Impact of Multidrug Resistance Protein-4 Inhibitors on Modulating Platelet Function and High on-Aspirin Treatment Platelet Reactivity.

Platelet multidrug resistance protein 4 (MRP4) plays a modulating role on platelet activation. Platelet function and thrombus formation are impaired in MRP4 knockout mice models, and, among aspirin-treated patients, high on-aspirin residual platelet reactivity (HARPR) positively correlates with MRP4 levels. To better understand the effects of MRP4 on platelet function, the aim of this investigation was to assess the impact of cilostazol-induced inhibition of MRP4-mediated transport and assess aspirin-induced antiplatelet effects and rates of HARPR in human subjects.Cilostazol-dependent inhibition of MRP4-mediated transport was assessed with the release of the fluorescent adduct bimane-glutathione and aspirin entrapment. Effect of Cilostazol on cAMP inhibition was evaluated by vasodilator-stimulated phosphoprotein (VASP). Platelet function was studied by collagen and TRAP-6-induced platelet aggregation and secretion.Cilostazol reduced the release of bimane-glutathione and enhanced aspirin entrapment demonstrating an inhibitory effect on MRP4 in platelets. VASP phosphorylation was absent until 10 seconds after addition of cilostazol, and becomes evident after 30 seconds. An inhibitory effect on platelet aggregation and secretion was found in activated platelets, with threshold concentration of agonists, 10 seconds after addition of cilostazol, supporting a role of MRP4 on platelet function that is cAMP independent. Cilostazol effects were also shown in aspirin-treated platelets. A reduction of platelet aggregation and secretion were observed in aspirin-treated patients with HARPR.This study supports the role of MRP4 on modulating platelet function which occurs through cAMP-independent mechanisms. Moreover, inhibition of MRP4 induced by cilostazol enhances aspirin-induced antiplatelet effects and reduces HARPR.

PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2.

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.

Characterization of scavengers of gamma-ketoaldehydes that do not inhibit prostaglandin biosynthesis.

Expression of cyclooxygenase-2 (COX-2) is associated with the development of many pathologic conditions. The product of COX-2, prostaglandin H(2) (PGH(2)), can spontaneously rearrange to form reactive gamma-ketoaldehydes called levuglandins (LGs). This gamma-ketoaldehyde structure confers a high degree of reactivity on the LGs, which rapidly form covalent adducts with primary amines of protein residues. Formation of LG adducts of proteins has been demonstrated in pathologic conditions (e.g., increased levels in the hippocampus in Alzheimer's disease) and during physiologic function (platelet activation). On the basis of knowledge that lipid modification of proteins is known to cause their translocation and to alter their function, we hypothesize that modification of proteins by LG could have functional consequences. Testing this hypothesis requires an experimental approach that discriminates between the effects of protein modification by LG and the effects of cyclooxygenase-derived prostanoids acting through their G-protein coupled receptors. To achieve this goal, we have synthesized and evaluated a series of scavengers that react with LG with a potency more than 2 orders of magnitude greater than that with the epsilon-amine of lysine. A subset of these scavengers are shown to block the formation of LG adducts of proteins in cells without inhibiting the catalytic activity of the cyclooxygenases. Ten of these selective scavengers did not produce cytotoxicity. These results demonstrate that small molecules can scavenge LGs in cells without interfering with the formation of prostaglandins. They also provide a working hypothesis for the development of pharmacologic agents that could be used in experimental animals in vivo to assess the pathophysiological contribution of levuglandins in diseases associated with cyclooxygenase up-regulation.

Structures of prostacyclin synthase and its complexes with substrate analog and inhibitor reveal a ligand-specific heme conformation change.

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H(2). PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H(2), leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels.

Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation.

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyl-lysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH(2). They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets.

Continuous spectrophotometric assay amenable to 96-well plate format for prostaglandin E synthase activity.

The measurement of prostaglandin E synthase (PGES) activity is cumbersome because the product of the reaction, PGE(2), is not readily quantitated by spectral means. The activity of isolated PGES is typically determined by PGE(2) immunoassay or by high-performance liquid chromatography using radiolabeled substrate. A relatively rapid continuous spectrophotometric assay which uses 15-hydroxyprostaglandin dehydrogenase (PGDH) to couple the oxidation of the 15-hydroxy group of PGE(2) to the formation of NADH was developed. PGDH is relatively specific for PGE(2) over the substrate for the PGES reaction, PGH(2), allowing a highly reproducible assay of PGES activity to be obtained.

Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice.

PGE(2) has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE(2) production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE(2) than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE(2) produced, macrophage-derived IL-12 and T cell-derived IFN-gamma production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A(2) mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE(2) by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE(2) synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE(2) by macrophages, which is regulated by secretory phospholipase A(2) and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania.

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.

Prostaglandin F synthase.

Prostaglandin (PG) F2 is synthesized via three pathways from PGE2, PGD2, or PGH2 by PGE 9-ketoreductase, PGD 11-ketoreductase, or PGH 9-, 11-endoperoxide reductase, respectively. The enzymological and molecular biological properties of these enzymes have been reported in work over the last 30 years. Here, these three pathways of PGF synthesis by these enzymes are reviewed, and the physiological roles of the enzymes are discussed.

Prostacyclin is an autocrine regulator in the contraction of oviductal smooth muscle.

BACKGROUND: It was recently discovered that prostacyclin constituted 40-50% of prostaglandins (PG) produced by minced human oviduct. It is well established that prostacyclin relaxes vascular smooth muscle, but whether oviductal smooth muscle synthesizes prostacyclin and whether its contraction is affected by prostacyclin remain unclear. METHODS: Smooth muscle microdissected from human oviducts was used for the study. The expression of prostacyclin synthase (PGIS) and prostacyclin receptor (IP) was confirmed by Western blot analysis. Metabolites of [(3)H]PGH(2) were analysed for prostacyclin. Functional coupling of IP to adenyl cyclase was assessed by the accumulation of intracellular cAMP upon prostacyclin challenge. The presence of saturable, specific binding sites for prostacyclin was confirmed by binding assay. The identity of IP was further confirmed by RT-PCR and nucleotide sequence analysis. Finally, the effects of prostacyclin on muscle contraction were studied. RESULTS: Human oviductal smooth muscle expresses functionally active PGIS and IP. The IP expressed is the same as that cloned from human lung tissue. The ED(50) of prostacyclin to increase intracellular cAMP was 16 nmol/l. Prostacyclin dose-dependently decreased the amplitude of muscle contraction. CONCLUSIONS: Human oviductal smooth muscle produces prostacyclin, which, in turn, decreases its contractility. Prostacyclin may regulate embryo transport.

Placental GH, IGF-I, IGF-binding protein-1, and leptin during a glucose challenge test in pregnant women: relation with maternal body weight, glucose tolerance, and birth weight.

The prediction of birth weight may be improved by the measurement of hormones or growth factors in the mother. We measured body weight (BW) and plasma levels of placental GH (PGH), IGF-I, IGF-binding protein-1 (IGFBP-1), and leptin at the time of the glucose challenge test (GCT) in 289 women, who were pregnant with a single fetus, between 24 and 29 wk gestational age (GA). Delivery occurred 12 +/- 2 (mean +/- SD) wk later. First, we examined which variables regulate these hormonal factors. Multiple regression showed that PGH concentrations were determined by GA at sampling and were negatively related to BW. IGF-I levels were mainly determined by PGH, and also by insulin, BW, and (negatively) age. IGFBP-1 concentrations were negatively determined by BW, insulin, and IGF-I. BW was also a powerful determinant of leptin levels, with insulin as a less robust determinant. Second, we examined the relation to glucose levels. PGH, IGF-I, and IGFBP-1 concentrations were not correlated with post-GCT glucose levels and were comparable in women with a normal or disturbed GCT (glucose >/=7.8 mmol/liter; n = 72). Finally, we examined the relation with birth weight and placental weight. Birth weight, corrected for GA and stratified into percentile groups, and the ponderal index at birth were strongly related to maternal BW, but not to maternal PGH, IGF-I, or IGFBP-1 levels. Neither was maternal leptin related to birth weight, but leptin concentrations were slightly higher in women who delivered obese babies. Placental weight was not related to any of the hormonal factors. This prospective study indicates that the variation in circulating PGH, IGF-I, IGFBP-1, and leptin between 24 and 29 wk of pregnancy is strongly dependent on maternal BW, but is unrelated to glucose tolerance. In addition, the measurement of PGH, IGF-I, IGFBP-1, or leptin at the time of the GCT is not useful clinically to predict birth weight.

Involvement of protein kinase C and Na+/K+-ATPase in the contractile response induced by myricetin in rat isolated aorta.

The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.NA:noradrenalineNA+/K+-ATPase pump:sodium-potassium-activated ATPasePKC:protein kinase CPMA:phorbol 12-myristate 13-acetateTXA2:thromboxane A2The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.

[Prostaglandin E2 synthases].

Prostaglandin E2 (PGE2) is widely distributed in various tissues, and exhibits various biologically important activities. PGE2 synthase (PGES) catalyzes conversion of COX-derived PGH2 to PGE2. It now appears that there are at least three distinct types of PGES in mammals. We identified two distinct glutathione-dependent PGESs. Cytosolic PGES (cPGES), known as p23, is constitutively and ubiquitously expressed and predominantly converts COX-1-derived PGH2 to PGE2. We find that the regulation of cPGES/p23 activity in cells depends on its association with hsp90. Microsomal PGES-1 (mPGES-1), identical to MGST1-L1, is an inducible perinuclear enzyme that is functionally linked with COX-2 in marked preference to COX-1. COX-2 and mPGES-1 are essential components for delayed PGE2 synthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer. Most recently, glutathione-nonspecific mPGES-2, homologous to glutaredoxin and thioredoxin, was identified. These PGESs seem to be a potential novel target for drug development.

Ifetroban sodium: an effective TxA2/PGH2 receptor antagonist.

This review presents a comprehensive discussion on the chemistry, pharmacokinetics, and pharmacodynamics of ifetroban sodium, a new thomboxane A2/prostaglandin H2 receptor antagonist. Thromboxane A2 is an arachidonic acid product, formed by the enzyme cyclooxygenase. In contrast to other cyclooxygenase products, thromboxane A2 has been shown to be involved in vascular contraction and has been implicated in platelet activation. In general, results of clinical studies and animal experiments indicate that hypertension is associated with hyperaggregability of platelets and increased thomboxane A2 levels in blood, urine, and tissues. The precursors to thromboxane A2, prostaglandin G2, and prostaglandin H2, also bind and activate the same receptors. Thus, a receptor antagonist was thought to be an improved strategy for reversing the actions of thromboxane A2/prostaglandin H2, rather than a thromboxane synthesis inhibitor. This review describes new methods for the synthesis and analysis of ifetroban, its tissue distribution, and its actions in a variety of animal models and disease states. We describe studies on the mechanisms of how ifetroban relaxes experimentally contracted isolated vascular tissue, and on the effects of ifetroban on myocardial ischemia, hypertension, stroke, thrombosis, and its effects on platelets. These experiments were conducted on several animal models, including dog, ferret, and rat, as well as on humans. Clinical studies are also described. These investigations show that ifetroban sodium is effective at reversing the effects of thromboxane A2- and prostaglandin H2-mediated processes.

Oxidation of prostaglandin H(2) and prostaglandin H(2) analogues by human cytochromes P450: analysis of omega-side chain hydroxy metabolites and four steroisomers of 5-hydroxyprostaglandin I(1) by mass spectrometry.

The objective was to examine the NADPH-dependent oxygenation of prostaglandin H(2) (PGH(2)) and three PGH(2) analogues, 9,11-diazo-15-deoxy-PGH(2) (U51605), 9,11-epoxymethano-PGH(2) (U44069), and 11,9-epoxymethano-PGH(2) (U46619), by cytochromes P450, and to characterize the metabolites by mass spectrometry. CYP2C19, CYP4A11, CYP4F8, and liver and renal cortical microsomes oxidized the omega-side chain of U44069, U46619, and U51605, whereas only CYP4F8 oxidized the omega-side chain of PGH(2). PGH(2) was transformed to four stereoisomers of 5-hydroxy-PGI(1) by recombinant cytochromes P450. CYP4F8 formed the 5-hydroxy-PGI(1) isomers in small amounts compared to the 19-hydroxy metabolites of PGH(2). Isomers of 5-hydroxy-PGI(1) and 6-keto-PGF(1 alpha) were detectable when PGH(2) decomposed in the presence of hemin, hemoglobin, or heat-inactivated microsomes. 5-Hydroxy-PGI(1) is likely formed from PGH(2) in a pseudo-enzymatic reaction involving homolytic scission of the endoperoxide and formation of an ether between C-9 and C-6 and a carbon-centered radical at C-5, which reacts with molecular oxygen. CYP4F8 catalyzes 19-hydroxylation of PGH(2), but the absolute configuration of the 19-hydroxy group is unknown, whereas human seminal fluid contains (19R)-hydroxy-PGE(2). CYP4F8 was found to metabolize U51605 to 90% of the (19R)-hydroxy metabolite, providing further evidence in favor of a role of CYP4F8 in biosynthesis of (19R)-hydroxy PGE in human seminal vesicles. We conclude that omega-side chain hydroxylation of PGH(2) analogues may be catalyzed by many different cytochromes P450, but only CYP4F8 oxidizes the omega-side chain of PGH(2) efficiently.

Cloning and characterization of CYP4F21: a prostaglandin E2 20-hydroxylase of ram seminal vesicles.

Ram semen contains high concentrations of PGE1, PGE2, 20-hydroxy-PGE1, and 20-hydroxy-PGE2, which mainly originate from the ram seminal vesicles. The 20-hydroxy-PGE compounds are formed by a tentatively identified cytochrome P450, designated PGE2 20-hydroxylase. Our aim was to clone the enzyme and express it in yeast. Total RNA was isolated from ram seminal vesicle. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers for the CYP4 family yielded a novel cDNA sequence of a cytochrome P450. The full coding region (1584 bp) was cloned by RT-PCR and designated CYP4F21. The deduced protein sequence of CYP4F21 contained 528 amino acids and showed 74% amino acid identity with CYP4F8 of human seminal vesicles. CYP4F21 was expressed in yeast, and its catalytic properties were studied by liquid chromatography-mass spectrometry. Recombinant CYP4F21 oxidizes three stable PGH2 analogs (U44069, U46619, and U51605) and PGE2 to their 20-hydroxy metabolites, whereas PGH1, PGH2, PGE1, and PGF2alpha appeared to be poor substrates. The apparent Km for hydroxylation of PGE2 was 0.05 mM. Microsomes of ram seminal vesicles and NADPH metabolized PGE2 and the three PGH2 analogs essentially in the same way as CYP4F21. Our results suggest that CYP4F21 might be a sheep homolog to CYP4F8 of human seminal vesicles. The reproductive function of CYP4F21 is likely to biosynthesize 20-hydroxy-PGE1 and 20-hydroxy-PGE2, which is excreted by the seminal vesicles.

Substrate binding is the rate-limiting step in thromboxane synthase catalysis.

Thromboxane synthase (TXAS) is a "non-classical" cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105-130 s(-1); 2) binding of cyanide, a carbon-based ligand, is a one-step process with k(on) of 2.4 M(-1) s(-1) and k(off) of 0.112 s(-1); and 3) both imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 x 10(4) M(-1) s(-1) and 1.5 x 10(5) M(-1) s(-1) for imidazole and clotrimazole, respectively, followed by a slow conformational change with off-rate constants of 8.8 s(-1) and 0.53 s(-1), respectively. The results of our binding study indicate that the TXAS active site is hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min(-1) and that the k(cat)/K(m) for PGH2 consumption was 3 x 10(6) M(-1) s(-1). Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, indicating minimal accumulation of any heme-derived intermediates. Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state. Further kinetic analysis using a combination of rapid-mixing chemical quench and computer simulation showed that the kinetic parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2-2.0 x 10(7) M(-1) s(-1); the rate of catalytic conversion of PGH2 to TXA2 or MDA was at least 15,000 s(-1) and the lower limit of the rates for products release was 4,000-6,000 s(-1). Given that the cellular PGH2 concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with "classical" P450 enzymes.

Mesangial cells release untransformed prostaglandin H2 as a major prostanoid.

BACKGROUND: Prostaglandin H2 (PGH2) is the precursor of the other prostanoids and exhibits a vasoconstricting activity. Glomerular mesangial cells are an important source of vasoactive prostanoids in kidney. Hence, the present investigation focused on the release of untransformed PGH2 by rat glomerular mesangial cells (RGMCs). METHODS: Synthesis of prostanoid by resting and interleukin-1beta (IL-1beta)-treated (overnight) RGMCs from exogenous or endogenous arachidonic acid (AA) was assessed by high-performance liquid chromtography or enzyme immunoassay, respectively. Cyclo-oxygenase isoforms were determined by Western blotting. Release of untransformed PGH2 from exogenous AA was evaluated in RGMCs and intact glomeruli as the difference of PGF2alpha formed in the incubations performed in the presence and in the absence of SnCl2 or measuring the ability of aspirin-treated platelets to form thromboxane B2 (TXB2) in mixed incubations of platelets and RGMCs or glomeruli. RESULTS: The prostanoids formed by RGMCs were PGE2, PGF2alpha, PGI2 and PGD2. SnCl2 totally deviated formation of PGE2 and PGD2 toward PGF2alpha in resting RGMCs, whereas PGE2 was only partially deviated toward PGF2alpha in IL-1beta-treated RGMCs. The PGE2/PGD2 ratio in resting RGMCs was similar to that expected for nonenzymatic isomerization of PGH2, whereas this ratio was higher in IL-1beta-treated RGMCs, suggesting the induction of PGE synthase by IL-1beta. Aspirin-treated platelets formed TXB2 when either RGMCs or intact glomeruli were present in the incubation and formation of TXB2 was approximately fourfold higher with IL-1beta-treated RGMCs or glomeruli. CONCLUSIONS: RGMCs and intact glomeruli released substantial amounts of untransformed PGH2, which was enhanced following exposure to IL-1beta.