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Biomedical subjects

U Walter

Publications and source records attributed to U Walter.

At least 127 records · Page 7Linked to original sources
PubMed1993

[The parasitization of Gadus morhua in the southern Baltic Sea].

During a survey of the working group 25 of BMB in the south of the Baltic Sea in October 1991 105 cod (Gadus morhua L.) were investigated for parasites (ICES Subdivision Areas 22, 24, 25 and 26). They were parasitized by 15 species. With increasing salinity (from east to west) and in the higher size groups more parasite species were present. Eimeria spec. in the liver and Schistocephalus spec. in the body cavity were registered for the first time in cod.

Animals↗
PubMed1992

Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP.

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells.

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets.

Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.

Adenosine Diphosphate↗
PubMed1992

The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.

Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.

Actin Cytoskeleton↗
PubMed1992

HIV-1 gp120 receptor on CD4-negative brain cells activates a tyrosine kinase.

Human immunodeficiency virus (HIV-1) infection in the human brain leads to characteristic neuropathological changes, which may result indirectly from interactions of the envelope glycoprotein gp120 with neurons and/or glial cells. We therefore investigated the binding of recombinant gp120 (rgp120) to human neural cells and its effect on intracellular signalling. Here we present evidence that rgp120, besides binding to galactocerebroside or galactosyl-sulfatide, specifically binds to a protein receptor of a relative molecular mass of approximately 180,000 Da (180 kDa) present on the CD4-negative glioma cells D-54, but not on Molt4 T lymphocytes. Binding of rgp120 to this receptor rapidly induced a tyrosine-specific protein kinase activity leading to tyrosine phosphorylation of 130- and 115-kDa proteins. The concentration of intracellular calcium was not affected by rgp120 in these cells. Our data suggest a novel signal transducing HIV-1 gp120 receptor on CD4-negative glial cells, which may contribute to the neuropathological changes observed in HIV-1-infected brains.

Brain↗
PubMed1992

Protein phosphorylation regulated by cyclic nucleotide-dependent protein kinases in cell extracts and in intact human lymphocytes.

A specific 46,000/50,000 molecular weight protein substrate for both cAMP-dependent protein kinase (cAK) and cGMP-dependent protein kinase (cGK) extensively characterized and purified from human platelets was found to be present also in human T-lymphocytes, B-lymphocytes and other cells and tumour cell lines. This protein termed vasodilator-stimulated phosphoprotein (VASP) was present in cytosol and membranes of lymphocytes. Addition of exogenous purified cAK or cGK to lymphocyte cytosol or membranes converted 80-90% of VASP to its phosphoform. Endogenous VASP phosphorylation in both cytosol and membranes was stimulated by the addition of cAMP but not by cGMP. With intact lymphocytes, prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) induced an increase of cAMP and converted 70% of VASP to its phosphoform. In contrast, an increase of cGMP was not associated with VASP phosphorylation although cGK was detected in lymphocytes. These data support the hypothesis that VASP phosphorylation may be an important component of cAMP-mediated regulation of lymphocyte function.

Cell Extracts↗
PubMed1992

Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells.

The phosphorylation of endogenous proteins was investigated in subcellular fractions prepared from isolated rabbit parietal cells incubated with either cimetidine (unstimulated) or a combination of histamine and forskolin (maximally stimulated). Phosphorylation of endogenous proteins in subfractions was then assessed in a post hoc assay using [gamma-32P]ATP as a phosphate donor in vitro. The Mg(2+)-dependent incorporation of [32P]phosphate into a 52-kDa protein (pp52M) was observed in the 4,000 g membrane fraction from stimulated but not unstimulated cells. The pp52M protein was identified as the type II regulatory subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (RII) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [32P]phosphate into RII in the in vitro assay in the presence of Zn2+ was apparent in the 4,000 g membrane from stimulated but not unstimulated cells. The results thus suggested that, on stimulation, RII in membrane was dephosphorylated. Incorporation of [32P]phosphate into membrane-associated RII was completely abolished in the presence of 10 microM cAMP. The decrease in RII phosphorylation in membrane from stimulated cells assayed in the presence of cAMP was due to a phosphoprotein phosphatase activity that was completely inhibited by okadaic acid (1 microM). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound RII by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [14C]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.

Adenosine Triphosphatases↗
PubMed1992

Localization of the human gene for the type I cyclic GMP-dependent protein kinase to chromosome 10.

We have recently characterized cDNAs and genomic DNA fragments for human type I cGMP-dependent protein kinase (cGK). By probing human x hamster hybrid cell lines with a 1.2-kb intron fragment from the human type I cGK gene, we identified a 5.9-kb BglII restriction fragment and localized it to human chromosome 10. In situ hybridization analyses using 3H-labeled cDNA and genomic DNA probes for the human type I cGK to human metaphase chromosomes supported the somatic cell hybrid data and indicated that the gene (PRKG1B; protein kinase, cGMP-dependent) maps to 10p11.2----q11.2.

Blotting, Southern↗
PubMed1991

Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells.

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

Animals↗
PubMed1991

Endothelial cell-dependent phosphorylation of a platelet protein mediated by cAMP- and cGMP-elevating factors.

We reported previously that a 46/50-kDa membrane-associated vasodilator-stimulated phosphoprotein (VASP) is phosphorylated in intact human platelets in response to both cGMP- and cAMP-elevating vasodilator drugs and presented evidence that this is mediated by cGMP- and cAMP-dependent protein kinases, respectively. VASP was recently purified and an antibody against it was developed which detects a phosphorylation-induced mobility change of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Halbrügge, M., Friedrich, C., Eigenthaler, M., Schanzenbächer, P., and Walter, U. (1990) J. Biol. Chem. 265, 3088-3093). We have now used these methods for the quantitative analysis of VASP phosphorylation during coincubations of human endothelial cells and human platelets. Endothelial cell-derived factors caused the rapid, stoichiometric, and reversible phosphorylation of platelet VASP during these coincubations. Other experiments indicated that the endothelium-derived factors which stimulate VASP phosphorylation are prostacyclin and endothelium-derived relaxing factor whose effects are mediated by cAMP/cAMP-dependent protein kinase and cGMP/cGMP-dependent protein kinase, respectively. The results suggest that VASP phosphorylation is an important component of the inhibitory effects of prostacyclin and endothelium-derived relaxing factor on platelet activation and that VASP phosphorylation is a useful biochemical marker for the interaction of endothelial cells and platelets.

Autoradiography↗
PubMed1991

[Primary coronary stent implantation in complex morphology of stenosis or unsatisfactory results of angioplasty].

Early and late results of coronary angioplasty are influenced by the morphology of the coronary artery stenosis. Primary implantation of a balloon-expandable Palmaz-Schatz stent was indicated in 37 patients (31 men and 6 women; mean age 57 +/- 2 years) with symptomatic coronary heart disease and complex morphology of the stenosis. Intracoronary stents were successfully placed in 30 of 37 patients, without complications in all but one. This patient developed a thrombotic occlusion of the vessel 3 days after the procedure which, despite high-dosage thrombolysis treatment, resulted in an anterior wall myocardial infarction. A repeat angiography has since been performed (3-7 months after the procedure) in 25 of the 29 patients with a complication-free course. In 19 of the 25 patients the site of implantation was smooth, while a silent re-occlusion had occurred in one after successful recanalization of a chronic occlusion. Two patients had a subtotal restenosis (12% angiographic restenosis rate). These data indicate that intracoronary stent implantation improves the morphological results after balloon dilatation and reduces the restenosis rate.

Angiography↗
PubMed1991

Comparison of vasodilatory prostaglandins with respect to cAMP-mediated phosphorylation of a target substrate in intact human platelets.

The recent purification of a vasodilator-stimulated phosphoprotein (VASP) from human platelets and the development of a specific antiserum against VASP made it possible to study the quantitative effects of cAMP-elevating prostaglandins on cAMP-mediated phosphorylation of VASP in intact human platelets. Prostacyclin (PG-I2), prostaglandin-E1 (PG-E1) and the stable prostacyclinanalog Iloprost, all agents used for the treatment of peripheral vascular disease, induced rapid, stoichiometric and reversible phosphorylation of VASP in human platelets mediated by the cAMP-dependent protein kinase. However, there were substantial differences between these three cAMP-elevating prostaglandins with respect to their effects on extent, duration and reversibility of VASP phosphorylation. Maximal VASP phosphorylation was induced both by PG-I2 and Iloprost, but the PG-I2 effect was only of short duration in comparison to that of Iloprost. The extent of PG-E1-induced VASP phosphorylation was less than that observed with PG-I2 and Iloprost. In endothelial cell-platelet coincubations, an endothelial cell-derived, indomethacin-sensitive factor caused a rapid elevation of platelet cAMP level and VASP phosphorylation. These results provided direct evidence that human endothelial cells are capable of producing biologically active quantities of cAMP-elevating prostaglandins sufficient to induce stoichiometric cAMP-mediated protein phosphorylation in human platelets. VASP-phosphorylation induced by PG-I2 and PG-E1 was completely reversible after removal of the prostaglandins whereas this was only partially the case with Iloprost. In addition, evidence is presented that the prostaglandin-regulated adenylate cyclase system but not the cAMP-mediated protein phosphorylation desensitizes in human platelets after prolonged treatment with cAMP-elevating prostaglandins. VASP phosphorylation is proposed as a marker for quantitating aspects of vessel wall-platelet interaction.

Alprostadil↗
PubMed1991

[Effects of high and low doses of acetylsalicylic acid on the restenosis rate after initially successful coronary angioplasty].

A comparison was made in 79 patients (63 men, 16 women: mean age 52 +/- 9 years) of the effect of high and low doses of aspirin on restenosis rate during the first six months after originally successful percutaneous transluminal coronary angioplasty (PTCA), 39 patients (group 1) received 1000 mg aspirin daily, while 40 (group 2) received 100 mg daily. All patients took 1000 mg aspirin as loading dose on the day before PTCA, and additionally calcium antagonists and slow-release nitrates in the post-PTCA period. Both groups were comparable with respect of localization of the dilated coronary artery stenosis and the morphological changes after dilatation. Intimal lesions after PTCA were demonstrated in 9 patients of group 1 and 10 of group 2. Within six months clinically significant restenosis had occurred in 8 patients of group 1 and 7 of group 2. 33 patients in group 2 and 31 in group 1 were free of symptoms and had no ischaemic reaction on the exercise ECG six months after the initial successful PTCA. These results demonstrate that high aspirin dosage does not reduce the restenosis rate more than low dosage.

Adult↗
PubMed1991

Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes.

Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

1-Methyl-3-isobutylxanthine↗