Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils

In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.     

Characterization of dihydro-A2PE: an intermediate in the A2E biosynthetic pathway

Bisretinoid lipofuscin pigments that accumulate in retinal pigment epithelial cells are implicated in the etiology of several forms of macular degeneration, including juvenile onset Stargardt disease, Best vitelliform macular degeneration, and age-related macular degeneration. One of these compounds, A2E, is generated by phosphate hydrolysis of a phosphatidyl-pyridinium bisretinoid (A2PE) that forms within photoreceptor outer segments. Here, we demonstrate that the formation of the aromatic pyridinium ring of A2PE follows from the oxidation of a dihydropyridinium intermediate. Time-dependent density functional theory calculation, based on the structure of dihydro-A2E, produced a simulated UV-visible absorbance spectrum characterized by maxima of 494 and 344 nm. Subsequently, a compound exhibiting similar UV-visible absorbance maxima (lambdamax 490 and 330 nm) was identified in the A2E biomimetic reaction mixture. By liquid chromatography-mass spectrometry (LC-MS) this bischromophore had the expected mass of the dihydro-pyridinium bisretinoid. The compound also exhibited the behavior of a biosynthetic intermediate since it formed in advance of the final product A2E and was consumed as A2E accumulated. Moreover, under deoxygenated conditions, conversion to the aromatic pyridinium bisretinoid was inhibited. Taken together, these findings indicate that A2E biosynthesis involves the oxidation of a dihydropyridinium intermediate dihydro-A2PE. An understanding of the biosynthetic pathways of retinal pigment epithelial lipofuscin pigments is critical to the development of therapies for macular degeneration that are based on limiting the formation of these damaging compounds.     

Insulin-induced peroxynitrite production in human platelet-rich plasma

Abstract

Recent data support the possible role of nitric oxide (NO^?) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO^? production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO^? by luminol is possible in the form of peroxynitrite produced in the reaction of NO^? with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 µmol/l of desferrioxamine mesylate. Insulin, in the range of 0.084–840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO^? synthase enzyme, Nω-nitro-L-arginine methyl ester (at concentrations of 2.0–4.0 mmol/l, P <0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72–144 IU/ml, P <0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO^? and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.     

Expression of prostaglandin D synthase and the prostaglandin D2 receptors DP and CRTH2 in human nasal mucosa

BACKGROUND: Prostaglandin D2 (PGD2) is released from mast cells during the allergic response. OBJECTIVE: Since PGD2 has been shown to induce nasal congestion in humans, we investigated the distribution of hematopoietic prostaglandin D synthase (PGDS) and the two PGD2 receptors, DP and CRTH2 in human nasal mucosa from healthy subjects and subjects suffering from polyposis, a severe form of chronic rhinosinusitis. METHODS: DP mRNA expression was detected by in situ hybridization while PGDS, CRTH2 and various leukocyte markers expression were revealed by immunohistochemistry. RESULTS: In the normal mucosa, PGDS was only detected in few resident mast cells while CRTH2 was undetectable. In contrast, DP receptor mRNA was detected in epithelial goblet cells, serous glands and in the vasculature. In the nasal mucosa of subjects suffering from polyposis: (1) PGDS was detected in mast cells and other large infiltrating inflammatory cells, (2) both DP mRNA and CRTH2 were detected in eosinophils and (3) CRTH2 was detected on a subset of infiltrating T cells. Although DP mRNA could not be detected in the T cells invading the nasal mucosa, it was found to be expressed in the T cells present in the lymph node and the thymus from normal individuals. CONCLUSION: This study indicates that cells capable of producing PGD2 are present in the nasal mucosa and that both PGD2 receptors, DP and CRTH2, might play a role in inflammatory disease of the upper airways.     

Insulin-induced peroxynitrite production in human platelet-rich plasma

Recent data support the possible role of nitric oxide (NO*) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO* production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO* by luminol is possible in the form of peroxynitrite produced in the reaction of NO* with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 micromol/l of desferrioxamine mesylate. Insulin, in the range of 0.084-840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO* synthase enzyme. N(omega)-nitro-L-arginine methyl ester (at concentrations of 2.0-4.0 mmol/l, P<0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72-144 IU/ml, P<0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO* and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.     

Characterization of the biosynthetic pathway of glucosylglycerate in the archaeon Methanococcoides burtonii

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50 degrees C. The maximal activity of GpgP was at 50 degrees C with GPG as the substrate and at 60 degrees C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.     

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.     

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.     

Agonist regulation of the human platelet prostaglandin D2 receptor.

The effect of exposing platelets to prostaglandin D₂ (PGD₂) on hormone binding was studied. Incubation of platelets with PGD₂ for 2 hr resulted in a decrease in [³H]PGD₂ binding that was dose dependent. Inhibition of binding was 14% after incubation with 10^?8M PGD₂, 19% after incubation with 10^?7M PGD₂, and 40% after exposure to 10^?6M PGD₂. This decreased binding (desensitization) was specific for [³H]PGD₂ as binding to platelets by [³H]PGE₁ and the α-adrenergic antagonist [³H] dihydroergocryptine (DHEC) was comparable to control platelets. Saturation of [³]PGD₂ binding to desensitization platelets was at 27 fmole ligand/10⁸ platelets compared to 43 fmoles/10⁸ platelets in control platelets. Half-maximal saturation occured at 20 nM PGD₂ both for desensitized and control platelets, suggesting that decreased binding sites rather than altered affinity between ligand and receptor accounted for these results. These platelets had a partial increase in [³H]PGD₂ binding a few hours after plasma was washed free of PGD₂ with complete resensitization after 24 hr. Since prostaglandins such as PGI₂, PGD₂, and PGE₁ are potent inhibitors of platelet aggregation, decreased binding of platelets to these hormones after prostaglandin exposure may provide a mechanism for altered responsiveness of platelets to aggregating stimuli.     

Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway.

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.     

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp.

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.     

Ability of human plasma to inhibit prostaglandin F2 alpha in asthma

Human plasma has been reported to inhibit the conversion of arachidonic acid into prostaglandin (PG) E2 and PGF2 alpha. In the present study the plasma inhibitory activity was determined in three groups (16 each) of plasma obtained from normal healthy volunteers, treated asthmatics and untreated asthmatic patients. The result showed that plasma from all three groups were equally effective in inhibiting the biosynthesis of PGE2. Plasma of normal volunteers and treated asthmatics also inhibited PGF2 alpha biosynthesis. In contrast the plasma obtained from untreated asthmatics was considerably less active in inhibiting the biosynthesis of PGF2 alpha than plasma from the other two groups.     

Characterization of the prostaglandin receptors in human osteoblasts in culture

Prostaglandins have complex actions on bone metabolism that depend on interactions with different types and subtypes of receptors. Our objective was to characterize the prostaglandins receptors present in primary cultures of human osteoblasts. RT-PCR analysis revealed the presence of DP, EP(4), IP, FP and TP receptor mRNA in primary cultures of human osteoblasts. FP receptor mRNA was detected only after 3 weeks of confluency, all the others were detected at every culture time tested. To verify the functionality of these receptors we challenged the cells with the prostanoids and synthetic analogues and determined the intracellular levels of cAMP. All receptors found by RT-PCR were coupled to second messengers except for the DP subtype. These results clearly show the presence of functional EP(4), IP, FP and TP receptors in human osteoblasts in culture.     

Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans- Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.