High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

Microsomal prostaglandin E synthase-1: the inducible synthase for prostaglandin E<Subscript>2</Subscript>

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.     

Inhibitors of the inducible microsomal prostaglandin E2 synthase (mPGES-1) derived from MK-886

A series of potent and selective inhibitors of the inducible microsomal PGE2 synthase (mPGES-1) has been developed based on the indole FLAP inhibitor MK-886. Compounds 23 and 30 inhibit mPGES-1 with potencies in the low nanomolar range and with selectivities of at least 100-fold compared to their inhibition of mPGES-2, thromboxane synthase and binding affinity to FLAP. They also block the production of PGE2 in cell based assays but with a decreased potency and more limited selectivity compared to the enzyme assays.     

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE(2) biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE(2) generation. Arg(110), a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A(2) allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE(2) biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.     

Lipopolysaccharide-induced increase of prostaglandin E(2) is mediated by inducible nitric oxide synthase activation of the constitutive cyclooxygenase and induction of membrane-associated prostaglandin E synthase

NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE(2) concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE(2) concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.     

Synthesis of tritium-labelled prostaglandin E1 and its binding to human platelets

A method has been developed that makes it possible to obtain [5,6-3H2]PGE1 with a yield of 35% and a molar radioactivity of 1.7-1.8 TBq/mmol. The binding of [5,6-3H2]PGE1 to native platelets proved to be specific, saturating and reversible. It is characterized by low values (approximately 10(-9) M) of dissociation constants for high-affinity sites, correlates with the inhibition of ADP-induced aggregation of platelets and can be considered as receptor binding. Specific binding of 10 +/- 2 molecules of PGE1 with one platelet was found to cause 50% inhibition of the ADP-induced aggregation.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Effect of changing [K+], [Na+] and [Cl-] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO3(-) but with 95% O2/5% CO2 in both cases. In both cases, changing from 4 to 40 mM K+ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO3(-). Changing from 102 to 8 mM Na+ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO3(-) was insignificant but with 25 mM HCO3(-) the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K+ and Na+ were largely related to (Na+ + K+)-ATPase pump. Changes in nutrient Cl- from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of (Na+ + K+)-ATPase pump and Na+/Cl- symport. Removal of HCO3(-) and concurrent reduction of pH modify resistance of these pathways.     

Characterization of the heparin binding sites in human apolipoprotein E

Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the C-terminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pK(a) values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan.     

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.     

Specific binding sites for prostaglandin D2 on human platelets.

³H-PGD₂ was biosynthesized from ³H-arachidonate and used to study the binding of PGD₂ to intact human platelets. The binding of ³H-PGD₂ to platelets was rapid, being essentially complete within two min. Bound ³H-PGD₂ PGD₂. Scatchard analysis of concentration-dependent binding indicated a single class of binding sites with a dissociation constant (K_D) of 4.12 × 10^?7M and a capacity of 760 sites per platelet. The relative ability of PGD₂, PGE₂, PGE₁ and PGI₂ to displace ³H-PGD₂ bound to these sites was 100:2:2<1. We conclude therefore, that these PGD₂ binding sites are specific for PGD₂ and independent of those previously demonstrated to recognize ³H-PGI₂ and ³H-PGE₁.     

Activated human dendritic cells express inducible cyclo-oxygenase and synthesize prostaglandin E2 but not prostaglandin D2

Prostaglandins (PG) are well known lipid mediators with important immunoregulatory properties. While exogenous PGE2 has the ability to modulate the function and maturation of antigen presenting cells, such as dendritic cells (DC), it is not clear whether human DC have the capacity to synthesize PGE2 and other prostaglandins themselves. We therefore examined the expression of inducible cyclo-oxygenase (COX-2) by monocyte derived DC and the production of PGE2 and PGD2. Both monocyte derived DC and freshly isolated blood myeloid DC expressed little COX-2 constitutively, though COX-2 expression was rapidly but transiently upregulated in response to lipopolysaccharide stimulation. COX-2 mRNA was detectable within 1 h of LPS exposure, peaked at 4-6 h, and rapidly declined thereafter. COX-2 expression was accompanied by DC synthesis of PGE2, with peak levels present at 6-18 h post-stimulation. In contrast, PGD2 synthesis was not detected at any time point. When DC were activated with LPS in the presence of nimesulide, a COX-2 selective inhibitor, IL-10 synthesis was inhibited, indicating that endogenous prostaglandins regulate DC cytokine production. PGE2 production by DC may therefore modulate DC and T-cell function, thereby shaping the character of the immune response.     

Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2 enzymes, cyclooxygenase (COX) enzymes, and various lineagespecific terminal prostanoid synthases. Prostaglandin E synthase (PGES), which isomerizes COX-derived PGH2 specifically to PGE2, occurs in multiple forms with distinct enzymatic properties, expressions, localizations and functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli, is down-regulated by antiinflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE2 production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes.     

Location of binding sites in small molecule rescue of human carbonic anhydrase II

Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp-64 approximately 8 A from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w(r) and w(p) rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.     

Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.     

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.     

Effect of prostaglandin E1 on low density lipoprotein apo-B-receptor binding in human, rat and swine liver in vitro.

The effect of PGE1 on low density lipoprotein (LDL) apo-B-receptor binding was examined in human, rat and swine liver. Autologous LDL (for humans and swines) and homologous LDL (for rats) were isolated by ultracentrifugation and labelled with 123I using Iodogen followed by purification with dialysis. LDL-concentrations of 0.1-6 micrograms protein/ml were used for direct binding assays investigating the specific binding of labelled LDL in presence of increasing PGE1-concentrations (100 pM to 100 microM). In separate experiments the effect of PGE1 on displacement of specifically bound 123I-LDL by unlabelled ones was studied. The binding capacities estimated by Scatchard analysis were similar for human and rat liver LDL-apo-B-receptor binding, however, swine liver exhibited a significantly (p less than 0.001) lower binding capacity for 123I-LDL. PGE1 significantly (p less than 0.01-0.001) increased the amount of 123I-LDL specifically bound to the liver apo-B-receptors and the binding affinity in all liver preparations of the 3 species in a dose-dependent manner. PGE1 also significantly increased competition of unlabelled LDL for 123I-LDL bound to its specific apo-B-receptors in a dose-dependent manner (p less than 0.01-0.001) with an ED50 of 123 +/- 64 nM for human liver, 901 +/- 102 nM for rat liver obtained during anaesthesia, 74 +/- 23 nM for rat liver obtained after decapitation and 941 +/- 121 nM for swine liver. In human liver iloprost (ED50 = 876 +/- 53 nM) and PGI2 (ED50 = 52 +/- 12 microM) were less effective than PGE1, PGE2 had no effect on LDL-induced competition. It is concluded that PGE1 renders LDL more sensitive for apo-B-receptor binding suggesting a potential hypolipidemic action of PGE1.