Simultaneous analyses of urinary eicosanoids and related mediators identified tetranor-prostaglandin E metabolite as a novel biomarker of diabetic nephropathy.

Diabetic nephropathy is a major complication of diabetes mellitus, and thus novel biomarkers are desired to evaluate the presence and progression of diabetic nephropathy. In this study, we sought to identify possible metabolites related to diabetic nephropathy among urinary eicosanoids and related mediators. Using liquid chromatogram-tandem mass spectrometry, we optimized the lipid extraction from urine using the Monospin C18 as a solid-phase extraction cartridge and measured the urinary lipid mediators in 111 subjects with type 2 diabetes mellitus as well as 33 healthy subjects. We observed that 14 metabolites differed significantly among the clinical stages of nephropathy. Among them, levels of tetranor-prostaglandin E metabolite (tetranor-PGEM), an arachidonic acid metabolite, were significantly higher in subjects with stage 1 nephropathy than in healthy subjects and increased with the progression of nephropathy. We also observed that levels of maresin-1, a docosahexaenoic acid metabolite, and leukotriene B4-ethanolamide, an arachidonoyl ethanolamide metabolite, were significantly lower in subjects with stage 3-4 nephropathy than in healthy subjects and those with stage 1-2 nephropathy. Finally, using a comprehensive analysis of urinary eicosanoids and related mediators, we concluded that tetranor-PGEM was capable of discriminating clinical stages of nephropathy and thus useful as a novel biomarker for diabetic nephropathy.

Discovery of an 8-aza-5-thiaProstaglandin E1 analog as a highly selective EP4 receptor agonist.

For the purpose of discovering an orally available EP4 subtype-selective agonist, a series of 8-aza prostaglandin E(1) (PGE(1)) analogs were synthesized and evaluated for their affinity for PGE(2) receptor subtypes. Additionally, the structure-activity relationships of these compounds were studied. Among the tested compounds, the 8-aza PGE(1) analog 6 and 8-aza-5-thiaPGE(1) analog 12 had highly potent EP4 receptor affinity, good functional activity, and excellent subtype-selectivity. Furthermore, these analogs demonstrated good stability in human liver microsomes. As a result, we concluded that these two series of 8-aza PGE(1) analogs could be promising chemical leads for an orally available EP4 subtype-selective agonist.

Upregulation of cyclooxygenase-2 by motorcycle exhaust particulate-induced reactive oxygen species enhances rat vascular smooth muscle cell proliferation.

Long-term exposure to particulate air pollution has been implicated as a risk factor for cardiovascular disease and mortality. Short-term exposure has also been suggested to contribute to complications of atherosclerosis. Aberrant regulation of smooth muscle cell proliferation is thought to associate with the pathophysiology of vascular disorders such as atherosclerosis. In this study, we investigate the influence of organic extracts of motorcycle exhaust particulates (MEPE) on rat vascular smooth muscle cell (VSMC) proliferation and related regulation signaling. Exposure of VSMCs to MEPE (10-100 microg/mL) enhanced serum-induced VSMC proliferation. The expression of proliferating cell nuclear antigen (PCNA) was also enhanced in the presence of MEPE. VSMCs treated with MEPE induced the increase in the extent of cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E 2 production, whereas the level of COX-1 protein was unchanged. Moreover, MEPE increased the production of reactive oxygen species (ROS) in VSMCs in a dose-dependent manner. MEPE could also trigger time-dependently extracellular signal-regulated kinase (ERK)1/2 phosphorylation in VSMCs, which was attenuated by antioxidants N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC). The level of translocation of nuclear factor (NF)-kappaB-p65 in the nuclei of VSMCs was also increased under MEPE exposure. The potentiating effect of MEPE on serum-induced VSMC proliferation could be abolished by COX-2 selective inhibitor NS-398, specific ERK inhibitor PD98059, and antioxidants NAC and PDTC. Taken together, these findings suggest that MEPE may contribute to the enhancement of the pathogenesis of cardiovascular diseases by augmenting proliferation of VSMCs through a ROS-regulated ERK1/2-activated COX-2 signaling pathway.

Regulation of mast cell development by inflammatory factors.

Mast cells are potent effectors playing a key role in IgE-associated hypersensitivity reactions, allergic disorders, inflammation and protective immune responses. Mast cell development in vivo occurs mainly in non-hematopoietic microenvironments and increased mast cell numbers can be seen in various inflammatory diseases and pathologic conditions. SCF (also known as kit ligand or KitL) and c-kit signaling are essential for both human and murine mast cell development, while IL-3 is required for murine mast cell hyperplasia that occurs in response to various stimuli. Besides SCF and IL-3, the cytokines IL-4, IL-9, IL-10 and IL-13 are also called mast cell growth factors due to their actions synergistically promoting mast cell proliferation and differentiation in the presence of SCF or IL-3. These cytokines alone however are unable to support neither the proliferation nor survival of mast cells. Most research has focused on examining the direct effects of the above cytokines on mast cells or their precursors. However, it is difficult to explain the process of mast cell development only in terms of the above mast cell growth factors. A series of experiments in our laboratory and by others has revealed that inflammatory mediators and cytokines, as triggers or regulators, are also crucial for mast cell development. This review summarizes recent progress in our understanding of how various inflammatory factors regulate mast cell development, with particular focus on the effects of prostaglandin E (PGE), TNF-alpha, IL-6, IFN-gamma and an unknown apoptosis-inducing factor produced by IL-4-stimulated macrophages.

Identification in human airways smooth muscle cells of the prostanoid receptor and signalling pathway through which PGE2 inhibits the release of GM-CSF.

1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse.

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.

Molecular characterization and ocular hypotensive properties of the prostanoid EP2 receptor.

The cloning of the genes that encode for prostaglandin (PG) receptors has resolved much of the complexity and controversy in this area by confirming the classification proposed by Coleman, et al. Two issues that remained unresolved were (1) the inability of the EP2 agonist butaprost to interact with the cloned putative EP2 receptor and (2) molecular biological confirmation of a fourth PGE2-sensitive receptor, which was pharmacologically designated EP4. In order to provide clarification, we attempted to clone further PGE2-sensitive receptors. By using a cDNA probe that encodes for the human EP3A receptor, a cDNA clone that encoded for a novel PGE2-sensitive receptor was obtained by screening a human placenta library. This cDNA clone was transfected into COS-7 cells for pharmacological studies. The cDNA clone obtained from human placenta had only about 30% amino acid identity with cDNAs for other PG receptors, including those that encode for the previously proposed murine and human EP2 receptors. Radioligand binding studies on the novel EP receptor expressed in COS-7 cells revealed that selective EP2 agonists such as butaprost, AH 13205, AY 23626 and 19(R)-OH PGE2 all competed with 3H-PGE2 for its binding sites, whereas selective agonists for other PG receptor subtypes had minimal or no effect. This receptor was coupled to adenylate cyclase and EP2 agonists caused dose-related increases in cAMP. It appears that the cDNA described herein encodes for the pharmacologically defined EP2 receptor. Ocular studies revealed that AH 13205 decreased intraocular pressure in normal and ocular hypertensive monkeys by a mechanism that does not appear to involve inhibition of aqueous humor secretion.

The EP2 receptor is the predominant prostanoid receptor in the human ciliary muscle.

Prostaglandins can reduce intraocular pressure by increasing uveoscleral outflow. We have previously demonstrated that the human ciliary muscle was a zone of concentration for binding sites (receptors) for prostaglandin F2 alpha and for prostaglandin E2. Here, we try to elucidate the types of prostanoid receptors in the ciliary muscle using competitive ligand binding studies in human eye sections and computer assisted autoradiographic densitometry. Saturation binding curves showed that the human ciliary muscle had a large number of binding sites with a high affinity for prostaglandin E2 compared with prostaglandin D2 and F2 alpha. The binding of tritiated prostaglandin E2 and F2 alpha in the ciliary muscle was displaced most effectively by prostaglandin E2 and 11-deoxy prostaglandin E1 (a selective EP2 prostanoid receptor agonist), whereas the binding of prostaglandin D2 was displaced most effectively by prostaglandin E2 and D2. These results indicate that the dominant prostanoid receptor in the human ciliary muscle is the EP2 subclass and that there is also a small number of DP receptors.

Metabolic fate of the new anti-ulcer drug enprostil in animals. 4th communication: effect on hepatic drug metabolizing enzyme system in rats.

Effects of multiple oral administration of enprostil ((+/-)-11 alpha,15 alpha-dihydroxy-9-oxo-16-phenoxy-17,18,19,20-tetranorprosta- 4,5,13(t)-trienoic acid methyl ester, TA 84135) (20 micrograms/kg/d) and its solvent propylene carbonate (PC, 100 microliters/kg/d) on body weight gain, liver weight, hepatic drug metabolizing enzyme system and hexobarbital sleeping time were investigated in male rats during a 14-day period. Cytochrome P-450 content (as compared to the untreated control) and cytochrome b5 content (as compared to PC treated group) were slightly, but significantly, reduced in the group given a single oral dose of enprostil. However, these slight reductions were not augmented significantly by repeated administrations of enprostil. Slight but significant increase in microsomal protein content was observed in the group given 14 oral doses of enprostil and PC. Enprostil did not affect the other indicators used to evaluate the status of the hepatic drug metabolizing enzyme system. Additionally, single or multiple oral doses of enprostil or PC showed no effect on the hexobarbital-induced sleeping time. It therefore may be safely concluded that multiple oral administration, both of enprostil and of PC, has very little effect on drug metabolizing enzyme inducing or inhibiting activity in rats.

Binding to prostaglandin (PG) receptors and activation of adenyl cyclase by 20-isopropylidene-PGS in rabbit platelet membranes.

20-Isopropylidene-PGE1 (Isop-PGE1) was about 10 times more potent than PGE1 in inhibition of thrombin-induced aggregation of rabbit washed platelets. Likewise, 20-isopropylidene-17(R)-methyl-carbacyclin (CS-570), a stable PGI2 analogue, was more potent than carbacyclin in the anti-aggregatory activity. In order to define the platelet-prostaglandin interactions, a binding assay was done using platelet membranes with [3H]-PGE1 as a radioligand. Isop-PGE1 (IC50 = 0.18 microM) bound to the PG receptors more potently than PGE1 (IC50 = 2.1 microM). CS-570 (IC50 = 0.39 microM) was more potent than carbacyclin (IC50 = 1.9 microM). These indicate that introduction of an isopropylidene group to the carbon 20 of PGs increases the binding ability to the receptors. These PGE1 and PGI2 analogues activated platelet membrane adenyl cyclase and increased intracellular cAMP levels with the same potency series obtained in the binding experiments. All these results suggest that the binding to the receptors by these PGs is coupled to the activation of adenyl cyclase, followed by the increase in cAMP levels in platelets and the inhibition of platelet aggregation. Thus, the increased anti-aggregatory activity of 20-isop-PGs may be explained by their increased affinity for the PG receptors and stimulation of adenyl cyclase. 15-Epimeric-20-isopropylidene-PGE1 (15-Epi-isop-PGE1), which has an unnatural configuration of the 15-hydroxyl group, was much less potent than isop-PGE1 in the binding experiment and the other three investigations. This indicates that the configuration of the 15-hydroxyl group is important for the binding to the PG receptors and the consequent activities in platelets.

Biliary, fecal and urinary excretion of [3H]-prostaglandins in the rat.

The profiles of biliary, fecal and urinary excretion of tritium labeled prostaglandins (PG's) of differing biological activity were investigated in the rat. The PG's (10 micrograms/kg: 2 to 50 microCi/rat, in 1 ml polyethylene glycol-400) were administered intragastrically. Excretion data were expressed as a percentage of the total administered radioactivity. For the orally administered PG's 11R-methyl-16R-fluoro-15R-hydroxy-9-oxoprosta-ci s-5-trans-13-dienoic acid and its methyl ester, excretion was equally divided between urine and feces. The fecal and urinary profile of excretion of 3H after prostacyclin (PGI2) was similar to that following administration of 11R, 16, 16-trimethyl-15R-hydroxy-9-oxoprosta-cis-5-trans-13-dienoic acid (trimoprostil), a PG with antisecretory-antiulcer potential. However, PGI2 was very poorly absorbed from the intestine, while the absorption of trimoprostil was very efficient. Biliary excretion, with little entero-porto-hepatic biliary circulation, was the main route of elimination of trimoprostil, thereby resulting in rapid elimination of drug-related products and diminishing the potential for systemic liability in the rat.

16,16-Dimethyl prostaglandin E2 increases survival in mice following irradiation.

16,16-Dimethyl prostaglandin E2 (DiPGE2), a stable analog of PGE2, increases the LD50/30 survival in CD2F1 male mice when given prior to ionizing radiation. Subcutaneous administration of 40 micrograms of DiPGE2 30 min prior to 60Co gamma irradiation extends the LD50/30 from 9.39 Gy in the control animals to 16.14 Gy in DiPGE2 treated, with a dose reduction factor of 1.72 [95% confidence limits: 1.62, 1.82]. The degree of protection is dependent on both the time of administration and the dose of the prostaglandin. Ten micrograms administered 5 min prior to receiving a lethal dose of 10 Gy provides 90% survival but only 10% survival if administered 30 min prior to irradiation. Experiments to determine the in vivo concentration of DiPGE2 in organs postinjection show increased levels over time, but these are not correlated with protection. At 30 min after injection, as much as 80% of the DiPGE2 present in the spleen and plasma is unmetabolized. These results suggest that the protection results from the physiologic action of DiPGE2 rather than direct in vivo detoxification of radicals.

Inflammatory bowel disease: the in vitro effect of sulphasalazine and other agents on prostaglandin synthesis by human rectal mucosa.

1. Prostaglandin (PG) E2 and F2 alpha synthesis in vitro by human rectal mucosa was significantly greater in patients with Inflammatory Bowel Disease (IBD) than in controls. 2. The addition of 250 microM sulphasalazine (SASP) significantly increased synthesis by rectal mucosa from patients with IBD, (ulcerative colitis and Crohn's disease) and controls after 1 h of incubation in Krebs' solution at 37 degrees C. 3. Flurbiprofen, indomethacin, sodium salicylate and 5-aminosalicylic acid decreased PGE2 and F2 alpha synthesis. 4. Sodium carbenoxolone decreased PGF2 alpha synthesis but had no effect on PGE2. Disodium cromoglycate, sulphapyridine, salicylazosulphadimidine and methylsulphasalazine had no effect on PG synthesis. 5. The data show marked variations in effect on PG synthesis in vitro of substances that have been investigated clinically for their actions on IBD.

Effect of enprostil, a synthetic prostaglandin E2 on 24 hour intragastric acidity, nocturnal acid and pepsin secretion.

We have studied the effect of a prostaglandin E2 analogue (enprostil), on intragastric acidity, gastric acid and pepsin outputs during a 24 hour period in nine patients with duodenal ulcer in remission. Enprostil 35 micrograms bd dose inhibited 24 hour intragastric acidity by 38% and a 70 micrograms nocturnal dose by 33%. Decrease in nocturnal pepsin secretion was both volume and concentration related.

Gastric mucosal binding studies with enprostil: a potent anti-ulcer prostaglandin.

The potent antiulcer prostaglandin enprostil binds with high affinity to porcine gastric mucosal tissues. This binding is saturable, dissociable and displaceable by compounds with similar structures. Various characteristics of binding such as pH optimum and displacement potencies suggest that enprostil binds to mucosal PGE2 sites. Structure-activity and gastric mucosal binding relationships were also examined.

Up-regulation of renal prostaglandin receptors in genetic salt-dependent hypertension.

Development of hypertension in Dahl salt-sensitive rats (DS) is accompanied by reduced renomedullary prostaglandin synthesis, which may be responsible for their lower natriuretic capacity. To examine the changes in renomedullary prostaglandin E2 synthesis, the effects of high (8.0%) and normal (0.6%) NaCl diets were examined in DS and in Dahl salt-resistant rats (DR). In response to an 8.0% NaCl diet, the number of prostaglandin E2 receptors in the renal outer medulla of DR increased (2.97 +/- 0.2 vs 2.18 +/- 0.2 pmol/mg on 0.6% NaCl diet) while no change was noted in their affinities (Kd, 9.5 +/- 0.2 vs 9.4 +/- 0.3 nM). Receptor number and affinity in the renal cortex, inner medulla, and liver of DR were not affected. In contrast, renomedullary receptors of DS had a lower affinity than those of age-matched DR (Kd, 13.9 +/- 0.2 nM on 0.6% NaCl diet and 14.0 +/- 0.3 nM on 8.0% NaCl diet) and did not increase in number after a high salt diet. This apparent inability of DS to modulate prostaglandin receptors may contribute to their susceptibility to salt-induced hypertension.

Identification of trimoprostil metabolites excreted in rat bile formed by oxidation and taurine conjugation.

14C-Trimoprostil was administered iv and orally to male rats. Radioactivity was excreted mainly via the feces after both routes of drug administration. Bile duct-cannulated rats excreted an average of 76% of an iv dose in the bile within 6 hr after dosing. Four biliary metabolites were isolated by HPLC and identified by proton NMR spectroscopy and mass spectrometry. These metabolites were taurine conjugates of: trimoprostil (approximately 11% of dose), 5,6-dihydro-2,3-dinor trimoprostil (approximately 5% of dose), 3,4-dehydro trimoprostil (less than 5% of dose) and 5,6-dihydro-2,3,4,5-tetranor trimoprostil (less than 5% of dose).

Metabolism of viprostol--a synthetic vasodilator PGE2 analog.

Metabolic studies were done with 14C-Viprostol (I) administered by various routes (I.V., oral and topical) to six animal species and to man. Total radioactivity and metabolic profiles were analyzed in plasma, tissues and excreta. The main metabolites were isolated and identified by capillary GC/MS. Plasma and urinary metabolic profiles were qualitatively similar across species, with two major metabolic reactions being predominant: rapid hydrolysis to the pharmacologically active free acid (II) and oxidation of the alpha-chain to dinor and tetranor acids (III, IV). In the monkey and man, reduction of the 9-keto group lead to PGF2 type metabolites (VI-VIII). In the rat, omega oxidation of the beta-chain occurred as well, resulting in the formation of dicarboxylic acids (V).