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V Ullrich

Publications and source records attributed to V Ullrich.

At least 91 records · Page 5Linked to original sources
PubMed1991

Porcine leukocyte 5- and 12-lipoxygenases are iron enzymes.

5- and 12-lipoxygenases isolated from porcine leukocytes were investigated by electron paramagnetic resonance at X-band and atomic absorption spectroscopy. For comparison potato 5-lipoxygenase was studied under identical experimental conditions. All three lipoxygenases contained between 0.7 and 0.9 Fe atoms/enzyme molecule. As isolated, both mammalian enzymes exhibited a characteristic EPR signal at low magnetic field with a maximum at g = 5.20 indicative of a high-spin ferric iron center. The signal was not affected by the oxidants 12-hydroperoxyeicosatetraenoic acid or arachidonic acid, nor was it affected by the reductant nordihydroguaiaretic acid. In the case of the potato enzyme an intense EPR signal with resonances at g = 7.50, 6.39 and 5.84 was only observed after addition of an oxidant, such as 9-hydroperoxyoctadecadienoic acid.

Animals↗
PubMed1991

Calcium mobilization in human platelets by receptor agonists and calcium-ATPase inhibitors.

Inhibitors of the endoplasmic reticulum Ca(2+)-ATPase like thapsigargin (TG) and 2,5-di (tert-butyl)-1,4-benzohydroquinone (tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 microM) mobilized 100% of sequestered Ca2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 microM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway (Ki approximately 7 microM). Therefore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca(2+)-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A2 to cause a similar response. We conclude that the calcium store sensitive to Ca(2+)-ATPase inhibitors is a subset of the receptor sensitive calcium pool.

Arachidonic Acids↗
PubMed1991

Ebselen affects calcium homeostasis in human platelets.

Ebselen (PZ 51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) is a selenoorganic compound with anti-inflammatory properties. Its pharmacological action is thought to originate from its peroxidase activity which could lower the peroxide tonus required for cyclooxygenase and lipoxygenase activations. From experiments with aspirin-treated human platelets we now present evidence that ebselen also affects intracellular calcium homeostasis by inhibiting the agonist-triggered increase in intracellular calcium. Using Mn2+ entry to quench the fura-2 fluorescence after cell stimulation, we could exclude an interaction of ebselen with receptor-operated calcium channels and therefore an inhibition of extracellular calcium influx. It became evident from whole cell experiments and by using isolated platelet microsomal vesicles that ebselen inhibits the inositol 1,4,5-trisphosphate (IP3) induced calcium release. Besides this inhibitory effect of ebselen on the calcium release higher concentrations of the compound (greater than or equal to 5 microM) induced a calcium release from our microsomal vesicles which also could be reversed by dithiothreitol. An activation of inflammatory cells is usually associated with increased cytosolic calcium concentrations. An inhibition of such calcium movements by ebselen may account for an up to now unidentified anti-inflammatory mechanism of ebselen action which is linked to a direct effect of this compound rather than to its peroxidase-like activity.

Anilides↗
PubMed1991

The involvement of extracellular calcium in the formation of 5-lipoxygenase metabolites by human polymorphonuclear leukocytes.

We have addressed the question why in the presence of a Ca2+ ionophore human polymorphonuclear leukocytes generate leukotrienes in high yields, but in only low amounts after stimulation by receptor agonists like fMLF (fM, formylmethionine), leukotriene B4 or platelet-activating factor (PAF), although a significant release of intracellular calcium can be measured. Using ionomycin we can show that from the two enzymes involved, phospholipase A2 and 5-lipoxygenase, the first requires a threshold level of about 350-400 nM calcium whereas 5-lipoxygenase shows a linear dependence on calcium and saturates at this concentration. Our data indicate that the Ca2+ requirement of phospholipase A2 can only be met by an additional influx of extracellular calcium, whereas 5-lipoxygenase will operate already at levels provided by intracellular stores. Consequently, the complexing of extracellular calcium by EGTA stops phospholipase A2 activity immediately, whereas added arachidonate can be still adequately metabolized by intracellular Ca2+ release triggered by fMLF or PAF. Interestingly, PAF shows a stronger extracellular component in its Ca2+ transient than fMLF, and also generates more 5-lipoxygenase metabolites. However, a clear correlation between the amount of 5-lipoxygenase metabolites and the extracellular Ca2+ signal was lacking, since maximal activity was achieved before the bulk of the extracellular calcium was monitored. Ca2+ influx after PAF stimulation could be blocked after 2 min by EGTA, but a further increase in the formation of 5-lipoxygenase metabolites was observed. In contrast ionomycin-elicited 5-lipoxygenase activity could be stopped at any time shortly after EGTA addition.

Arachidonate 5-Lipoxygenase↗
PubMed1991

Carbon monoxide relaxes ileal smooth muscle through activation of guanylate cyclase.

The reported relaxing effect of CO on various smooth muscle tissues could also be found in guinea pig ileal strips. The effect was pronounced after precontraction with 10-100 nM acetylcholine and rather small with KCl. Based on the photoreversibility of the CO-dependent relaxation, a photochemical action spectrum was established which showed a maximum at around 422 nm. This definitely rules out the participation of a cytochrome P450 dependent process as postulated for the CO induced relaxation of lamb ductus arteriosus. With regard to the potency of KCN and antimycin A to relax ileal smooth muscle, the involvement of respiratory chain inhibition was reinvestigated, but no indication for such a mechanism could be obtained. In analogy to the mechanism of CO-inhibition of platelet activation we found that CO about doubles cGMP levels in guinea pig ileal strips. This is similar to NO which also leads to effective relaxation. We propose that CO can be considered and experimentally used as a convenient activator of soluble G-cyclase in smooth muscle and platelets.

Acetylcholine↗
PubMed1991

Carbon monoxide inhibits depolarization-induced Ca rise and increases cyclic GMP in visceral smooth muscle cells.

Monocytes were isolated from the urinary bladder of the guinea-pig. By means of the voltage clamp technique, whole cells were depolarized from -65 to +10 mV in order to increase the intracellular calcium concentration [Ca2+]i and to monitor this increase by means of the calcium activated potassium current IK.Ca. Superfusion of the cells with carbon monoxide-containing solutions for 2 min inhibited the signal to about 50% of the control suggesting depression of the depolarization-induced increase in [Ca2+]i. The CO-mediated inhibition of IK.Ca was partially reversed by wash-off of CO; flashes of high light intensity accelerated the rate of recovery. Sodium nitroprusside (0.01-1 mM) depressed the depolarization-induced increase in [Ca2+]i similar to CO. In multicellular preparations of the urinary bladder, CO-containing media were shown to increase the cGMP concentration by a factor of 2 in the absence and by a factor of 3 in the presence of 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). According to our previous work, CO binds to and activates soluble guanylate cyclase [Brüne B and Ullrich V, Mol Pharmacol 32: 497-504, 1987; Utz J and Ullrich V, Naunyn Schmiedebergs Arch Pharmacol 337 (Suppl): 299, 1988] and the rise in cGMP could thus effect [Ca2+]i by still unknown mechanisms.

Animals↗
PubMed1991

Influence of selective phosphodiesterase inhibitors on human neutrophil functions and levels of cAMP and Cai.

Chromatographic analysis of 3',5'-cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the cytosol of human neutrophils shows the predominant presence of PDE IV (cAMP specific) and PDE V (cGMP specific). PDE IV is characterized by (1) cAMP selectivity, (2) a KM for cAMP of 1.2 microM and (3) a typical rank order of IC50-values for PDE inhibitors: 0.13, 0.17, 47 and 9.5 microM for PDE IV selective rolipram, PDE III/IV selective zardaverine, PDE III selective motapizone and unselective 3-isobutyl-1-methylxanthine (IBMX), respectively. Functions of polymorphonuclear leukocytes (PMN) such as N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide release and fMLP/thimerosal elicited leukotriene (LT) biosynthesis are inhibited by these PDE inhibitors with the same rank order and even lower IC50-values. Measurements of changes in cytosolic Cai in Fura-2 loaded PMN demonstrate a transient Cai increase after stimulation with 0.1 microM fMLP and an additional sustained elevation of Cai levels in the presence of thimerosal. PDE inhibitors suppress this sustained phase of Cai release with the same rank order of IC50-values as LT biosynthesis. The correlation between fMLP/thimerosal-induced LT biosynthesis and Cai levels reveal a Cai threshold of 150 nM for arachidonic acid metabolism. cAMP levels in PMN were elevated by PDE inhibitors alone by less than 2-fold. In the presence of fMLP however, cAMP was increased up to 10-fold and the efficacy of PDE inhibitors to increase cAMP paralleled their potency to inhibit PDE IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonate 5-Lipoxygenase↗
PubMed1991

12-hydroperoxyeicosatetraenoic acid inhibits main platelet functions by activation of soluble guanylate cyclase.

12-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) as well as several other fatty acid hydroperoxides are potent inhibitors of platelet activation. 12-HPETE but not 12-hydroxy-5,8,10,14-eicosatetraenoic acid blocks the U46619- and the thrombin-triggered aggregation of aspirin-treated platelets, dose dependently. 12-HPETE suppresses thromboxane production by inhibiting platelet cyclooxygenase and stimulates its own production by increasing lipoxygenase activity, although this effect does not explain the inhibitory activity of 12-HPETE during the initial phase of cell activation. The inhibitory effect is related to altered calcium homeostasis during platelet activation. 12-HPETE inhibits calcium release from intracellular stores and modifies the influx of extracellular calcium. The inhibitory effect on calcium mobilization is explained by activation of soluble guanylate cyclase. These inhibitory properties are shared by sodium nitroprusside, a compound known to activate soluble guanylate cyclase. Fatty acid hydroperoxides, especially 12-HPETE, produce a rapid and dose-dependent activation of soluble guanylate cyclase, using intact human platelets as a detection system. Activation of the enzyme shows a position isomer specificity, with 12-HPETE being the most potent activator. The generation of the labile lipoxygenase product 12-HPETE during platelet activation may modulate platelet reactivity by increasing cyclic GMP. This pathway may contribute to a physiological feedback mechanism to limit the size of a growing platelet plug.

Blood Platelets↗
PubMed1991

Low levels of hydrogen peroxide enhance platelet aggregation by cyclooxygenase activation.

Platelet aggregation can be triggered by addition of exogenous arachidonate owing to its conversion to endoperoxides and thromboxane A2. The dose-response curve of arachidonate-induced platelet aggregation exhibited a very steep slope. Simultaneous addition of H2O2 (1-200 microM) significantly shifted this curve to the left. H2O2 alone did not induce aggregation up to a concentration of 1 mM; however, a reversible increase of cytoplasmic Ca2+ and a small increase of the thromboxane levels could be observed. In the presence of exogenous arachidonate H2O2 led to an increased formation of arachidonate metabolites. Our data demonstrate that at threshold levels of 20:4 H2O2 is able to promote conversion of 20:4 to proaggregatory prostaglandin endoperoxides, and subsequently platelet activation is facilitated. Thus we support the evidence for a role of H2O2 in the activation of cyclooxygenase possibly by providing an adequate peroxide tone.

Dose-Response Relationship, Drug↗
PubMed1990

Soybean lipoxygenases-1, -2a, -2b and -2c do not contain PQQ.

Soybean isoenzymes lipoxygenases-1, -2a, -2b and -2c were examined spectroscopically for the presence of covalently bound pyrrolo quinoline quinone (PQQ) after derivatization by phenylhydrazine (PH), 2,4-dinitrophenylhydrazine (DNPH) and 3-methyl-2-benzothiazolinone hydrazone (MBTH). DNPH derivatization of PQQ after a pronase digestion step of lipoxygenase-1 in the presence of an anion exchange gel fixing the cofactor was also investigated. None of these experiments provided evidence for the presence of PQQ contrary to previous report by Van der Meer et al (1). We have checked, by EPR spectroscopy, that the three reactants used were able to reduce the active site ferric iron. Our results were confirmed by the absence of enzyme inhibition by cis- and trans-1,2-diaminocyclohexane or benzylamine in the presence of NaBH3CN which have been reported to react with PQQ and to inactivate quinoproteins (2,3).

Benzothiazoles↗
PubMed1990

Activation of soluble guanylate cyclase by carbon monoxide and inhibition by superoxide anion.

Human platelet soluble guanylate cyclase activity was studied with respect to the function of its heme-containing regulatory subunit. As an enzyme source, the 10,000 x g supernatant was used and, since its specific activity proved to be too low for inhibition studies, also a partially purified preparation was employed. The partially purified enzyme was stimulated about 2.5-fold by carbon monoxide and this effect was abolished by illumination with visible light. Sodium nitroprusside also increased the basal activity about fourfold, which, however, is much less than the greater than 100-fold stimulation seen with the supernatant. Superoxide anions generated by the xanthine/xanthine-oxidase system were strongly inhibitory in the enriched preparation as well as in the CO-stimulated platelet supernatant (median effector concentration = 0.1 mU/ml). Unlike CO and NO, the effect of superoxide cannot be mediated through the heme-containing regulatory subunit, since heme-free enzyme, which could not be activated by NO or CO, was inhibited to the same extent as the heme-containing enzyme. Superoxide dismutase did not influence the basal activity, but resulted in a synergistic stimulation in the presence of CO. When Mn2+ replaced Mg2+ as a cofactor, the basal activity was higher but superoxide could not inhibit the enzyme, possibly due to the superoxide-dismutase-like activity of Mn2+. Superoxide turned out to be a potent and reversible inhibitor of soluble guanylate cyclase which, together with endothelium-derived relaxing factor, recently identified as NO, could form a physiologically relevant regulatory effector system.

Blood Platelets↗
PubMed1990

Immunoaffinity purification of human thromboxane synthase.

A recently produced monoclonal antibody against human thromboxane synthase was used to purify the enzyme from platelets in a one-step procedure with good yields. The isolated protein exhibited a single band of about 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained one heme/mol. Although the visible spectrum of the oxidized enzyme displayed a peak at 418 nm like the previously isolated enzyme after dithionite reduction and CO addition, it shifted to 419 nm but not to 450 nm where only a small shoulder could be detected. Its catalytic activity was only 1-5% of the previous preparations, but with the same Km of about 10 microM and a ratio of thromboxane B2: 12-hydroxyheptadecatrienoic acid of 1:1. Studies with EPR spectrometry and inhibitors confirmed that only a minor part of the enzyme was in its native heme-thiolate conformation, whereas the major part had been converted to the inactive P420 form by the elution procedure. The amino acid analysis revealed 46% hydrophobic residues. According to the sequence of 26 amino acids from the N-terminus and two tryptic peptides no homology to one of the cytochrome P450 monooxygenases, or to cyclooxygenase, or to prostacyclin synthase was detected.

Amino Acid Sequence↗
PubMed1990

Production and characterization of polyclonal and monoclonal antibodies against human thromboxane synthase.

Polyclonal and monoclonal antibodies (MoAbs) were raised against human platelet thromboxane (Tx) synthase. Neither the antiserum nor the MoAbs inhibited the enzyme activity significantly. Three MoAbs, Tü 300, Kon 6, and Kon 7, were purified and further characterized. They are monospecific as shown by activity precipitation or Western blot analysis, and recognized different epitopes on Tx-synthase. Tü 300 could precipitate the enzyme and recognized conformational epitopes, whereas Kon 6 and Kon 7 only reacted in Western blots. Antibody Tü 300 can be used in immunohistology but shows no crossreactivity with Tx-synthase from other species. In human lung tissue staining with peroxidase, coupled Tü 300 was only found in alveolar macrophages.

Antibodies↗
PubMed1990

Involvement of calcium in the thimerosal-stimulated formation of leukotriene by fMLP in human polymorphonuclear leukocytes.

Only small amounts of leukotrienes could be detected by reverse-phase HPLC analysis after stimulation of human polymorphonuclear leukocytes (PMN) by the receptor agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). Preincubation of the cells with the organomercury compound thimerosal prior to fMLP-addition, however, resulted in the formation of significant amounts of 5-lipoxygenase derived metabolites. This effect was dose-dependent with respect both to fMLP and thimerosal. Thimerosal alone did neither lead to the formation of HPLC-detectable leukotrienes nor to the release of arachidonic acid in [1-14C]arachidonic acid prelabelled cells. The formation of leukotrienes by fMLP/thimerosal required extracellular Ca2+. Measurements of intracellular Ca2(+)-levels revealed that (i) thimerosal alone is able to release Ca2+ from internal stores and (ii) thimerosal causes a persistent accumulation of Ca2+ within the cells after stimulation by fMLP. We conclude that by the synergistic action of fMLP and thimerosal the Ca2(+)-levels exceed the threshold for phospholipase A2 activation resulting in the liberation of arachidonic acid and subsequently in the formation of 5-lipoxygenase products. Our results suggest that thimerosal may provide a model for leukotriene formation under pathophysiological conditions when SH-group oxidation leads to increased intracellular Ca2(+)-levels.

Arachidonate 5-Lipoxygenase↗
PubMed1990

Immunohistochemical localization of thromboxane synthase in human tissues.

Thromboxane A2 is formed mainly in platelets where we have isolated thromboxane synthase as a cytochrome P450-like haemoprotein. A rabbit antiserum prepared against this enzyme was monospecific according to Western blot analysis and was used for immunostaining of human tissues. In liver the Kupffer cells were clearly stained and in the connective tissue, histiocytes and monocytic cells were positive. In lung, alveolar macrophages contained most of the enzyme, whereas Type I and II cells were negative. Kidney mesangial cells were stained and in lymphatic tissue dendritic reticular cells as well as invading macrophages were positive. These were also seen in the crypts of tonsils and surprisingly also the epithelium. Placenta again showed staining of the Hofbauer cells and of the endothelial lining of the vessels. Umbilical cord and uterus only contained weak antigenicity in dendritic cells.

Female↗
PubMed1990

Immunoquantitation of thromboxane synthase in human tissues.

Although the formation of TxB2 has been reported for different cells such as monocytes, lymphocytes, granulocytes or fibroblasts and tissues like lung and liver, the corresponding enzyme has not yet been measured. We have established an enzyme-linked immunosorbent assay (ELISA) for measurement of human Tx-synthase with a monospecific polyclonal and a purified monoclonal antibody. ELISA data were verified by either western blots or immunohistochemistry. Next to platelets, blood monocytes were found to have the highest content of Tx-synthase, whereas lung fibroblasts and promyelocytic cells possess only very low levels. In granulocytes and lymphocytes neither Tx-synthase nor its activity could be demonstrated. Among different tissues, lung and liver showed the highest content of Tx-synthase. These data correlate TxB2-synthase activity in a given human tissue with its enzyme content of Tx-synthase for the first time. In view of the frequent involvement of TxA2 in pathological conditions, the technique developed may prove valuable for the characterization of disease states, particularly inflammatory events.

Blotting, Western↗