The enzymology of the human prostanoid pathway.

Prostanoids are short-lived autocrine and paracrine signaling molecules involved in a wide range of biological functions. They have been shown to be intimately involved in many different disease states when their regulation becomes dysfunctional. In order to fully understand the progression of any disease state or the biological functions of the well state, a complete evaluation of the genomics, proteomics, and metabolomics of the system is necessary. This review is focused on the enzymology for the enzymes involved in the synthesis of the prostanoids (prostaglandins, prostacyclins and thromboxanes). In particular, the isolation and purification of the enzymes, their enzymatic parameters and catalytic mechanisms are presented.

[Interaction of prostacyclin synthase with cytochromes P450]. / Vzaimodeistvie prostatsiklinsintazy s tsitokhromami P450.

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.

Dominant Role for Regulatory T Cells in Protecting Females Against Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females. OBJECTIVE: To evaluate whether and how Treg deficiency differentially affects male and female rats in experimental PH. METHODS AND RESULTS: Male and female athymic rnu/rnu rats, lacking Tregs, were treated with the VEGFR2 (vascular endothelial growth factor receptor 2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution before SU5416 administration. Plasma PGI2 (prostacyclin) levels were measured. Lung and right ventricles were assessed for the expression of the vasoprotective proteins COX-2 (cyclooxygenase 2), PTGIS (prostacyclin synthase), PDL-1 (programmed death ligand 1), and HO-1 (heme oxygenase 1). Inhibitors of these pathways were administered to athymic rats undergoing Treg immune reconstitution. Finally, human cardiac microvascular endothelial cells cocultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and ER (estrogen receptor) expression, and culture supernatants were assayed for PGI2 and IL (interleukin)-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented right ventricular fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1, and PDL-1 expression and reduced right ventricular PDL-1 levels. In both sexes, Treg immune reconstitution protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and PD-1 (programmed death 1)/PDL-1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL-1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10. CONCLUSIONS: In 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

No effects of atorvastatin (10 mg/d or 80 mg/d) on nitric oxide, prostacyclin, thromboxane and oxidative stress in type 2 diabetes mellitus patients of the DALI study.

The present study describes the effects of atorvastatin on whole body synthesis of nitric oxide (NO), prostacyclin (PGI2), and thromboxane A2 (TxA2), on oxidative stress and nitrite/nitrate-related renal carbonic anhydrase (CA) activity in patients with type 2 diabetes mellitus (T2DM). A double-blind, randomized, placebo-controlled parallel-group trial (the DALI study group) on 217 patients with T2DM and dyslipidemia was performed. Urinary samples were collected before and after administration of a standard dose (10 mg/d, n=73), a maximal dose atorvastatin (80 mg/d, n=72) or placebo (n=72) for 30 weeks. Urinary nitrite and nitrate were measured to assess whole body NO synthesis. The urinary molar ratio of nitrate to nitrite (UNOxR) served as a measure of renal CA activity. Free radical- and cyclooxygenase (COX)-catalyzed lipid peroxidation was estimated by measuring urinary 8-iso-prostaglandin F2α (8-iso-PGF2α). In subgroups, systemic PGI2 and TxA2 synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-6-keto-prostaglandin F1α and 2,3-dinor-thromboxane B2, respectively. All biochemical parameters were measured by GC-MS and GC-MS/MS methods. T2DM patients had elevated levels of nitrate, nitrite, UNOxR, and 8-iso-PGF2α compared to healthy non-diabetic and normolipidemic subjects. Thirty-week treatment with atorvastatin (10 or 80 mg/d) did not significantly alter NO, PGI2, TxA2 and 8-iso-PGF2α synthesis and did not improve the renal reabsorption of nitrite which is considered an important reservoir of NO. Our study suggests that atorvastatin (10 or 80 mg/d) does not provide cardiovascular benefit beyond its cholesterol lowering effect in patients with T2DM.

Elevated prostacyclin biosynthesis in mice impacts memory and anxiety-like behavior.

Prostacyclin is an endogenous lipid metabolite with properties of vasodilation and anti-platelet aggregation. While the effects of prostacyclin on the vascular protection have been well-documented, the role of this eicosanoid in the central nervous system has not been extensively studied. Recently, a transgenic mouse containing a hybrid enzyme, of cyclooxygenase-1 linked to prostacyclin synthase, was developed that produces elevated levels of prostacyclin in vivo. The goal of this study was to investigate whether increased prostacyclin biosynthesis could affect behavioral phenotypes in mice. Our results uncovered that elevated levels of prostacyclin broadly affect both cognitive and non-cognitive behaviors, including decreased anxiety-like behavior and improved learning in the fear-conditioning memory test. This study demonstrates that prostacyclin plays an important, but previously unrecognized, role in central nervous system function and behavior.

The effects of calcineurin inhibitors on prostanoid synthesis: a randomized cross-over study in healthy humans.

The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus (Tac) are implicated in post-transplant complications such as cardiovascular morbidity. Prostanoids are fatty acid-derived compounds essential for controlling cardiovascular homeostasis. We tested the hypothesis that CNIs suppress cyclooxygenase (COX)-2-derived prostacyclin (PGI) and increase thromboxane synthesis in humans. Ten healthy men underwent 5-h infusions of CsA, Tac, and saline in a randomized, double-blind, cross-over study. Blood and urine samples were collected before and after the infusion of each drug/saline, to measure PGI and thromboxane metabolites. CsA decreased whole-blood COX-2 activity by 39% (P = 0.05) and basal plasma 6-keto-PGF(1α) levels by 31%, only nonsignificantly. Urine excretion of PGI-M and TxB(2) did not change significantly after CsA infusion. Tac decreased TxB(2) in the COX-1 ex vivo assay by 30% (P = 0.03), while no changes were seen in urinary levels of PGI-M or TxB(2) . Urinary TxB(2) excretion was 15% lower after saline infusion (P = 0.03). These within-treatment differences in prostanoid synthesis did not differ significantly between the treatments (anova). Mean blood levels of CNIs were 486 µg/l for CsA and 12.8 µg/l for Tac. Clinically relevant doses of CsA and Tac induce acute differential changes in prostanoid levels in healthy human subjects. CsA suppresses COX-2 activity, while Tac decreases platelet activity.

A biological rationale for the cardiotoxic effects of rofecoxib: comparative analysis with other COX-2 selective agents and NSAids.

Clinical investigations have demonstrated a relationship between the extended use of rofecoxib and increased risk for atherothrombotic events. This has led to the removal of rofecoxib from the market and explicit cardiovascular safety warnings for other COX-2 selective and non-selective agents that remain on the market. Early explanations for the cardiotoxicity of rofecoxib, such as the relative cardioprotective effect of comparator agents (naproxen) or an "imbalance" between thromboxane and prostacyclin biosynthesis due to an absence of concomitant aspirin use, have not been substantiated by the evidence. New experimental findings indicate that the cardiotoxicity of rofecoxib is not a general class effect but may be due to its intrinsic chemical structure and unique primary metabolism. Specifically, rofecoxib has been shown to increase the susceptibility of human LDL and cell membrane lipids to oxidative modification, a hallmark feature of atherosclerosis. Rofecoxib was also found to promote the non-enzymatic formation of isoprostanes from biological lipids, which act as important mediators of inflammation in the atherosclerotic plaque. The explanation for such cardiotoxicity is that rofecoxib forms a reactive maleic anhydride in the presence of oxygen due to its chemical structure and primary metabolism (cytoplasmic reductase). By contrast, adverse effects on rates of LDL and membrane lipid oxidation were not observed with other chemically distinct (sulfonamide) COX-2 inhibitors under identical conditions. These findings provide a compelling rationale for distinguishing the differences in cardiovascular risk among COX-selective inhibitors on the basis of their intrinsic physico-chemical properties.

Nitric oxide reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin production in mice.

We examined whether nitric oxide (NO), derived from constitutive NO synthase (NOS) and/or inducible NOS (iNOS), could contribute to endotoxin-induced inflammatory hyperalgesia via interacting with nuclear factor-kappaB (NF-kappaB), inducible cyclooxygenase (COX-2) and/or polyADP-ribose synthase (PARS). Injection of endotoxin (10 mg kg(-1), i.p.) to mice elicited hyperalgesia, determined by hot plate test, which is prevented by NO precursor (L-arginine), cNOS/iNOS inhibitor (N(G)-nitro-L-arginine methyl ester; L-NAME), NF-kappaB inhibitor (N-acetylserotonin), COX inhibitor (indomethacin), COX-2 inhibitor (DFU) and PARS inhibitor (3-aminobenzamide). Endotoxin caused a decrease in serum nitrite levels prevented by N-acetylserotonin, L-arginine, indomethacin, DFU or 3-aminobenzamide. Endotoxin increased serum 6-keto-PGF(1alpha) levels diminished by L-arginine or aminoguanidine (iNOS inhibitor). L-Arginine, L-NAME, aminoguanidine, DFU or 3-aminobenzamide prevented endotoxin-induced decrease in heart, lungs, kidneys and brain nitrite and malonedialdehyde levels and myeloperoxidase activity. In conclusion, NO reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin production, and also contributes to the analgesic effect of NF-kappaB, COX or PARS inhibitors.

Signaling via the angiotensin-converting enzyme enhances the expression of cyclooxygenase-2 in endothelial cells.

Angiotensin-converting enzyme (ACE) inhibitors elicit outside-in signaling via ACE in endothelial cells. This involves the CK2-mediated phosphorylation of ACE on Ser1270 and the activation of the c-Jun N-terminal kinase (JNK)/c-Jun pathway, resulting in an enhanced endothelial ACE expression. Because cyclooxygenase-2 (COX-2) expression is reported to be increased in subjects treated with ACE inhibitors, we determined the role of ACE signaling in this phenomenon and the transcription factors involved. In lungs from mice treated with the ACE inhibitor ramipril for 5 days, COX-2 expression was increased. A similar (1.5- to 2-fold) increase in COX-2 protein was detected in primary cultures of human endothelial cells treated with ramiprilat. In an endothelial cell line stably expressing human somatic ACE, ramiprilat increased COX-2 promoter activity, an effect not observed in ACE-deficient cells or cells expressing a nonphosphorylatable ACE mutant (S1270A). The ramiprilat-induced, ACE-dependent increase in COX-2 expression and promoter activity (both 1.5- to 2-fold greater than control) was prevented by the inhibition of JNK. Ramiprilat significantly enhanced the DNA binding activity of activator protein-1 in cells expressing ACE but not S1270A ACE. Activator protein-1 decoy oligonucleotides prevented the ACE inhibitor-induced increase in COX-2 promoter activity and protein expression. As a consequence of the ramiprilat-induced increase in COX-2 expression, prostacyclin and prostaglandin E2, but not thromboxane A2, production was increased and was inhibited by the COX-2 inhibitor celecoxib. These results indicate that ACE signaling may underlie the increase in COX-2 and prostacyclin levels in patients treated with ACE inhibitors.

Clinical pharmacology of platelet, monocyte, and vascular cyclooxygenase inhibition by naproxen and low-dose aspirin in healthy subjects.

BACKGROUND: The current controversy on the potential cardioprotective effect of naproxen prompted us to evaluate the extent and duration of platelet, monocyte, and vascular cyclooxygenase (COX) inhibition by naproxen compared with low-dose aspirin. METHODS AND RESULTS: We performed a crossover, open-label study of low-dose aspirin (100 mg/d) or naproxen (500 mg BID) administered to 9 healthy subjects for 6 days. The effects on thromboxane (TX) and prostacyclin biosynthesis were assessed up to 24 hours after oral dosing. Serum TXB2, plasma prostaglandin (PG) E2, and urinary 11-dehydro-TXB2 and 2,3-dinor-6-keto-PGF(1alpha) were measured by previously validated radioimmunoassays. The administration of naproxen or aspirin caused a similar suppression of whole-blood TXB2 production, an index of platelet COX-1 activity ex vivo, by 94+/-3% and 99+/-0.3% (mean+/-SD), respectively, and of the urinary excretion of 11-dehydro-TXB2, an index of systemic biosynthesis of TXA2 in vivo, by 85+/-8% and 78+/-7%, respectively, that persisted throughout the dosing interval. Naproxen, in contrast to aspirin, significantly reduced systemic prostacyclin biosynthesis by 77+/-19%, consistent with differential inhibition of monocyte COX-2 activity measured ex vivo. CONCLUSIONS: The regular administration of naproxen 500 mg BID can mimic the antiplatelet COX-1 effect of low-dose aspirin. Naproxen, unlike aspirin, decreased prostacyclin biosynthesis in vivo.

Prostacyclin production in rat aortic smooth muscle cells: role of protein kinase C, phospholipase D and cyclooxygenase-2 expression.

OBJECTIVE: The present study was designed to investigate the role of protein kinase C (PKC) and phospholipase D (PLD) in angiotensin II (AngII)- and phorbol ester (PMA)-induced cyclooxygenase-2 (COX-2) expression and prostacyclin (PGI(2)) production in rat aortic smooth muscle cells (VSMC). METHODS: Prostacyclin production in cultured VSMC was determined by radioimmunoassay. PKC activity was examined by measuring the transfer of 32P from (gamma-32P)ATP to histone III-S. COX-2 expression was determined by Western blotting. To measure PLD activity, thin layer chromatography was used. RESULTS: AngII (50 nM) and PMA (100 nM) promoted the translocation of PKC activity from the cytosol to the membranes within 30 min, followed by a strong increase in PLD activity as well as COX-2 expression and PGI(2) production. After 48 h exposure to PMA, PKC was downregulated resulting in a complete suppression of its activity. PKC-downregulation and the PKC inhibitor CGP41251 abolished PMA- and AngII-induced PLD activation, suppressed the stimulatory effect of PMA on COX-2 expression and PGI(2) production and strongly inhibited that of AngII. Furthermore, AngII- and PMA-induced PGI(2) production depended on protein synthesis and COX-2 but not COX-1 activity. Inhibition of PLD-mediated phosphatidic acid (PA) formation by 1% 1-butanol abolished AngII-induced COX-2 expression and PGI(2) secretion, while dioctanoyl PA increased COX-2 expression and PGI(2) production in a time- and concentration-dependent manner. CONCLUSION: Our results indicate that in VSMC, AngII promotes PGI(2) production to a large extent through a rise in COX-2 expression which is mediated by PA generated from increased PKC-dependent PLD activity.

[Effect of ligustrazine on cell cycle and prostacyclin of injured vascular endothelial cells induced by hypoxia and lack of glucose].

OBJECTIVE: To study the effects of ligustrazine on cell cycle and prostacyclin of injured human umbilical vein vascular endothelial cell line induced by hypoxia and lack of glucose. METHODS: The experiments were performed by culturing vascular endothelial cell induced by hypoxia and lack of glucose in vitro. Cell viability was assessed by MTT assay and cell cycle was observed by flow cytometry. Radioimmunoassay (RIA) was used to assess the amount of prostacyclin (PGI2). RESULTS: The vascular endothelial cell viability and PGI2 were decreased in condition of hypoxia and lack of glucose-injury. Preincubation of vascular endothelial cell with Ligustrazine for 24 h significantly increased the cell viability, S-Phase cell, G2M-phase cell and PGI2 level. CONCLUSION: These results demonstrated that ligustrazine had protective effect on hypoxia and lack of glucose-injured vascular endothelial cell and the effect may be related to increasing cell viability, S-phase cell, G2M-phase cell and level of PGI2.