Biomedical subjects

Ulrich Walter

Publications and source records attributed to Ulrich Walter.

65 records · Page 4Linked to original sources

PubMed2002

Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits.

OBJECTIVE: With the present studies we sought to determine how treatment with nitroglycerin (NTG) affects endothelial function, oxidative stress and nitric oxide (NO)-downstream signaling in Watanabe heritable hyperlipidemic rabbits (WHHL). BACKGROUND: In vitro experiments have demonstrated potent antiatherosclerotic effects of NO suggesting that treatment with NO-donors such as NTG could compensate for the diminished availability of endothelial NO. Nitric oxide may, however, not only be scavenged by reaction with endothelium-derived superoxide but also form the potent oxidant and inhibitor of vascular function, peroxynitrite (ONOO(-)). METHODS: Watanabe heritable hyperlipidemic rabbits were treated for three days with NTG patches. Normolipidemic New Zealand White rabbits (NZWR) served as controls. Endothelial function was assessed ex vivo with organ chamber experiments and vascular superoxide was quantified using lucigenin (5 and 250 microM) and CLA-enhanced chemiluminescence. Vascular ONOO(-) formation was determined using nitrotyrosine antibodies. The activity of the cGMP-dependent kinase (cGK-I) was assessed by determining the phosphorylation of vasodilator-stimulated phosphoprotein VASP (P-VASP). RESULTS: Nitroglycerin treatment caused endothelial dysfunction in NZWR and WHHL, associated with an increase in superoxide and ONOO(-) production and a substantial drop in cGK-I activity. In vivo NTG-treatment decreased lipophilic antioxidants (alpha- and beta-carotene) in NZWR and WHHL. Treatment of NZWR with NTG also decreased plasma extracellular superoxide dismutase (EC-SOD)-activity. CONCLUSIONS: Nitroglycerin treatment of WHHL with exogenous NO worsens rather than improves endothelial dysfunction secondary to increased formation of superoxide and/or peroxynitrite leading to decreased cGK-I activity. The decrease in plasma levels of alpha- and beta-carotene may be at least in part due to a decrease in EC-SOD activity.

Animals↗

PubMed2002

Relaxation, equilibrium oligomerization, and molecular symmetry of the VASP (336-380) EVH2 tetramer.

An investigation of the structural and dynamic properties of the C-terminal fragment of the human protein VASP (VASP 336-380) has been performed. Full length VASP has been shown to be tetrameric in solution, and the C-terminal 45 residues of the protein have been suggested to be responsible for the oligomerization. We have expressed and purified a C-terminal fragment of the human VASP protein from residue 336-380. It was found to form a stable domain in its own right. The fragment was shown by CD spectroscopy to form a helical structure, stable under a wide range of temperature and pH conditions. A (15)N-HSQC-experiment exhibits only one set of peaks, suggesting a high degree of symmetry for a putative oligomer. Measurements of the rotational correlation time tau(C) of the molecule and analytical ultracentrifugation data show VASP (336-380) to be entirely tetrameric in solution. The secondary structure was confirmed from a (15)N-NOESY-HSQC experiment and is completely alpha-helical. We conclude that VASP (336-380) forms a tetramer in solution via a coiled coil arrangement and is solely responsible for tetramerization of full-length VASP.

Amino Acid Sequence↗

PubMed2002

Inhibition of platelet P2Y12 and alpha2A receptor signaling by cGMP-dependent protein kinase.

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G(i)-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G(i)-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G(i)-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G(i)-signaling for platelet activation, we propose that inhibition of G(i)-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.

Blood Platelets↗

PubMed2002

CBS 844ins68, MTHFR TT677 and EPCR 4031ins23 genotypes in patients with deep-vein thrombosis.

The role of methylenetetrahydrofolate reductase (MTHFR) TT677 genotype, cystathionine beta-synthase (CBS) 844ins68 mutation and endothelial cell protein C receptor (EPCR) 4031ins23 in the development of deep-vein thrombosis (DVT) was investigated in 300 consecutive DVT patients and 410 healthy blood donors. MTHFR TT677 was found in 40 (13.3%) patients and in 59 (14.4%) controls (OR 0.92; 95% C.I. 0.54-1.41); CBS 844ins68 in 20 (6.7%) patients and in 56 (13.7%) control subjects (OR 0.45; 95% C.I. 0.27-0.77); and the combination of MTHFR TT677 with CBS 844ins68 in 4 (1.3%) patients and in 7 (1.7%) controls (OR 0.78; 95% C.I. 0.23-2.68). Logistic regression analysis did not show a further increase of risk for MTHFR TT677 or CBS 844ins68 in combination with the factor V Leiden or the prothrombin gene G20210A mutations. The EPCR 4031ins23 was observed in 2 patients (0.66%) and none of the controls. In conclusion, MTHFR TT677 does not appear to be an important risk factor for DVT, EPCR 403ins23 seems to be very rare, its role in the development of DVT unclear. A putative protective effect of CBS 844ins68 should be further investigated.

Adolescent↗

PubMed2002

Increased spreading, Rac/p21-activated kinase (PAK) activity, and compromised cell motility in cells deficient in vasodilator-stimulated phosphoprotein (VASP).

Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) proteins are key regulators that promote or inhibit actin-based motility, cell adhesion, and various aspects of axon guidance. However, a conclusive concept of Ena/VASP functions remains elusive. Here, we report that VASP-deficient fibroblasts, despite normal mammalian Enabled (Mena) and Ena-VASP-like (Evl) expression levels, are highly spread. VASP(-/-) cells cover about twice the substrate surface area as wild type cells, while cell volumes are unchanged. In accordance with these observations, activation of the Rac/p21-activated kinase (PAK) pathway, a crucial element in the regulation of cell spreading, is markedly enhanced in VASP(-/-) cells. Thus, in the absence of VASP Rac activation is dramatically prolonged, and PAK activity is elevated after stimulation with platelet-derived growth factor or serum, respectively. Moreover, VASP-deficient cells show compromised migration and reorientation in a wound healing assay. Collectively, our results reveal a VASP-dependent modulation of the Rac/PAK pathway and Rac/PAK-regulated processes, like cell motility and polarization.

Animals↗

PubMed2002

Functional and biochemical analysis of endothelial (dys)function and NO/cGMP signaling in human blood vessels with and without nitroglycerin pretreatment.

BACKGROUND: In experimental animal models, long-term in vivo treatment with nitroglycerin (NTG) induces both endothelial dysfunction and tolerance to nitrates. However, it is still controversial whether nitrate tolerance in humans is associated with both endothelial dysfunction and impaired vascular response to nitrovasodilator-derived NO. METHODS AND RESULTS: Patients undergoing elective bypass surgery were randomized to receive 48 hours of continuous NTG infusion (NTG group) or no nitrate therapy (control group). Segments of surgically removed arteria mammaria, vena saphena, and arteria radialis not required for the bypass procedure were used to examine (1) the vascular responsiveness to NTG and the endothelium-dependent vasodilator acetylcholine; (2) the expression of the NO target, the soluble guanylyl cyclase; (3) the expression of the soluble guanylyl cyclase/cGMP effector target, the cGMP-dependent protein kinase (cGK); and (4) the cGK activity as assessed by the phosphorylation state of its vascular substrate, the vasodilator-stimulated phosphoprotein at serine(239) (P-VASP). NTG treatment caused a marked degree of nitrate tolerance in all 3 vessel types studied and a significant cross-tolerance to the endothelium-dependent vasodilator acetylcholine in A. mammaria and A. radialis. Although soluble guanylyl cyclase, cGK-I, and VASP expression levels were not modified by NTG treatment, a marked decrease of P-VASP, a surrogate parameter for in-vivo cGK-I activity, was observed. CONCLUSIONS: We conclude that long-term NTG treatment induces endothelial dysfunction and impaired vascular NO/cGMP signaling in humans, which can be monitored by measuring P-VASP levels.

Aged↗

PubMed2002

Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling.

Angiotensin II infusion causes endothelial dysfunction by increasing NAD(P)H oxidase-mediated vascular superoxide production. However, it remains to be elucidated how in vivo angiotensin II treatment may alter the expression of the gp91(phox) isoforms and the endothelial nitric oxide synthase (NOS III) and subsequent signaling events and whether, in addition to the NAD(P)H oxidase, NOS III contributes to vascular superoxide formation. We therefore studied the influence of in vivo angiotensin II treatment (7 days) in rats on endothelial function and on the expression of the NAD(P)H oxidase subunits p22(phox), nox1, nox4, and gp91(phox) and NOS III. Further analysis included the expression of NO-downstream targets, the soluble guanylyl cyclase (sGC), the cGMP-dependent protein kinase I (cGK-I), and the expression and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-VASP). Angiotensin II caused endothelial dysfunction and increased vascular superoxide. Likewise, we found an increase in vascular protein kinase C (PKC) activity, in the expression of nox1 (6- to 7-fold), gp91(phox) (3-fold), p22(phox) (3-fold), NOS III mRNA, and protein. NOS-inhibition with N(G)-nitro-L-arginine decreased superoxide in vessels from angiotensin II-treated animals, compatible with NOS-uncoupling. Vascular NO assessed with electron paramagnetic resonance was markedly reduced. Likewise, a decrease in sGC-expression and P-VASP levels was found. In vivo PKC-inhibition with chelerythrine reduced angiotensin II-induced superoxide production and markedly inhibited upregulation of NAD(P)H oxidase subunits. We therefore conclude that angiotensin II-induced increases in the activity and the expression of NAD(P)H oxidase are at least in part PKC-dependent. NADPH oxidase-induced superoxide production may trigger NOS III uncoupling, leading to impaired NO/cGMP signaling and to endothelial dysfunction in this animal model. The full text of this article is available at http://www.circresaha.org.

Angiotensin II↗

PubMed2002

EVH1 domains: structure, function and interactions.

Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue protein-protein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1-containing proteins are associated closely with actin-based structures and are involved in re-organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline-rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3-6 residue) 'core' PRS in the target protein binds a 'recognition pocket' on the domain surface. Further affinity- and specificity-increasing interactions are then formed between additional domain epitopes and peptide 'core-flanking' residues. The three-dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes.

Amino Acid Sequence↗

PubMed2002

Normalization of nomenclature for peptide motifs as ligands of modular protein domains.

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer-readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.

Binding Sites↗

PubMed2002

Fine mapping of the H-kininogen binding site in plasma prekallikrein apple domain 2.

Plasma prekallikrein (PPK), the zymogen of the contact phase protease plasma kallikrein, forms a non-covalent complex with its substrate H-kininogen (HK). HK binds to cell surface proteoglycans, indirectly anchoring this bradykinin-generating protease to endothelial cells. The heavy chain of PPK consisting of four apple domains designated A1 to A4. Previous studies indicated that a major HK binding site on PPK is within the A2 domain, with additional contributions to binding provided by the N-terminal portion of Al and the central part of A4. To precisely map the relevant binding segments in A2, we employed a monoclonal anti-PPK antibody (PKH6) that binds to A2 and blocks HK-PPK complex formation with an apparent IC50 of 8 nM. Using recombinant A2 C-terminal deletion mutants, we mapped the target epitope of PKH6 to the N-terminal portion of A2, residues 92-153. C-terminal deletion of A2 to residue 145 resulted in a loss of PKH6 binding, as did proteolytic cleavage of A2 at Lys140-Arg141. A comparison of HK binding to various A2 deletion mutants revealed that the major HK binding site is localized to residues 145-153 in the central portion of A2, where it overlaps with the PKH6 epitope. This sequence is conserved in the A2 domain of the related protease factor XI, explaining the unusual strong cross-reactivity of PHK6 with factor XI, as well as the similar HK-binding characteristics of PPK and factor XI.

Antibodies, Monoclonal↗

PubMed2002

Calcium-dependent membrane association sensitizes soluble guanylyl cyclase to nitric oxide.

Nitric oxide (NO) is a ubiquitous, cell-permeable intercellular messenger. The current concept assumes that NO diffuses freely through the plasma membrane into the cytoplasm of a target cell, where it activates its cytosolic receptor enzyme, soluble guanylyl cyclase (sGC). Recent evidence, however, suggests that cellular membranes are not only the predominant site of calcium-dependent NO synthesis, but also the site of its distribution and binding. Here we extend this concept to NO signalling to show that active sGC is partially associated with the plasma membrane in a state of enhanced NO sensitivity. After cellular activation, sGC further translocates to the membrane fraction in human platelets and associates with the NO-synthase-containing caveolar fraction in rat lung endothelial cells, in a manner that is dependent on the concentration of intracellular calcium. Our data suggest that the entire NO signalling pathway is more spatially confined than previously assumed and that sGC dynamically translocates to the plasma membrane, where it is sensitized to NO.

Animals↗