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V Ullrich

Publications and source records attributed to V Ullrich.

At least 73 records · Page 4Linked to original sources
PubMed1994

The localization of thromboxane synthase in normal and pathological human kidney tissue using a monoclonal antibody Tü 300.

Thromboxane, excreted in the urine in increased amounts in glomerular, vascular and tubulo-interstitial diseases, is considered to originate from the kidney. The localization of thromboxane synthase, a key enzyme of arachidonic acid metabolism, was studied in the human kidney by immunohistology using the monoclonal antibody Tü 300. In the interstitial tissue dendritic reticulum cells surrounding the tubules expressed high concentrations of the enzyme. In glomeruli the enzyme was weakly expressed in podocytes. This was confirmed by co-localization with an antiserum directed to podocalyxin, a marker of the visceral epithelial cells. In the study of various kidney diseases, massive accumulation of thromboxane synthase containing cells was observed in interstitial diseases, whereas in glomerular diseases there were no differences from normal kidney; in a case of thrombotic microangiopathy podocytes exhibited an increase in thromboxane-synthase. The thromboxane-synthase positive infiltrating interstitial cells were shown by conventional light microscopy to be mononuclear phagocytic cells. The physiological sources of renal thromboxane are dendritic reticular cells and podocytes. In interstitial renal disease infiltrating cells of the monocyte/macrophage system constitute the major site of thromboxane synthesis. In glomerular disease, a characteristic alteration of thromboxane-synthase was not found.

Antibodies, Monoclonal↗
PubMed1994

Eicosanoid biosynthetic enzymes in placental and decidual tissues from preeclamptic pregnancies: increased expression of thromboxane-A2 synthase gene.

Preeclampsia is a disease of late pregnancy characterized by hypertension, edema, and proteinuria, in which vasoconstriction, platelet aggregation, and reduced uteroplacental blood flow contribute to preterm delivery, perinatal morbidity, and mortality. Increased thromboxane-A2 (TXA2) and/or decreased prostacyclin (PGI2) have been implicated as causative factors of this disease. The present studies investigated the expression of TXA2 synthase gene along with those of TXA2 receptors, PGI2 synthase, cyclooxygenase-1 (COX-1), and COX-2 in placental and decidual tissue from preeclamptic and normal pregnancies. In situ hybridization and immunocytochemistry showed that primarily trophoblast layer and decidual cells express TXA2 synthase, COX-1, and COX-2 enzymes. Immunocytochemistry for PGI2 synthase and in situ hybridization for TXA2 receptors showed similar results. Trophoblast layer and decidua from preeclamptic pregnancies contained a greater abundance of mRNA and protein of TXA2 synthase than the matched normal pregnancies. In summary, our findings suggest that an increased local expression of TXA2 synthase could be responsible for local and/or peripheral vascular changes in preeclampsia.

Decidua↗
PubMed1994

Inhibition of NADPH-oxidase activity in human polymorphonuclear neutrophils by lipophilic ascorbic acid derivatives.

As recently reported, B-003 (6-S-hexadecyl-2-methoxythioascorbic acid) shows strong inhibition of the N-formylmethionylleucyl phenylalanine (fMLP)-stimulated neutrophil superoxide production and degranulation ex vivo, which is not correlated with its antioxidant properties. Structure-activity studies with 12 derivatives. together with permeation studies, pointed to a process for uptake of B-003 but not its regioisomer B-015 into neutrophils and revealed the importance of the free acidic enolic hydroxyl group in the 3-position of ascorbic acid and of a long chain alkyl group having a chain length of C16-C18 for effective inhibition. We now report that B-003 also strongly suppressed C5a-, concanavalin A-, and calcium ionophore A23187-stimulated superoxide formation, whereas protein kinase C-mediated activation by phorbol ester remained unaffected. The fMLP- or C5a-induced calcium mobilization form intracellular stores of fura-2-loaded cells, as well as the fMLP- or A23187-triggered release of [14C] arachidonate from prelabeled neutrophils, was not affected by B-003. The observed release of GSH was not causally related to inhibition of the oxidative burst, because GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the fMLP-stimulated superoxide formation or on the inhibitory effect of B-003. In a cell-free system, consisting of a light membrane fraction and a cytosol fraction from resting neutrophils, B-003 inhibited the arachidonate-induced assembly of the NADPH-oxidase under conditions where particulate NADPH-oxidase from phorbol ester-preactivated neutrophils and catalytically active cell-free assembled oxidase were not affected. The inhibitory effect was more pronounced when the system was incubated in the presence of the G protein activator guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). [35S]GTP gamma S binding studies excluded displacement of the G protein activator from guanine nucleotide binding sites by B-003. In vitro assembly/co-sedimentation experiments in the presence of GTP gamma S revealed a 2-fold increase in a small cytosolic G protein with a molecular mass of 21 kDa (p21) in pelleted membranes, as detected by [35S]GTP gamma S protein blot probing, that was not affected by B-003. Structure-activity relationship studies of the effects of various 6-S-alkylascorbyl derivatives on the GTP gamma S/arachidonate-triggered assembly of the NADPH-oxidase showed strong dependence of the inhibition on the alkyl chain length, with long chain alkyl derivatives (C16 and C18) being most effective.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Antioxidant and neutrophil-inhibiting properties of new 2-O-methyl-6-(alkylthio)ascorbic acid derivatives.

A series of new 6-(alkylthio)ascorbic acids was synthesized, and their inhibitory effects on lipid peroxidation and the oxidative burst of human neutrophils were tested. Of 12 structurally different lipophilic ascorbic acid derivatives 6-S-n-hexadecyl-2-O-methyl-6-deoxy-6-thio-L-ascorbic acid (7b; B-003) inhibited the Fe2+/ADP-induced lipid peroxidation of rat liver microsomes with an IC50 value of 2 microM. In human neutrophils, 7b most potently inhibited the fMLP-induced oxidative burst in a cell density-dependent manner with an IC50 value of 0.6 microM at 5 x 10(5) cells/mL. Shorter alkyl chain lengths decreased the inhibitory potency for both lipid peroxidation and oxidative burst, but in general no correlation was found between the two parameters. Likewise, 6-S-n-hexadecyl-3-O-methyl-6-thio-L-ascorbic acid (7c; B-015), the regioisomer of 7b, was a potent antioxidant but did not affect the oxidative burst. Since superoxide anions generated by xanthine/xanthine oxidase were not quenched by 7b, it became evident that its target was somewhere between receptor stimulation and NADPH-oxidase activation. By measuring the cellular concentrations of 7b and 7c, an accumulation of the first was found explaining its potency and the dependence on cell density. Expecting a pKa value of 3.3 for 7b and 7.7 for 7c a protonophore action of 7b was likely and could be verified by the drop in intracellular pH (pHi) which did not occur with 7c. Ionophores such as nigericin, CCCP, or propionic acid also lowered the pHi but did not inhibit the oxidative burst, indicating that the pHi drop was not the cause for this inhibition. 7b also strongly inhibited the fMLP-induced secretion of azurophilic (IC50 = 7 microM) and specific (IC50 = 2.5 microM) granules.

Animals↗
PubMed1993

Selective eicosanoid formation during HL-60 macrophage differentiation. Regulation of thromboxane synthase.

Earlier studies on HL-60 cells induced to differentiate into macrophages by phorbol esters have shown a selective stimulation of thromboxane (Tx) formation from endoperoxide prostaglandin (PG) H2, indicating that Tx synthesis is regulated at the level of Tx synthase (TxS), one of the peripheral enzymes of the PGH-synthase pathway. We now report on the regulation of TxS during HL-60 macrophage differentiation using monoclonal anti-TxS serum and comparing turnover rates of TxS and its biological activity with those of other enzymes of arachidonic acid metabolism. Western-blot analysis, enzyme-linked immunosorbent assay, immunohistochemical staining and [35S]methionine-labeling experiments suggested a phorbol-ester-dependent early induction of synthesis of TxS. [35S]Methionine incorporation into TxS was stimulated within 4 h after initiation of differentiation and was associated with a major rise in the TxS catalytical activity. Pulse-chase experiments showed a half life for the TxS protein of 16.4 h in both control and phorbol-ester-treated cells. The biological half life of TxS was 10.5 h, as determined by PGH2 incorporation into TxB2 after cycloheximide treatment. In contrast, the biological half lives of PGH synthase, prostacyclin synthase and 5-lipoxygenase were significantly shorter and were 3, 2.5 and 2.5 h, respectively. These results reveal that Tx synthesis in macrophages is mediated by at least two distinct mechanisms; a protein-kinase-C-dependent induction of de novo synthesis of TxS and the selective resistance of the enzyme against the activity of protein kinase C.

Cell Differentiation↗
PubMed1993

Expression of human thromboxane synthase using a baculovirus system.

Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479-1484]. A recombinant baculovirus TXS7 was expressed in Spodoptera frugiperda Sf9 insect cells. The expressed protein was recognized by monoclonal antibody, Kon 7 raised against human TX synthase [(1990) Blood 76, 80-85]. The recombinant TX synthase catalyzed the conversion of prostaglandin (PG) H2 to TXA2 and 12-hydroxy-heptadecatrienoic acid (HHT). Both conversions of PGH2 to TXA2 and HHT by the expressed TX synthase were completely inhibited by a specific TX synthase inhibitor, OKY-046 (5 microM).

Animals↗
PubMed1993

Efflux of cyclic GMP from activated human platelets.

Activation of human platelets is associated with an increased level of cGMP, when total cGMP in individual samples is measured. However, by discriminating between intracellular and extracellular cGMP we were able to demonstrate that cGMP accumulates in the extracellular space only, whereas the level of intraplatelet cGMP actually decreases. Therefore, during the first minutes of platelet aggregation cGMP is released from the cell, and it thereby escapes hydrolysis by intracellular phosphodiesterases. In contrast, during direct activation of soluble guanylyl cyclase by nitrovasodilators, such as sodium nitroprusside, the newly synthesized cGMP remains mainly inside the cells. Elevation of intracellular calcium and activation of protein kinase C are likely to be involved in promoting cGMP efflux. Our results are discussed in contrast to the general hypothesis that the cGMP increase associated with platelet aggregation may represent a feedback mechanism designed to terminate early events of activating signal transduction. According to our data the apparent cGMP increase results from cGMP release from thrombocytes, rather than soluble guanylyl cyclase activation. This cGMP efflux provides a mechanism of decreasing the intracellular cGMP level upon stimulation with platelet agonists and thus favors platelet activation.

Blood Platelets↗
PubMed1992

Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester.

The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the mRNA levels of two enzymes, thromboxane synthase (TXS) and prostaglandin endoperoxide synthase (PES), responsible for the synthesis of thromboxane A2 from arachidonic acid, were studied in human erythroleukemia (HEL) cells by RNA blot analysis. TPA induced both TXS and PES mRNAs in HEL cells in a dose-dependent manner at 36 h. The half-maximal and maximal effects for the induction of both mRNAs were at approximately 3 x 10(-9) M and at 10(-8) M, respectively. TXS and PES mRNA levels increased in a time-dependent fashion by TPA, and reached to 7- and 3.5-fold of the control, respectively after 48 h of TPA treatment. These results suggest that expression of TXS and PES genes in HEL cells were simultaneously stimulated by TPA.

Cyclooxygenase Inhibitors↗
PubMed1992

Cyclic nucleotides and intracellular-calcium homeostasis in human platelets.

The relationship between agonist-sensitive calcium compartments and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese influx was investigated in relation to the effect of altered cyclic-nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self-amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin-sensitive compartments play only a minor role in inducing manganese influx compared to the receptor-sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist-elevated platelet calcium into the thapsigargin-sensitive compartment, from which calcium can be released by inhibition of the Ca(2+)-ATPase. In human platelets, thapsigargin-induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane-A2-receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self-amplification mechanism of platelets to operate.

Blood Platelets↗
PubMed1992

Regulation of cyclooxygenase and thromboxane synthase in human monocytes.

Stimulation of human monocytes by lipopolysaccharide or phorbol ester resulted in an increase in thromboxane-B2 and prostaglandin-E2 production, whereas interleukin 1, tumour necrosis factor alpha and leukotriene C4 exerted no effects. Inhibitors of protein kinase C suppressed these increases. The activity of cyclooxygenase was induced 3.2-fold by an 8-h stimulation, whereas thromboxane-synthase and prostaglandin-E-isomerase activities remained unchanged. A glucocorticoid, dexamethasone, blocked both basal and induced prostanoid release, as well as cyclooxygenase activity. By immunoprecipitation, we were able to demonstrate an enhanced de novo synthesis of cyclooxygenase protein induced by lipopolysaccharide and phorbol ester. Dexamethasone suppressed cyclooxygenase synthesis, whereas thromboxane synthase was induced. For cyclooxygenase, we calculated a half-life of 3.2 h in human monocytes, and for thromboxane synthase, a half-life of 28 h. These results suggest that the regulation of differential prostanoid production mainly occurs by up and down regulation of cyclooxygenase.

Cells, Cultured↗
PubMed1992

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.

Alkaloids↗
PubMed1992

Localization of thromboxane synthase in human tissues by monoclonal antibody Tü 300.

Using the monoclonal antibody Tü 300 we localized thromboxane synthase, a secondary enzyme of the arachidonic acid cascade, employing the alkaline phosphatase anti-alkaline phosphatase method and indirect double labelling immunofluorescence in frozen sections of human tissues. Aside from platelets, the source of the antigen, all cells of the mononuclear phagocytic system were positive, including epithelioid cells and associated giant cells, starry sky macrophages, dendritic cells of T-cell areas, Langerhans cells and Kupffer cells. In addition, some epithelial cells such as epithelia of tonsillar crypts, reticular epithelia of the thymic cortex and ductular epithelia in liver, pancreas, female breast and salivary glands showed occasional focal reactivity for thromboxane synthase. We suggest that the mAb Tü 300 is a key marker for the macrophage system and the thromboxane generating system in normal and pathological conditions. It may detect functional activities of as yet unknown significance in some specialized epithelial cells.

Blood Platelets↗
PubMed1992

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.

Blood Platelets↗
PubMed1992

Regulation of leukotriene biosynthesis in human polymorphonuclear leukocytes.

Since the formation of leukotrienes in human polymorphonuclear leukocytes is Ca-dependent we have determined the Ca-pools involved and the enzymes affected. With PAF, fMLP and LTB4 as agonists the Ca-transients measured with Fura-2 showed the same height of the intracellular Ca-signal but an external Ca-component in decreasing magnitude. In parallel the activity of 5-lipoxygenase was also decreasing. 5-lipoxygenase was linearly dependent on Ca with saturation of around 350 nMol/l already provided by the release of intracellular Ca. Phospholipase A2, however, started to activate at about this concentration. Hence arachidonate is a limiting factor in stimulated leukocytes due to a limited influx of extracellular Ca.

Arachidonate 5-Lipoxygenase↗
PubMed1991

Different calcium pools in human platelets and their role in thromboxane A2 formation.

Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.

Antioxidants↗
PubMed1991

Molecular cloning of human platelet thromboxane A synthase.

Complementary DNA coding for thromboxane A synthase was amplified by polymerase chain reaction using primers synthesized according to the partial amino acid sequences of human platelet thromboxane A synthase (Nüsing, R., Schneider-Voss, S., and Ullrich, V. (1990) Arch. Biochem. Biophys. 280, 325-330) and cloned into pBluescript SK II(-). The primary structure of human platelet enzyme was deduced from the nucleotide sequence of the cDNA. The enzyme is composed of 533 amino acids with a molecular weight of 60,487. The primary structure of the enzyme exhibited a 34-36% homology to the amino acid sequences of cytochrome P450s classified in the P450 III gene family. The highly conserved cysteine-containing sequence involved in the heme-binding site of P450 was found near the carboxyl terminus (residues 472-492). The size of the major thromboxane A synthase mRNA from human platelets and human erythroleukemia cells was estimated to be approximately 2.2 kilobases by RNA blot analysis.

Amino Acid Sequence↗