid secretion in endothelial cells by amphotericin B acting synergistically with interleukin-1beta: possible explanation of proinflammatory effects.

Human umbilical vein endothelial cells were exposed to free-form and lipid-complexed versions of amphotericin B (alone or in combination with human recombinant interleukin [IL]-1 beta) and to culture medium from the human macrophage cell line THP-1 that had been exposed to amphotericin B. Endothelial cells were then incubated with exogenous-labeled arachidonic acid or were stimulated with histamine. Measurement of the resulting prostanoids indicated that amphotericin B and IL-1 beta acted synergistically to increase the ability of endothelial cells to synthesize prostanoids from endogenous and exogenous substrate and to increase expression of cyclooxygenase-2. This resulted in an increase of the ratio of untransformed prostaglandin (PG) H2 to PGI2 released by endothelial cells. Culture medium from amphotericin B-activated macrophages caused similar effects in endothelial cells. The synergistic effect with IL-1 beta was observed with free-form amphotericin B and, to a lesser extent, with lipid-complexed amphotericin B (Abelcet). Differences between Abelcet and the lipisome carrier (AmBisome) were not significantly different with respect to any of the parameters analyzed.

TP receptors regulate renal hemodynamics during angiotensin II slow pressor response.

We investigated the hypothesis that thromboxane A2 (TxA2)-prostaglandin H2 receptors (TP-Rs) mediate the hemodynamic responses and increase in reactive oxygen species (ROS) to ANG II (400 ng x kg(-1) x min(-1) sc for 14 days) using TP-R knockout (TP -/-) and wild-type (+/+) mice. TP -/- had normal basal mean arterial blood pressure (MAP) and glomerular filtration rate but reduced renal blood flow and increased filtration fraction (FF) and renal vascular resistance (RVR) and markers of ROS (thiobarbituric acid-reactive substances and 8-isoprostane PGF2alpha) and nitric oxide (NOx). Infusion of ANG II into TP +/+ increased ROS and thromboxane B2 (TxB2) and increased RVR and FF. ANG II infusion into TP -/- mice reduced ANG I and increased aldosterone but caused a blunted increase in MAP (TP -/- : +6 +/- 2 vs. TP +/+: +15 +/- 3 mmHg) and failed to increase FF, ROS, or TxB2 but increased NOx and paradoxically decreased RVR (-2.1 +/- 1.7 vs. +2.6 +/- 0.8 mmHg x ml(-1) x min(-1) x g(-1)). Blockade of AT1 receptor of TP -/- mice infused with ANG II reduced MAP (-8 mmHg) and aldosterone but did not change the RVR or ROS. In conclusion, during an ANG II slow pressor response, AT1 receptors activate TP-Rs that generate ROS and prostaglandins but inhibit NO. TP-Rs mediate all of the increase in RVR and FF, part of the increase in MAP, but are not implicated in the suppression of ANG I or increase in aldosterone. TP -/- mice have a basal increase in RVR and FF associated with ROS.

Downregulation of COX-2 and JNK expression after induction of ischemic tolerance in the gerbil brain

The response of the inducible isoform of the prostaglandin H2 synthase (COX-2) and the c-Jun N-terminal kinase (JNK) in post-ischemic neuronal damage was assessed in a model of ischemic tolerance in Mongolian Gerbils. After a single 6-min bilateral carotid occlusion, histological damage was evident in the CA1 region of hippocampus, correlated with a high expression of JNK and COX-2 mRNA. However, in the group of animals with a 2-min ischemia and the tolerance group, in which a 2-min bilateral carotid occlusion was followed 3 days later by a 6-min ischemia, no hippocampal or cortical damage was detected. Accordingly, the JNK and COX-2 mRNA levels remained unaffected. We suggest that the level of JNK and COX-2 expression may determine the outcome as either post-ischemic cell death or tolerance.

Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus.

Macro and microvascular diseases are the principal causes of morbidity and mortality in patients with type I and II diabetes mellitus. Growing evidence implicates reactive nitrogen species (RNS), such as peroxynitrite (ONOO-), derived from nitric oxide (NO) and superoxide anion (O2*-), are important in diabetes. The mechanisms by which diabetes increases RNS, and those by which RNS modifies vascular function, are poorly understood. The authors recently discovered that physiologically relevant concentrations of ONOO- oxidize the zinc thiolate center in endothelial nitric oxide synthase (eNOS). In active eNOS dimers, a tetracoordinated zinc ion is held by four thiols, two from each 135-kDa monomer. Because it remains partially positively charged, the zinc thiolate center is subject to attack by the ONOO-. This oxidant disrupts the zinc thiolate center, releasing zinc, and oxidizing the thiols. Upon thiol reduction, eNOS dimers dissociate into monomers. This modification of eNOS results in reduced NO bioactivity and enhanced endothelial O2*- production, which reacts with NO, further generating ONOO- (eNOS uncoupling). In addition, the authors' studies also demonstrate that low concentrations of ONOO- selectively nitrate and inactivate prostacyclin synthase (PGIS), which not only eliminates the vasodilatory, growth-inhibiting, antithrombotic, and antiadhesive effects of prostacyclin (PGI2), but also increases release of the potent vasoconstrictor, prothrombotic, growth- and adhesion-promoting agents, prostaglandin H2 (PGH2) and thromboxane A2 (TxA2). In diabetic mice and rats, eNOS is uncoupled resulting in an increased tyrosine nitration of PGIS. The authors' studies indicate that in diabetes the synthetic enzymes of the two major endogenous vasodilators undergo oxidative inactivation by different mechanisms, which are, however, tightly interdependent.