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V Ullrich

Publications and source records attributed to V Ullrich.

At least 55 records · Page 3Linked to original sources
PubMed1996

Site-directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys-pocket, and Glu347 and Arg350 of the EXXR motif.

The possible active site Cys441 in the Cys-pocket and Glu347 and Arg350 of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441 Ala, Cys441 Ser, Cys441 His, Glu347 Ala and Arg350 Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1alpha/min per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys441 in the Cys-pocket, and Glu347 and Arg350 of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

Amino Acid Sequence↗
PubMed1996

Peroxynitrite formed by simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase.

The effect of various oxidants on bovine aortic prostacyclin synthase was tested with 14C-labelled prostaglandin endoperoxide as substrate. No sensitivity against hydrogen peroxide, superoxide or hydroxyl radicals was observed but hypochlorite inhibited with an IC50 value of 7 microM. Among the reactive nitrogen species nitric oxide and nitrogen dioxide radicals were ineffective, but peroxynitrite irreversibly blocked prostacyclin biosynthesis with an IC50 value of 50 nM. Peroxynitrite acted within seconds whereas hypochlorite required up to 30 min for completion. Simultaneous generation of nitric oxide and superoxide also caused inhibition which suggested that under pathological conditions like ischemia-reperfusion not only the vasodilatory effects of nitric oxide but also those of prostacyclin could be eliminated.

Animals↗
PubMed1996

Mechanistic studies on the selective inhibition of cyclooxygenase-2 by indanone derivatives.

The cyclooxygenase step in the conversion of arachidonic acid is a key point in the biosynthesis of prostanoids, managed by two enzymatic isoforms. In the following study we focused on the mechanism of the inhibitory action of CGP 28238 and structurally-related indanone derivatives using purified enzymes. Consistent with our earlier studies on cell systems, CGP 28238 revealed selective inhibition of cyclooxygenase-2. The process affects the bisoxygenase subunit time-dependently, and is reversible in the early phase of inhibition. From structure-activity relationships, we propose the formation of a Schiff base between the oxo-groups of CGP 28238 and an amino group at the active site providing additional binding forces for an effective inhibition of cyclooxygenase-2.

Animals↗
PubMed1996

Effect of cyclic AMP and prostaglandin E2 on the induction of nitric oxide- and prostanoid-forming pathways in cultured rat mesangial cells.

Cyclic AMP (cAMP) represents an important cellular signalling molecule. We analysed the effect of dibutyryl cAMP (db-cAMP), a cell-permeable and stable derivative of cAMP, on the regulation and expression of cyclo-oxygenase 2, inducible NO synthase and argininosuccinate synthetase. We observed different transcriptional regulation of these enzymes depending on the db-cAMP concentration used. Low concentrations of db-cAMP in the range 10-50 microM elevated levels of cyclo-oxygenase 2 mRNA, protein and activity, but not the respective mRNA and protein concentrations of the inducible NO synthase or argininosuccinate synthetase. At higher concentrations a massive induction of the latter two enzymes was also apparent. Expression of prostacyclin synthase and argininosuccinate lyase, secondary enzymes of NO- and prostanoid-forming pathways, was not stimulated by db-cAMP. Prostaglandin E2, known to be an intracellular physiological trigger of cAMP formation, stimulated only cyclooxygenase 2 expression and activity at a concentration of 10 microM, and not inducible NO synthase. The induction of the mRNA for the transcription factors JunB and p65, a component of the NF kappa B complex, by prostaglandin treatment of the cells might be a possible mechanistic explanation for this observation.

Animals↗
PubMed1996

Peroxynitrite inhibition of nitric oxide synthases.

Peroxynitrite (PN) can be formed under mainly pathophysiological conditions from nitric oxide (NO) and superoxide anion and may be responsible for oxidative modifications of biomolecules. Preparations of nitric oxide synthases from porcine cerebellum (nNOS), bovine aortic endothelium (eNOS) and cytokine-treated murine macrophages (iNOS) were inhibited by PN in their ability to transform arginine to citrulline and nitric oxide with IC50 values of 15, 28, and 10 microM, respectively. Glutathione, bovine serum albumin and tyrosine provided varying degrees of protection in the three preparations. Intact endothelial cells, upon exposure to PN, rapidly lost their glutathione content but protein-SH groups and eNOS activity remained largely unaffected. Destruction of the heme-thiolate catalytic site was observed when nNOS was exposed to PN suggesting that the irreversible oxidation of this bond may be the common mechanism of NOS inhibition.

Animals↗
PubMed1996

Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices.

Contraction of airways of different size can be studied in viable lung slices by videomicroscopy. However, at present, application of this technique is limited by the heterogeneous responses obtained. We investigated the use of precision-cut lung slices to examine contraction of individual airways. Lung slices of 250 +/- 20 microns were prepared from Wistar rats and cultured in a roller incubator in serum-free minimum essential medium (MEM). Under these conditions, the slices were viable for at least 70 h, as indicated by leakage of lactate dehydrogenase into the supernatant, thymidine incorporation and ciliary beating. The slices were placed in a newly developed incubation chamber and mounted by a nylon thread that was fixed to a platinum wire. The whole chamber was positioned on a microscope stage, and contraction of single airways was followed under a microscope that was coupled to a CCD-camera. Reduction in airway area was taken as an index of bronchoconstriction and was determined by a computer program. Addition of methacholine resulted in a concentration-dependent (concentration producing half the maximal effect (EC50) = 0.64 +/- 0.08 (mean +/- SD) microM; n = 64) contraction of single airways. In the presence of hydrocortisone, the EC50 was about six times greater, i.e. 3.7 +/- 0.9 microM (n = 7), and the effect of the steroid was largely abolished by propanolol (EC50 = 1.1 +/- 0.1 microM; n = 7). Airways with an area smaller than 35,000 microns2 were nearly nine times more sensitive to methacholine (EC50 = 0.1 +/- 0.03 microM; n = 20) than larger ones (EC50 = 87 +/- 0.27 microM; n = 22). We conclude that cultured precision-cut lung slices are a useful model for routine study of contraction of individual airways of various sizes. The measurements were precise and reproducible and showed that smaller airways are more sensitive to methacholine than larger ones.

Animals↗
PubMed1996

Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin.

BACKGROUND: Lipopolysaccharides (LPS), widely used to study the mechanisms of gram-negative sepsis, increase airway resistance by constriction of terminal bronchioles. The role of the cyclooxygenase (COX) isoenzymes and their prostanoid metabolites in this process was studied. MATERIALS AND METHODS: Pulmonary resistance, the release of thromboxane (TX) and the expression of COX-2 mRNA were measured in isolated blood-free perfused rat lungs exposed to LPS. RESULTS: LPS induced the release of TX and caused increased airway resistance after about 30 min. Both TX formation and LPS-induced bronchoconstriction were prevented by treatment with the unspecific COX inhibitor acetyl salicylic acid, the specific COX-2 inhibitor CGP-28238, dexamethasone, actinomycin D, or cycloheximide. LPS-induced bronchoconstriction was also inhibited by the TX receptor antagonist BM-13177. The TX-mimetic compound, U-46619, increased airway resistance predominantly by constricting terminal bronchioles. COX-2-specific mRNA in lung tissue was elevated after LPS exposure, and this increase was attenuated by addition of dexamethasone or of actinomycin D. In contrast to LPS, platelet-activating factor (PAF) induced immediate TX release and bronchoconstriction that was prevented by acetyl salicylic acid, but not by CGP-28238. CONCLUSIONS: LPS elicits the following biochemical and functional changes in rat lungs: (i) induction of COX-2; (ii) formation of prostaglandins and TX; (iii) activation of the TX receptor on airway smooth muscle cells; (iv) constriction of terminal bronchioles; and (v) increased airway resistance. In contrast to LPS, the PAF-induced TX release is likely to depend on COX-1.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

Inhibition of cyclooxygenase-1 and -2 by R(-)- and S(+)-ibuprofen.

Since the discovery of a cytokine-inducible isozyme of cyclooxygenase (COX-2), its pharmacologic inhibition has been the subject of recent investigations. These include tests for the selectivity of known nonsteroidal antiinflammatory drugs (NSAIDs) on the constitutive enzyme of cyclooxygenase (COX-1) compared with the inducible enzyme COX-2. The interesting question arose whether the R(-)- and S(+)-isomers exhibited different inhibitory potencies for ibuprofen. Results with isolated COX-1 and COX-2 isozymes confirmed the known higher efficacy of S(+)-compared with R(-)-ibuprofen. The R(-)-isomer is almost inactive in inhibiting COX-2. In addition, the S(+) form has a several times lower potency with COX-2 than with COX-1. These data were evaluated in platelets containing mainly the constitutive COX-1, with interleukin-1, pretreated, rat mesangial cells which almost exclusively express COX-2.

Animals↗
PubMed1995

Activin A and retinoic acid synergize in cyclooxygenase-1 and thromboxane synthase induction during differentiation of J774.1 macrophages.

The murine macrophage cell line J774.1 was used to study the development of prostanoid biosynthesis under the influence of activin A and retinoic acid. Treatment of cells with 3 nM activin A for 48 h increased the biosynthesis of the prostaglandins E2, D2, F2 alpha and thromboxane A2 more than fourfold due to an induction of cyclooxygenase-1 while cyclooxygenase-2 was unaffected. Transforming growth factor-beta acted in a similar way. Retinoic acid, when present alone, was without effect on the total cyclooxygenase products and only slightly changed the pattern of prostanoids. However, when coincubated with activin A, retinoic acid specifically induced the synthesis of thromboxane-A-synthase-specific mRNA and induced an increase in enzyme activity with a synergistic effect on cyclooxygenase-1 protein and mRNA. JunB, but not c-jun, mRNA expression was found under these conditions in addition to a transient c-fos mRNA increase. The combination of activin A and retinoid acid may be regarded as a differentiation model to study the development of cell-specific prostanoid patterns in macrophages and possibly other differentiating cells.

Activins↗
PubMed1995

Regulation of prostanoid-synthesis in the cardiovascular system.

Thromboxane A2 and prostacyclin are the two prostanoids involved in the regulation of the vascular tone. Their release is controlled by the activity of cyclooxygenase which has made this enzyme a preferred pharmacological target. We here report on the distribution of the two isoforms of cyclooxygenase in cultured mesangial cells and on a selective inhibitor of the cytokine-inducible cyclooxygenase-2. We also comment on the structure of thromboxane and prostacyclin synthase and their regulation under physiological and pathophysiological conditions.

Amino Acid Sequence↗
PubMed1994

Receptor occupancy regulates Ca2+ entry and intracellular Ca2+ redistribution in activated human platelets.

Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Selective inhibition of cyclooxygenase 2.

Cyclooxygenase (COX), a key enzyme in the formation of prostanoids, is known to exist in two isoforms: an inducible enzyme (COX 2) and a constitutive from (COX 1). Both enzymes are inhibited by non-steroidal anti-inflammatory drugs (NSAID), but only marginal selectivity has thus far been reported. In this study, we report on a novel selective inhibitor of COX 2, CGP 28238 (6-(2,4-difluorophenoxy)-5-methyl-sulfonylamino-1-indanon e). Human washed platelets were used as a source of COX 1. For IL-1 stimulated rat mesangial cells we demonstrated the almost exclusive presence of COX 2 in western blot and mRNA analysis. Therefore these two model systems were chosen for selectivity testing. With an IC50 value of 15 nM, CGP 28238 blocked COX 2 activity in a similar concentration range to that of other potent NSAID such as indomethacin and diclofenac (IC50 = 1.17-8.9 nM). However, in contrast to these reference NSAIDs, CGP 28238 was at least 1000-fold less potent in inhibiting COX 1. Using other cell systems reported to express COX 1 or COX 2, we obtained a similar selectivity for COX 2. Thus, on the basis of our findings, CGP 28238 is a novel, highly potent and selective inhibitor of COX 2 and may be a lead compound for a new generation of potent anti-inflammatory drugs with an improved side-effect profile.

Animals↗
PubMed1994

Isolation and molecular cloning of prostacyclin synthase from bovine endothelial cells.

Prostacyclin synthase catalyzes the conversion of prostaglandin H2 to prostacyclin, which is a powerful vasodilator and the most potent natural occurring inhibitor of platelet aggregation. In the present study, we determined the amino acid sequence of bovine prostacyclin synthase by combined protein chemical and molecular cloning techniques. The enzyme was purified and characterized from bovine aorta microsomes, and the partial amino acid sequences were determined with the native enzyme and endoproteinase Lys-C-cleaved peptides. Using primers synthesized according to the amino acid sequences, cDNA coding for prostacyclin synthase was amplified by polymerase chain reaction with bovine endothelial cell poly(A)+ RNA and cloned into pBluescript II. Nucleotide sequence analyses of the cloned cDNA inserts revealed that cDNA for this enzyme contained a 1500-base pair open reading frame coding for a 500-amino acid polypeptide with a M(r) of 56,628. COS-7 cells transfected with an expression plasmid harboring this cDNA clone expressed prostacyclin synthase activity. The primary structure of the enzyme showed structural characteristics of cytochrome P450 and exhibited a 32% identity to that of human cholesterol 7 alpha-hydroxylase. However, the identity between the amino acid sequences of bovine prostacyclin synthase and human thromboxane synthase was only 16%, and no P450 showed an identity higher than 40%, suggesting that prostacyclin synthase represents a new family in the P450 superfamily. RNA blot analysis indicated that the mRNA for prostacyclin synthase from bovine endothelial cells showed a size of approximately 2.7 kilobases and that the mRNA level increased about 3-fold by treatment of tumor necrosis factor-alpha.

Amino Acid Sequence↗
PubMed1994

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase.

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.

Animals↗
PubMed1994

Molecular cloning and expression of human prostacyclin synthase.

The cDNA for human prostacyclin synthase was cloned by polymerase chain reaction using poly(A)+ RNA from human aortic endothelial cells according to the partial nucleotide sequence of prostacyclin synthase gene. The cloned cDNA with a size of 1977 base pairs contained a 1500 base pairs open reading frame which encoded a 500 amino acid protein sharing an 88% identity with bovine prostacyclin synthase. RNA blot analysis indicated that the size of major prostacyclin synthase mRNA of human aortic endothelial cells was approximately 6 kilobases and that its mRNA level was increased by interleukin 1 or interleukin 6 treatment. Moreover, tissue distribution study demonstrated that prostacyclin synthase mRNA is widely expressed in human tissues and is particularly abundant in ovary, heart, skeletal muscle, lung, and prostate. These results suggest a variety of physiological roles of prostacyclin in addition to the implications in the cardiovascular system.

Amino Acid Sequence↗