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Biomedical subjects

U Walter

Publications and source records attributed to U Walter.

At least 19 recordsLinked to original sources
PubMed1999

An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment.

The LIM domain protein zyxin is a component of adherens type junctions, stress fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we demonstrate that human zyxin, like the avian protein, specifically interacts with alpha-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin. This motif is both necessary and sufficient for alpha-actinin binding, whereas a downstream region, which is related in sequence, appears to be dispensable. A synthetic peptide comprising human zyxin residues 21-42 specifically binds to alpha-actinin in solid phase binding assays. In contrast to full-length zyxin, constructs lacking this motif do not interact with alpha-actinin in blot overlays and fail to recruit alpha-actinin in living cells. When zyxin lacking the alpha-actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting to focal adhesions is grossly impaired. Our results suggest a crucial role for the alpha-actinin-zyxin interaction in subcellular zyxin localization and microfilament organization.

Actinin↗
PubMed1999

Identification of profilin and src homology 3 domains as binding partners for Drosophila enabled.

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly-(L-proline) residues with similarity to both profilin and src homology 3 binding sites. Here, we show that Ena can bind directly to the Drosophila homolog of profilin, chickadee. Furthermore, Ena and profilin were colocalized in spreading cultured cells. We report that the proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing Drosophila embryo.

Animals↗
PubMed1999

Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes.

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.

Animals↗
PubMed1998

Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody.

The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP- and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrated the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs showed that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells.

Animals↗
PubMed1998

Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells.

An overactive renin-angiotensin-aldosterone system (RAAS) has a central role in the pathogenesis of hypertension and cardiac hypertrophy, precursors of cardiac failure. Natriuretic peptides and NO acting through their second messenger, cGMP, increase natriuresis and diuresis, and inhibit renin release; however the mechanism by which this inhibition of the RAAS system functions is obscure. We recently reported cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), elucidated its first known function of inhibiting the cystic fibrosis transmembrane conductance regulator in rat intestine, and initially described its location in rat kidney juxtaglomerular (JG) cells, the ascending thin limb, and the brush border of proximal tubules. Here, we demonstrate inhibition of isoproterenol- or forskolin-stimulated renin release by 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP), a selective activator of cGK, and prevention of this inhibition by a selective inhibitor of cGK, Rp-8-pCPT-cGMPS. In systems of differing complexity, inhibition by 8-pCPT-cGMP was nearly complete in isolated perfused kidney and microdissected afferent arterioles but only approximately 25% in isolated JG cells. Expression of either cGK II or cGK I in JG cells by using adenoviral vectors enhanced the inhibition of forskolin-stimulated renin release by 8-pCPT-cGMP to 50%. Our results indicate that cGK II, and possibly cGK I, can mediate cGMP inhibitory effects on renin release and are physiological components of the cGMP signal transduction system which opposes the RAAS.

Animals↗
PubMed1998

The Silicate Garden Reaction in Microgravity: A Fluid Interfacial Instability.

In the "silicate garden" reaction, crystals of a metal salt are placed in sodium silicate solution. The crystals become coated with a semipermeable membrane of metal silicate reaction product, from which hollow tubes of metal silicate rise convectively upward, against gravity. In the absence of gravity, and free of convective influences, the reaction might be expected to reveal more fundamental organizing principles. Accordingly, we have grown silicate gardens in microgravity, from salts of calcium, cobalt, and magnesium with added nickel. Even in these isotropic conditions, complex structures developed. They included tubes and hollow spheroids, and also novel dendritic "fingers" which grew by continuous plastic deformation. This new mode of growth is favored by the slow, diffusion-limited rate of reaction in microgravity, which greatly reduces the rate of hardening of the reaction products. The magnesium-nickel garden grew almost entirely as a fluid interfacial instability between the metal salt solution inside and the silicate solution outside, by deformation of the semipermeable fluid membrane between them. The resulting shape had similarities to that of a solid front advancing through a supercooled melt. The morphology of such a solid is determined by the diffusion of released latent heat away from it, according to the Laplacian diffusion equation. We suggest that Laplacian-growth morphology arises in a microgravity silicate garden when its development is controlled by the analogous diffusion of dissolved ions away from it. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Ligand specificity and ticlopidine effects distinguish three human platelet ADP receptors.

Human platelets express adenosine 5'-diphosphate (ADP)-specific purinoceptors of the P2X and P2Y receptor superfamily, but their structure, diversity, and precise pharmacological profile is not well understood. Here, functional assays with intact platelets and well-characterized nucleotide derivatives were performed in order to characterize the ligand specificity of these platelet-specific purinoceptors. For the signalling pathways investigated (aggregation, rapid Ca2+-influx, desensitization of Ca2+-influx, Ca2+-mobilization, inhibition of adenylyl cyclase), significant differences in ligand specificity were demonstrated. ADP activated all purinoceptors of human platelets, while adenosine 5'-triphosphate (ATP) was a weak agonist for the P2X receptor and an antagonist for the P2Y receptors. The ADP-receptor pathway-antagonist ticlopidine inhibited ADP-evoked aggregation and adenylyl cyclase inhibition but did not affect platelet purinoceptors associated with Ca2+-influx and Ca2+-mobilization. These results indicate the presence of three distinct ADP-selective purinoceptors on human platelets.

Adenosine Diphosphate↗
PubMed1998

Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery.

Endothelial cells in normal blood vessels might prevent the unscheduled proliferation of smooth muscle cells (SMCs) by the expression of cell migration and growth inhibitors. NO, a potent vasodilator, generated by endothelium-specific constitutive NO synthase (ecNOS) might be such an inhibitor. To test this hypothesis, we overexpressed human ecNOS in syngeneic rat arterial SMCs using retrovirus-mediated gene transfer. Compared with SMCs transduced with vector alone (LXSN SMCs), DNA synthesis and cell proliferation were inhibited in the ecNOS-expressing SMCs (LCNSN SMCs). Basal and stimulated (by the calcium ionophore A23187) secretion of NO and intracellular cGMP were increased in LCNSN SMCs. Nomega-Nitro-L-arginine (L-NA), an inhibitor of NO synthesis, enhanced the proliferation of LCNSN SMCs but had no effect on LXSN SMCs. LCNSN SMCs seeded onto the luminal surface of balloon-injured rat carotid arteries inhibited neointimal formation by 37% and induced marked dilatation (3-fold increase in vessel diameter) at 2 weeks compared with LXSN SMC-seeded arteries. Orally administered L-NA blocked these changes. Phosphorylation of vasodilator-stimulated phosphoprotein, which is regulated in part by NO, was elevated in LCNSN SMCs and in LCNSN SMC-seeded arteries. This study demonstrates that NO generation by ecNOS inhibits SMC proliferation in vitro and modulates vascular tone locally in vivo.

Angioplasty, Balloon↗
PubMed1998

Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects.

NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs. Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various "specific cGK inhibitors" (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these "specific cGK inhibitors" have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.

Animals↗
PubMed1998

Expression of cGMP-dependent protein kinase I and its substrate VASP in neointimal cells of the injured rat carotid artery.

BACKGROUND: Neointimal fibroproliferative lesions after balloon angioplasty remain a major clinical problem, frequently leading to restenosis of initially successfully dilated coronary arteries. The cGMP-dependent protein kinase type I (cGMP-PK I) and its substrate vasodilator-stimulated phosphoprotein (VASP), molecular targets of the atrial natriuretic factor (ANF) and nitric oxide (NO) signalling pathways, are likely to be involved in various aspects of vascular wall regulation and restenosis formation. METHODS: To investigate the occurrence of cGMP-PK I and VASP in neointimal cells in situ, we performed immunohistochemistry and immunoblotting experiments on denuded rat carotid arteries. RESULTS: Although the soluble cGMP-PK I showed a homogeneous distribution throughout the neointima, VASP apparently was more concentrated in smooth muscle cells (SMCs) lining the artery lumen, possibly reflecting enhanced growth factor stimulation of luminal SMCs. The membrane-associated cGMP-PK type II could not be detected in both the non-injured vessel wall and the restenotic tissue. CONCLUSION: The presence of both cGMP-PK I and VASP, major regulators of the actin cytoskeleton and cell motility, in neointimal tissue suggest that this emerging signal transduction pathway could be a target for the regulation and control of restenosis.

Animals↗
PubMed1998

Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

Amino Acid Sequence↗
PubMed1998

YC-1 potentiates nitric oxide- and carbon monoxide-induced cyclic GMP effects in human platelets.

Nitric oxide (NO), the physiological activator of soluble guanylyl cyclase (sGC), induces inhibitory effects on platelet activation via elevation of cGMP levels and stimulation of the cGMP-dependent protein kinase. YC-1, a benzylindazole derivative, was shown to activate sGC in intact platelets, resulting in inhibition of platelet aggregation. In a previous study, we demonstrated that YC-1 not only stimulates purified sGC but also potentiates the stimulatory action of submaximally effective NO and carbon monoxide (CO) concentrations. Here, we investigated the potentiating effect of YC-1 in intact platelets. YC-1 together with NO or CO led to complete inhibition of platelet aggregation at concentrations that were ineffective by themselves. Maximally effective 2, 2-diethyl-1-nitroso-oxyhydrazine (3 microM) and YC-1 (100 microM) concentrations each elevated the cGMP levels in intact platelets approximately 13-fold, and administration of the two drugs together resulted in enormous potentiation of cGMP formation, which greatly exceeded the effect on the purified enzyme and yielded a >1300-fold increase in cGMP levels. Similar results were obtained using CO instead of NO. Furthermore, YC-1 not only stimulated sGC but also inhibited cGMP-hydrolyzing phosphodiesterases in platelets. The enormous elevation of cGMP levels led to enhanced phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein. Thus, by the combination of two effects (i.e., potentiation of NO-induced sGC stimulation and phosphodiesterase inhibition), YC-1-like substances are potent activators of the sGC/cGMP pathways and are therefore interesting candidates to act as modulators of cGMP-mediated effects, especially within the cardiovascular system.

1-Methyl-3-isobutylxanthine↗
PubMed1997

A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family.

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.

Amino Acid Sequence↗
PubMed1997

cDNA cloning of porcine p42IP4, a membrane-associated and cytosolic 42 kDa inositol(1,3,4,5)tetrakisphosphate receptor from pig brain with similarly high affinity for phosphatidylinositol (3,4,5)P3.

We previously identified a 42 kDa Ins(1,3,4,5)P4 (InsP4) receptor protein (p42IP4) in brain membranes from several species. Here the cDNA sequence of p42IP4 was obtained by PCR using degenerate primers derived from peptide sequences of proteolytic fragments of the porcine protein and by subsequent screening of a pig brain cDNA library. The derived peptide sequence of 374 amino acids for porcine p42IP4 is 45 amino acids shorter at the C-terminus than centaurin-alpha from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP4 binding activity present in brain tissue homogenate about 25% is found in the cytosolic fraction and 75% associated with microsomes. Both activities are due to p42IP4 since (i) a peptide-specific antiserum recognizing specifically p42IP4 labels the InsP4 receptor protein in membranes and in the cytosol, (ii) the antiserum immunoprecipitates both the membrane protein and the cytosolic protein of 42 kDa, (iii) the InsP4 binding activity released by high salt or by alkaline extraction from membranes is identified immunologically as the 42 kDa protein, and (iv) the affinity for InsP4 and specificity for various inositolphosphates are similar for the membrane-associated and for the soluble p42IP4. The functional importance of p42IP4 is highlighted by the identical affinity for InsP4 and for phosphatidylinositol (3,4,5)P3 (Ki = 1.6 and 0.9 nM, respectively). Thus, the InsP4 receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP4 or by PtdInsP3 between membranes and cytosolic compartment.

Amino Acid Sequence↗
PubMed1997

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta.

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Adenoviridae↗